Herpes simplex virus types 1 and 2 in organotypic cultures of mouse central and peripheral nervous system. 2. Electron microscopic observations of myelin degeneration. Organotypic cultures of mouse spinal cord with attached dorsal root ganglia, which contain both central and peripheral myelin in the one unit of tissue, were infected with HSV 1 or HSV 2 and studied using electron microscopy. Intranuclear viral nucleocapsids and intracytoplasmic enveloped particles were found in the Schwann cells associated with peripheral myelin and in oligodendroglia associated with central myelin. Degeneration of peripheral myelin most commonly involved an asymmetrical swelling of the myelin lamellae, whereas degeneration of central myelin was characterized by a more generalized swelling resulting in separation of the myelin lamellae. Degeneration of both central and peripheral myelin was found in the presence of intact axons which were indistinguishable from those in controls.


Cerebellar ganglioglioma in a child.

Cerebellar ganglioglioma in a child. A cerebellar ganglioglioma was surgically removed from a two-year old boy, who had developed manifestations of increased intracranial pressure and cerebellar symptoms. At surgery, the tumor presented as a firm nodular mass displacing the cerebellar cortex. By light microscopy, its architecture differed distinctly from that of hamartomatous diffuse hypertrophy of the cerebellar cortex (Lhermitte-Duclos' disease). Mature ganglion cells were grouped in clusters and linked by thick bundles of nerve cell processes. Nerve cells and processes were enmeshed in a rich network of fibrillary connective tissue. Electron microscopy disclosed typical neuronal perikarya as well as numerous asymmetric chemical synapses. The bulk of the tumor consisted of tightly grouped, (non-myelinated) nerve cell processes arranged in parallel. One of the most prominent features of the tumor consisted of numerous dilatations of these processes. The largest ones contained microfilaments, while the smaller ones were entirely filled with dense bodies (most probably derived from degenerating mitochondria). Only scattered dense core vesicles were seen, which probably did not represent neurosecretory granules. A second cell type consisted probably of astrocytes. Most neuroepithelial cell processes could not be identified with certainty as being of either neuronal or glial origin. A third cell type consisted of numerous slender cells which were probably mesenchymal. They were surrounded by a network of basement membrane which extended between the surrounding nerve cell processes.


Acoustic input to single neurons in pulvinar-posterior complex of cat thalamus. 1. Extracellular microelectrode recordings have been made of 429 single neurons in the pulvinar-posterior (Pul-PO) complex and adjacent regions of the thalamus of cats anesthetized with either sodium pentobarbital or alpha-chloralose. Controlled acoustic stimuli were presented by sealed systems incorporating probe microphone assemblies. 2. Neurons in pulvinar, lateralis posterior, and nucleus posterior were unresponsive to acoustic stimulation. Few cells in medial PO were observed to receive acoustic input, while sensitivity to tonal stimuli was a general feature of driven cells in other PO divisions. 3. Cells in lateral PO were generally sharply tuned to stimulus frequency, while the majority of cells in magnocellular medial geniculate and intermediate division of PO were broadly tuned. 4. Neurons in lateral PO and magnocellular medial geniculate had short response latencies to acoustic stimulation. Cells in intermediate division of PO were more often long latency. 5. Divisions of PO could not be differentiated on the basis of their binaural properties. Cells receiving excitatory input from solely the contralateral ear (E/O) or fros with onset discharge patterns showed occlusive binaural interaction properties. For cells with multiple-component discharge patterns, individual response components frequently had different patterns of binaural input and/or interaction. 6. On the basis of their discharge patterns, short latency, and frequency-tuning properties, it is suggested that lateral PO and magnocellular medial geniculate might derive their acoustic input from different divisions of the inferior colliculus. In contrast, the long latencies of units in PO intermediate division suggests a corticofugal input. 7. These data support anatomical parcelations of the Pul-PO complex, and the suggestion that this complex might provide acoustic input to the association cortices is evaluated.


Effects of stimulating in raphe nuclei and in reticular formation on response of spinothalamic neurons to mechanical stimuli. 1. The purpose of these experiments was to compare effects of electrical stimuli applied in two regions of the brain stem that are the sites of origin of descending bulbospinal systems; namely, the nucleus gigantocellularis of Brodal (7) and the nucleus raphe magnus, on the responses of lumbosacral spinothalamic neurons to mechanical stimuli. 2. In cats anesthetized with alpha-chloralose, stimulating in either of these structures with single pulses of current while the spinothalamic neuron was tonically activated by a sustained mechanical pressure resulted in an increase in the excitability of the cell followed by a prolonged suppression of its impulse activity. 3. For different neurons, the latency of the excitation ranged from 4 to 18 ms following the brain stem stimulus, while the latency of the suppression ranged from 16 to 34 ms. 4. In general, the effects of stimulating in the reticular formation and in the raphe nuclei were similar. although quantitative differences were found in the effects of each on different spinothalamic neurons. On the basis of these two studies, it is argued that the reticulospinal and raphe-spinal systems exert qualitatively similar effects on the responses of spinothalamic neurons evaluated in this experiment. 5. A comparison of the magnitudes of the suppression phase evoked from several different sites in the ipsilateral reticular formation and nucleus raphe magnus suggests that the descending systems arising from both these structures may be quite heterogeneous. 6. Stimulation of both regions of the brain stem produced a much greater suppression of the response of the spinothalamic neurons to slowly changing or sustained mechanical stimuli than to transient stimuli. It is suggested that the effects of descending systems arising both in the raphe nuclei and in the reticular formation on the responses of spinothalamic neurons to a mechanical stimulus are at least as dependent on the time course of the mechanical stimulus as they are on its intensity.


Brain stem gigantocellular neurons: patterns of activity during behavior and sleep in the freely moving rat. 1. The activity of 44 single brain stem gigantocellular neurons was recorded in the freely moving rat during the following four states: quiet waking (W), waking with movement (W-M), slow-wave sleep (SWS), and rapid eye movement (REM) sleep. 2. Cells were classified into three groups on the basis of the states in which they maintained their highest rate of discharge. The three cell categories were: movement-REM (MOV-REM), movement (MOV), and quiet waking (QW) neurons. The MOV-REM neurons, comprising 68% of the cell population, discharged significantly more during waking-movement and REM sleep than during either W or SWS. The MOV neurons, 16% of the cells, showed significant increases in activity only when the rat moved. The QW neurons, also 16% of the cells, typically maintained high rates of discharge in the absence of movement. 3. The MOV-REM neurons were further divided into two subclasses of cells--phasically and tonically discharging neurons. The phasic MOV-REM cells appeared to participate in phasic motor events of REM sleep and corresponding movements during waking. The pattern of activity of the tonic MOV-REM neurons suggested that they may be involved in the generation and maintenance of the theta rhythm of the hippocampus during waking-movement and REM sleep. 4. No cells were found to discharge significantly more in REM sleep or SWS sleep than in the other states, (i.e., no REM or SWS selective cells were seen).


Comparison of receptive-field properties of X and Y ganglion cells with X and Y lateral geniculate cells in the cat. 1. To examine the transmission of visual information through the lateral geniculate nucleus, we have studied the receptive-field properties of 65 X and Y optic tract axons and compared them with the receptive-field properties of X and Y LGN cells in paralyzed cats anesthetized with N2O/O2 (70/30%). The same experimental conditions and quantitative methods have been used as in the preceding study of LGN cells (2). 2. The spatiotemporal organization of the receptive fields of X and Y retinal axons are similar to those of X and Y LGN cells. X ganglion cell receptive fields show a simple center-surround organization, whereas Y ganglion cell receptive fields show a more complex organization with three concentric regions: a central region of center-type response, a region of mixed center-type and surround-type responses, and a region of surround-type response. 3. The inhibitory strengh of the surrounding region was tested with a centrally located flashing light spot of successively increased diameter. As in the LGN, the inhibitory strenght of the surrounding region was stronger in retinal X-cells than in retinal Y-cells, and the strength of the inhibition decreased as the diameter of the receptive-field center increased. 4. The decrease of the inhibitory strength of the surrounding region with increasing distance from the receptive-field center was similar in the retina and in the LGN for cells belonging to the same class (X or Y) and having the same receptive-field center size. 5. The differences in properties in the LGN between small-field X-, large-field X-, and Y-cells are best explained by assuming that they are driven, respectively, by small-field X, large-field X, and Y retinal ganglion cells. There does not appear to be a significant mixing of properties either between cells having different receptive-field center sizes. 6. The principal transformation we found between retinal and LGN units is that X LGN cells have sharply lower spontaneous activities and driven activities, as compared with X ganglion cells. Y LGN units show only a small decrease in spontaneous activity in comparison with Y ganglion cells. 7. We conclude that there is a significant alteration in the LGN only in the properties of X-cells, possibly by way of a strong inhibitory pool converging on X LGN units. We further suggest that this inhibitory pool plays a role in the modulation of transmission of information through the LGN only in the X channel, while the Y channel appears to be relatively unaffected.


Electrical interaction between motoneurons and afferent terminals in cat spinal cord. 1. The electrical threshold of the terminations of single group Ia and II afferent fibers was transiently reduced by the antidromic discharge of the initial segment of spinal motoneurons in the cat. 2. This depolarization of the terminals of afferent fibers known to synapse on motoneurons confirms previous observations of antidromic electrical interaction between these structures.


Emission-computed tomography and its application to imaging of acute myocardial infarction in intact dogs using Tc-99m pyrophosphate. The techniques of emission-computed tomography have been used to obtain in vivo quantitative estimates of the three-dimensional distribution of gamma-emitting radionuclides in dog hearts. Conjugate views, obtained for 60 equiangular projections around 360 degrees by rotating the object in front of a gamma camera, were used to reconstruct multiple-level emission transaxial images for various test objects, and for dogs with surgically induced acute myocardial infarcts. Corrections for attenuation were performed in the backprojection step of the convolution algorithm used for reconstruction. Quantitative estimates of the spatial extent and concentration of activity were obtained to within 10--15% rms error. Correlations were obtained between the radionuclide and histopathologic estimates of the extent and location of infarction.


Differential renal function using technetium-99m dimercaptosuccinic acid (DMSA): in vitro correlation. The relationship between differential renal uptake of Tc-99m 2-3 dimercaptosuccinic acid (DMSA) and differential renal function was examined in normal and abnormal dogs by correlating Tc-99m DMSA localization with relative renal blood flow and creatining clearance. There was close correlation of fractional Tc-99m DMSA localization with relative renal function.


Radiation decomposition of technetium-99m radiopharmaceuticals. Technetium-99m radiopharmaceuticals are shown to be subject to autoradiation-induced decomposition, which results in increasing abundance of pertechnetate in the preparation. This autodecomposition is catalyzed by the presence of oxygen, although the removal of oxygen does not prevent its occurrence. The initial appearance of pretechnetate in the radiopharmaceutical is shown to be a function of the amount of radioactivity, the quantity of stannous ion used, and the ratio of Tc-99m to total technetium in the preparation.


Juvenile oral angiofibroma in a female.

Juvenile oral angiofibroma in a female. A case of oral angiofibroma in a 14-year-old girl is presented. Unlike in earlier reports, the patient was confirmed as female by gynecological examination and other specific examinations. The possibility of the origin of the angiofibroma from the Meckel's cartilage is postulated. Any exuberant, sessile growth with a tendency toward spontaneous bleeding should be viewed with suspicion in terms of angiofibroma. Lesions such as pyogenic granulomas, hemangiomas, myxomas, and fibromyxomas should be kept in mind while making a diagnosis of angiofibroma. A biopsy examination as a routine office procedure should not be done on benign-looking masses with tendencies to bleeding.


Enhancement by disulfiram (Antabuse) of toxic effects of 95 to 97% O2 on the rat lung. Disulfiram (Antabuse), a drug used in alcohol aversion therapy, has been demonstrated to protect various species against hyperbaric O2 toxicity. In contrast, we have found that disulfiram accelerates the onset of pulmonary edema and death of rats exposed to normobaric 95 to 97% O2. When rats were given 200 mg of disulfiram per kg b.wt., 100% of the rats died at 24 to 48 hr of O2 exposure whereas only 5% of the rats died when exposed to O2 without disulfiram. This effect was not seen with an equal dose of diethyldithiocarbamate, the reduced monomer of disulfiram. The toxic effect was not due to an inhibition of superoxide dismutase, nor did disulfiram significantly affect the level of glutathione or change the reduced to oxidized glutathione ratio in the lung. Concurrent administration of 200 mg per kg b.wt. of ascorbate, vitamin E or reduced glutathione or 100 mg/kg of catalase did not affect the toxic response.


Effect of polybrominated biphenyls on the development of hepatic excretory function. Polybrominated biphenyls (PBBs) stimulate hepatic drug metabolism in adult and developing rats. The purpose of this investigation was to determine the influence of PBBs on the development of the liver as an organ for chemical excretion. Exposure of developing rats to polybrominated biphenyls (PBBs) by feeding 50 ppm of PBBs to pregnant or lactating mothers and rat weanlings did not produce overt toxicity when compared to controls over a 49-day postnatal period. However, prenatal and postnatal dietary exposure to PBBs resulted in elevated liver weight. In 15-day-old rats, increased liver weight after PBBs was correlated with enhanced ouabain transport from plasma into bile. Liver weight was also elevated in 21-, 35- and 49-day-old rats treated with PBBs, but this effect was not associated with stimulation of ouabain transport in these animals. Stimulation of ouabain transport after PBBs in 15-day-old rats was associated with increased hepatic uptake of ouabain. Stimulation in 15-day-old rats and not older rats may be attributed to the relative importance of uptake for ouabain transport in 15-day-old rats.


Control of phenylethanolamine N-methyltransferase by glucocorticoids in cultured bovine adrenal medullary cells. A procedure for the disaggregation of bovine adrenal medullary cells and a system for their maintenance in primary culture is described. The effect of glucocorticoids on the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), was examined. The initial level of PNMT activity in cultured cells was maintained by cortisol for 5 days. PNMT activity in untreated cultures declined steadily. After 5 days in culture the response of PNMT activity to cortisol was lost. The EC50 for maintenance of PNMT activity was about 15 ng/ml or 4 x 10(-8) M cortisol. Steroid structural and stereochemical requirements were similar to other glucocorticoid responses. Changes in the functional group or steric configuration of position 11 of cortisol abolished activity. Progesterone inhibited the maintenance of PNMT activity by cortisol in a competitive fashion. It was concluded that the response of PNMT activity to glucocorticoids is specific and sensitive, with strict structural and stereochemical requirements.


Modulation of the turnover rate of hippocampal acetylcholine by neuropeptides: possible site of action of alpha-melanocyte-stimulating hormone, adrenocorticotrophic hormone and somatostatin. The intraventricular injection of alpha-melanocyte-stimulating hormone (alpha-MSH), adrenocorticotrophic hormone (ACTH1--24) or somatostatin increases the acetylcholine turnover rate (TRACh) in the hippocampus of rats. Two to 3 weeks after surgical transection of the projections from the cingulum of the entorhinal cortex to the hippocampus the injections of these peptides can still activate hippocampal TRACh. alpha-MSH, ACTH1--24 and somatostatin also increase hippocampal TRACh when injected two to 3 hr after section of the fimbria. In contrast, the intraseptal administration of these peptides fails to change the hippocampal TRACh. The results suggest that the increase in hippocampal TRACh elicited by the three polypeptides may be caused by their interaction with receptors located in the hippocampus. Moreover, the data exclude the possibility that these peptide receptors may be located in septum or in other telencephalic areas that contain neurons projecting to the hippocampus. In addition, this study shows that the septal-hippocampal cholinergic pathway is necessary to elicit a specific stretching-yawning syndrome described by Ferrari et al. (Ann. N.Y. Acad. Sci. 104: 330--345,  1963) after injection of alpha-MSH or ACTH1--24.


Acute renal haemodynamic and renin-angiotensin system responses to graded renal artery stenosis in the dog. 1. The acute renal haemodynamic and renin-angiotensin system responses to graded renal artery stenosis were studied in chronically instrumented, unanaesthetized dogs. 2. Stenosis was induced over 30 sec by inflation of a cuff around the renal artery to lower distal pressure to 60, 40 or 20 mmHg, with stenosis maintained for 1 hr. This resulted in an immediate fall in renal vascular resistance, but over the next 5--30 min both resistance and renal artery pressure were restored back towards prestenosis values. Only transient increases in systemic arterial blood pressure and plasma renin and angiotensin levels were seen with the two milder stenoses. Despite restoration of renal artery pressure, renal blood flow remained reduced at all grades of stenosis. 3. Pre-treatment with angiotensin I converting enzyme inhibitor or sarosine1, isoleucone8 angiotensin II greatly attenuated or abolished the restoration of renal artery pressure and renal vascular resistance after stenosis, and plasma renin and angiotensin II levels remained high. Renal dilatation was indefinitely maintained, but the normal restoration of resistance and pressure could be simulated by infusing angiotensin II into the renal artery. 4. The effective resistance to blood flow by the stenosis did not remain constant but varied with changes in the renal vascular resistance.


The effect of hypoxia on evoked potentials in the in vitro hippocampus. 1. We have studied the effect of hypoxia on transmission of electrical activity between the perforant path and the dentate granule cells in the in vitro guinea-pig hippocampus. 2. Hypoxia abolishes the evoked field potential within about 3 min, a time similar to that occurring in vivo (Andersen, 1960). 3. The evoked potential is very rapidly abolished by extracellular K+ concentrations greater than 13.4 mM; it is abolished by ouabain concentrations greater than 10(-5) M. The rate at which it is abolished increases with increasing ouabain concentrations: concentrations of about 8 x 10(-5) M abolish the evoked potential at the same rate as does hypoxia. 4. The time required to abolish the evoked potential during hypoxia decreases markedly as the extracellular K+ concentration is elevated from 4.4 to 13.4 mM. The time to abolish the potential during hypoxia is also decreased by partial replacement of the Cl- in the bathing medium by less permeant anions and by the presence of a low (10(-7) M) concentration of ouabain. All these are conditions which are expected to depolarize neuronal cell membranes. None of these alterations in the perfusing medium affect the concentrations of ATP or creatine phosphate in the hippocampal slice. Increasing extracellular Mg2+/Ca2+ to levels which reduce the evoked response by about 50% has no effect upon the time required to abolish the evoked potential during hypoxia at any concentration of extracellular [K+]. 5. These results provide evidence that the basis for the hypoxic block of the evoked potential is a depolarization of neuronal processes. They are consistent with the hypothesis that this depolarization is a result of inhibition of the Na+/K+ pump.


On the effect of ionophoretically applied dopamine on salivary gland cells of Nauphoeta cinerea. 1. Responses to ionophoretically applied dopamine ('ionophoretic responses') have been recorded with an intracellular micro-electrode from acini of isolated salivary glands of the cockroach, Nauphoeta cinerea. They resembled responses to nerve stimulation ('neural responses') but they usually decayed more slowly and were sometimes more rapid in onset. 2. As already described for neural resonses, ionophoretic responses (i) included a secondary phase of depolarization (although more rarely than neural responses), (ii) increased in duration with increased amplitude and (iii) when small, added superlinearly. These features therefore reflect post-synaptic processes. By contrast, tachyphylaxis was much more marked for neural responses and is therefore mainly of presynaptic origin. 3. Ionophoretic and neural responses were affected similarly by increase in temperature. The times-to-peak of both decreased with values for Q10 of about 3. 4. The differences between the onset and decay of ionophoretic and neural responses can probably be explained by the inherently different conditions of application of the agonist. The similarities therfore provide further support for dopamine as the neurotransmitter.


On the time course of the electrical response of salivary gland cells of Nauphoeta cinerea to ionophoretically applied dopamine. 1. An attempt has been made to account for the latency and slow time course of the electrical responses of salivary gland cells of Nauphoeta cinerea (Olivier) to nerve stimulation and ionophoretically applied dopamine from a pipette placed as close as possible to the acinar surface. 2. The effects have been investigated on the time course of the ionophoretic response of changes in the distance of the pipette from the acinar surface and of the amount of dopamine ejected. 3. The observed changes were smaller than those predicted by models in which the rate limiting step was assumed to be diffusion or slow receptor kinetics. Indirect evidence suggests that the time course of the response is determined by processes subsequent to receptor activation.


3-Substituted pyrazole derivatives as inhibitors and inactivators of liver alcohol dehydrogenase. 3-Substituted pyrazoles, HOCH2 (1), HOCH2CH2 (2), HOCH2CH2CH2 (3), ClCH2 (4), ClCH2CH2 (5), ClCH2CH2CH2 (6), and CH3CO (7), were synthesized and evaluated in vitro on horse liver alcohol dehydrogenase for their potential as inhibitors of ethanol metabolism. 1 to 6 bound to the enzyme-NAD+ complex with dissociation constants of 40 to 200 microM, much higher than the constants for the corresponding 4-substituted pyrazoles, but with the same absorption maximum at 295 nm. 4 inactivated the enzyme within a few minutes, but NAD+ protected against reaction, and 4 nonspecifically alkylated many sulfur atoms in the protein. The isomer, 4-(chloromethyl)pyrazole, behaved similarly, 5 and 6 strongly inhibited the enzyme in the presence of NAD+, due to formation of the slowly dissociable (10(-3)s-1) enzyme-NAD+-pyrazole complex, but did not irreversibly inactivate the enzyme. 7 inhibits the enzyme weakly (Kp = 5 mM). It appears that the 3-substituted pyrazoles bind to the enzyme-NAD+ complex with the reactive functional group improperly positioned for specific irreversible reaction.


Effects of amorphous silicon dioxides on drug dissolution. The dissolution profiles of prednisone, digoxin, and griseofulvin in simulated GI fluids were determined after solvent deposition or ball milling with three commercially available grades of amorphous silicon dioxide. The former procedure resulted in adsorbates showing evidence of drug entrapment by the two grades with larger average pore diameters. Ball milling the drugs with the grade possessing the largest average particle diameter produced triturations with the slowest dissolution rates. A relationship between drug dissolution and extent of dilution with the amorphous silicon dioxides was shown. Particle-size measurements revealed that the ball milling procedure was more apt to broaden the size distribution as compared with the solvent-deposition method of drug incorporation.


Differential pulse polarographic determination of 2,3-dimercaptosuccinic acid and tin (II) in radiopharmaceuticals. A differential pulse polarographic procedure was developed for the assay of dimercaptosuccinic acid and tin(II), components of a commercially available pharmaceutical kit for kidney scintigraphy. The method is quantitative and qualitative for both the chelated and unchelated forms of dimercaptosuccinic acid and tin(II) in a mixture of the two.


[Topographic diagnosis of lesions of the acoustico-facial nerve complex using the winking reflex]. An electrophysiological study in close to 1,500 cases of various lesions of the trigeminal nerve, the acoustico-facial complex and the brain stem was presented. It was concluded that following a detailed clinical examination which, most often, allows precise topographical diagnosis, the study of the trigemino-facial and facial reflexes appears to be an essential diagnostic tool to determine the topography of a lesion. Moreover, this method may give prognostic evaluation as well as facilitate the choice of surgical approach to the lesions of the acoustico-facial complex.


Characterization of an Epstein-Barr virus-induced DNA polymerase. The addition of iododeoxyuridine to P3HR-I cell cultures led to a large increase in both Epstein-Barr virus (EBV)-induced DNA polymerase activity and early antigen-positive cells. This EBV-induced DNA polymerase was separated from the cellular alpha- and beta-polymerases by sequential column chromatography on Sepharose 6B, DEAE-cellulose, and phosphocellulose, resulting in partial purification of about 320-fold. The partially purified-EBV DNA polymerase could be distinguished from the cellular DNA polymerases by its activation by salts, its catalytic properties, and its degree of sensitivity to N-ethylmaleimide, phosphonoacetic acid, araATP, and araCTP. The viral polymerase showed properteis similar to those reported for other herpesvirus DNA polymerases. The enzyme exhibited optimal activity for copying activated calf DNA in the presence of 50 mH (NH4)2SO4 and was resistant to 150 mM (NH4)2SO4. It utilized with high efficiency template-primer poly(dC)-oligo(dG)12-18 or poly(dA)-oligo(dT)12-18, but failed to copy poly(rA)-oligo(dT)10 and oligo(dT)10, indicating that this enzyme has characters distinct from DNA polymerase gamma, reverse transcriptase, and terminal deoxynucleotidyl transferase. Phosphonacetic acid inhibited not only EBV DNA polymerase, but also, to a lesser degree, the cellular polymerase alpha. AraATP did not severely inhibit viral activity, whereas the polymerase alpha was inhibited most effectively. Both EBV polymerase and polymerase alpha were inhibited at a comparable level by araCTP.


Tryptic peptide analysis of gag gene proteins of endogenous mouse type C viruses. Tryptic digests of the internal proteins p30, p15, p12, and p10 of mouse xenotropic, ecotropic, and amphotropic type C viruses were subjected to cation-exchange chromatography. Analysis of these maps revealed that the p30 proteins from representative isolates of all three viral subgroups were distinguishable. The p15 proteins were all unique. The p12 proteins of NZB xenotropic and wild-mouse amphotropic viruses were not identical and yielded peptide maps remarkably different from that of the ecotropic virus. The p10 proteins of xenotropic and ecotropic viruses were identical and were dissimilar to that of the wild-mouse amphotropic virus.


Selective inhibition of protein synthesis in virus-infected mammalian cells. A number of translation inhibitors were tested for their effects on both control and encephalomyocarditis virus-infected mouse 3T6 cells. The virus-infected cells were specifically inhibited by gougerotin, edeine, and blasticidin S, whereas these drugs failed to penetrate into uninfected cells. Inhibition of infected cells by gougerotin became apparent when the synthesis of viral proteins commenced, suggesting that the latter process is accompanied by a permeability change in the cells that allows uptake of the drug. This permeability change was not observed in cells treated with cycloheximide soon after viral infection, although treatment with actinomycin D did not prevent inhibition of gougerotin. It is possible, therefore, that a specific viral protein is involved in the permeability change of the plasma membrane. Moreover, gougerotin was unable to inhibit protein synthesis in the presence of zinc ions, thus preventing gougerotin from entering into the infected cell. Membrane leakiness was not restricted to the encephalomyocarditis virus-3T6 system; it was also observed in mengovirus-infected 3T6 cells, Semliki Forest virus-infected BHK cells, and simian virus 40-infected CVI1 cells at the time in which the synthesis of late proteins is maximal.


Envelope proteins and replication of vesicular stomatitis virus: in vivo effects of RNA+ temperature-sensitive mutations on viral RNA synthesis. Temperature-sensitive (ts) mutants of vesicular stomatitis virus belonging to complementation groups III and V were investigated for their in vivo RNA synthesis. The sucrose gradient patterns of the RNA species which they produced at nonpermissive temperature (39.2 degrees C) were systematically compared under different experimental conditions: variation of input multiplicity and of time of infection, superinfection with T particles, and temperature shifts. Finally, a more precise analysis of the various RNA species synthesized was carried out. It appeared that the characteristics of RNA synthesis specified at 39.2 degrees C by tsIII or tsV mutants differed from the normal RNA synthesis of vesicular stomatitis virus wild type. Their common depression at 39.2 degrees C in virion-like RNA (38S) production--i.e., so-called genome replication--was tentatively paralleled with the concomitant ts events which have been previously shown to affect the two viral envelope proteins. An overproduction of the RNA transcripts was described for mutants in group III and posed the question of a regulation process to determine the amount of RNA to be transcribed.


Role of the membrane (M) protein in endogenous inhibition of in vitro transcription by vesicular stomatitis virus. An endogenous transcriptase inhibitor active at high concentrations of vesicular stomatitis (VS) virus was present in trypsinized whole virions but was absent from ribonucleoprotein cores containing only the L, N, and NS proteins. Poly(L-glutamic acid) effectively reversed the transcriptase inhibition. Transcription under noninhibited, inhibited, and poly(L-glutamic acid)-reversed conditions did not appear to greatly affect the nature of the RNA transcription product. The VS virion matrix (M) protein was purified to greater than 98% homogeneity and was found to have an isoelectric point of approximately 9.0. Purified M protein inhibited transcription by ribonucleoprotein cores, an effect that was partially reversed by poly(L-glutamic acid). Two group III temperature-sensitive (ts) mutants of VS virus (tsO23 and ts G31) with lesions in the M protein exhibited little or no endogenous inhibitor activity compared with two wild-type strains and a group V mutant (tsO45) with a lesion in the G protein. The data presented strongly suggest that the virion M protein is responsible for the endogenous inhibition of in vitro RNA synthesis seen at high concentrations of VS virus.


Altered restriction endonuclease cleavage pattern of simian virus 40 DNA. Three different groups of temperature-sensitive mutants of simian virus 40, isolated and characterized by Chou and Martin (J. Virol. 13:1101--1109, 1974), have been analyzed by using restriction endonucleases. Differences between the restriction endonuclease cleavage pattern of these mutants and that of the standard simian virus 40 strain have been mapped. These include the following observations: (i) tsD202 carries a defective HaeIII cleavage site at position 0.9 map units; (ii) tsB204 exhibits a defective HaIII site at position 0.21 and a defective HinIII site at 0.655 map units, and (iii) tsC219 carries a new HinIII site at position 0.15. We have isolated a few wild-type revertants from each of the temperature-sensitive mutant strains; each displays the endonuclease cleavage pattern of its parental temperature-sensitive strain.


Effect of actinomycin D on the expression of herpes simplex virus-common surface antigen in cells transformed by herpes simplex virus type 2. Using rabbit antiserum hyperimmune to herpes simplex virus (HSV) type 1, the expression of HSV-common surface antigen(s) was studied by indirect immunofluorescence tests in cells transformed by HSV type 2 and in derived tumor cells. The following results were obtained. (i) Antiserum to HSV type 1 reacted specifically with surface antigen present on the plasma membrane of both HSV type 2-infected and HSV type 2-transformed hamster cells. (ii) The expression of this antigen was enhanced in the absence of active protein synthesis in transformed cells, but not in tumor cells, after culture for 3 to 5 h at 37 degrees C. (iii) This enhancement of expression was maintained for 20 h in the presence of actinomycin D, but this prolonged expression required active protein synthesis. (iv) The enhancing effect observed in the presence of actinomycin D continued for some time after removal of the drug, for example, for 20 h after 5 h of treatment with 2 microgram/ml of actinomycin D per ml. Actinomycin D had no detectable effect on antigen expression in tumor cells. (v) The protease inhibitor antipain inhibited the actinomycin D-enhanced expression without causing significant cell damage but did not modify the transient enhanced expression of antigen when cells were seeded in the absence of actinomycin D. These results indicate that in transformed cells antigen expression can be enhanced in at least two ways.


Analysis of integrated avian RNA tumor virus DNA in transformed chicken, duck and quail fibroblasts. The state of integration of avian sarcoma virus DNA in the genomes of transformed chicken, duck, and quail fibroblasts was deduced by means of restriction enzyme digestion of total cell DNA, gel electrophoresis, and subsequent analysis by the procedure of Southern. The cells used in these studies were either mass-infected cultures or clones of infected cells selected by their ability to form colonies in agar. For both mass-infected cultures and clones of cells of all three species, we found that integration occurred at a specific site on the viral genome but appeared to occur at many sites on the cell genome. At least some of the integrated viral DNA existed as intact nonpermuted species flanked by direct terminal repeats of at least 0.134 megadalton (217 base pairs). For each of 12 transformed quail clones studied, it was possible to detect, after digestion with Kpn I, unique junctions between viral and cellular DNA. That is, at our level of analysis, the integration site on the cell genome for each clone was different. However, within each of the 17 chicken and 9 duck clones of transformed cells, a heterogeneity presumably occurred during the outgrowth of the cell clone population, in that we could not readily detect identifiable cell-virus junction fragments.


Morphological and biochemical characterization of viral particles produced by the tsO45 mutant of vesicular stomatitis virus at restrictive temperature. The growth at restrictive temperature of tsO45, a group V (glycoprotein) conditional lethal mutant of vesicular stomatitis virus (VSV), was demonstrated to result in the production of large numbers of noninfectious viral particles. The infectivity of these tsO45 particles could be enhanced by procedures known to promote membrane fusion. Morphologically and biochemically these particles differed from wild-type VSV by their lack of viral glycoprotein. The other structural proteins of VSV were present and indistinguishable by size and relative proportion from those of virus grown at the permissive temperature. Examination of glycoprotein maturation at the restrictive temperature (39.5 degrees C) in tsO45-infected cells demonstrated the synthesis of normal viral glycoprotein but failed to demonstrate the presence of this glycoprotein in either the cell membrane or the envelope of free virions. The further absence of soluble viral glycoprotein from the supernatants of such cells strongly suggests that viral glycoprotein may not be necessary for the successful budding of VSV.


Mutational alterations within the simian virus 40 leader segment generate altered 16S and 19S mRNA's. We have analyzed the structure of the late cytoplasmic RNAs made after infection with wild-type simian virus 40 and a set of viable mutants, four of which have deletions and one an insertion within the nucleotide sequence specifying the leader segment of the 16S and 19S mRNA's. The principal findings are: (i) simian virus 40 16S and 19S mRNA's made during infections with wild-type virnds and possibly in the nucleotide sequence comprising the "leader" segments. (II) "Spliced" 16S and 19S mRNA's are made during infections with each of the mutants although, in some cases, the ratio of 19S to 16S mRNA species is reduced. (iii) The deletion or insertion of nucleotides within the DNA segment defined by map position 0.70 to 0.75 causes striking alterations in the types of leader structures in the late mRNAs. (iv) Many of the late RNA leader segments produced after infection with the mutants appear to be multiply spliced, i.e., instead of the major 200- to 205-nucleotide-long leader segment present in wild-type 16S mRNA, the RNAs produced by several of the deletion mutants have leaders with whort discontiguous segments.


Association of simian virus 40 T antigen with simian virus 40 nucleoprotein complexes. Viral nucleoprotein complexes were extracted from the nuclei of simian virus 40 (SV40)-infected TC7 cells by low-salt treatment in the absence of detergent, followed by sedimentation on neutral sucrose gradients. Two forms of SV40 nucleoprotein complexes, those containing SV40 replicative intermediate DNA and those containing SV40 (I) DNA, were separated from one another and were found to have sedimentation values of 125 and 93S, respectively. [(35)S]methioninelabeled proteins in the nucleoprotein complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to VP1, VP3, and histones, a protein with a molecular weight of 100,000 (100K) is present in the nucleoprotein complexes containing SV40 (I) DNA. The 100K protein was confirmed as SV40 100K T antigen, both by immunoprecipitation with SV40 anti-T serum and by tryptic peptide mapping. The 100K T antigen is predominantly associated with the SV40 (I) DNA-containing complexes. The 17K T antigen, however, is not associated with the SV40 (I) DNA-containing nucleoprotein complexes. The functional significance of the SV40 100K T antigen in the SV40 (I) DNA-containing nucleoprotein complexes was examined by immunoprecipitation of complexes from tsA58-infected TC7 cells. The 100K T antigen is present in nucleoprotein complexes extracted from cells grown at the permissive temperature but is clearly absent from complexes extracted from cells grown at the permissive temperature and shifted up to the nonpermissive temperature for 1 h before extraction, suggesting that the association of the 100K T antigen with the SV40 nucleoprotein complexes is involved in the initiation of SV40 DNA synthesis.


Adenovirus type 12 gene 401 function and maintenance of transformation. Rat (3Y1) and hamster embryo brain cells were transformed by wild-type adenovirus type 12 or the DNA-minus temperature-sensitive mutant ts401. The ts401-transformed 3Y1 cells, but not the wild-type transformants, displayed a temperature-sensitive response with respect to the following characteristics of the transformed phenotype: morphology, saturation density, growth rate, cloning in soft agar, colony formation on plastic at low cell densities in 1% serum medium, and the T antigen(s). Temperature shift-down experiments showed that the density-dependent inhibition of growth of the ts401-transformed cells was reversible, as was, to some extent, the low efficiency of colony formation at low cell densities in 1% serum. Examination of hamster transformants for their ability to clone in soft agar at permissive and nonpermissive temperatures showed that this property was temperature dependent, again only in the ts401 transformants and not in the wild-type transformants. Alteration in uptake of 2-deoxyglucose or in intracellular cyclic AMP content was not a characteristic of the adenovirus-transformed phenotype in the 3Y1 cells. The findings suggest that an active 401 function is required for maintenance of the adenovirus-transformed cell pheno-type.


Epstein-Barr virus-specific RNA. III. Mapping of DNA encoding viral RNA in restringent infection. Namalwa and Raji cells, originally obtained from a Burkitt tumor biopsy, grow as continuous cell lines in vitro and contain the Epstein-Barr virus (EBV)-related nuclear antigen EBNA (B. M. Reedman and G. Klein, Int. J. Cancer 11:499-520, 1973) and RNA homologous to at least 17 and 30% of the EBV genome, respectively (S. D. Hayward and E. Kieff, J. Virol. 18:518-525, 1976; T. Orellana and E. Kieff, J. Virol. 22:321-330, 1977). The polyribosomal and polyadenylated [poly(A)+] RNA fractions of Namalwa and Raji cells are enriched for a class of viral RNA homologous to 5 to 7% of EBV DNA (Hayward and Kieff, J. Virol. 18:518-525, 1976; Orellana and Kieff, J. Virol. 22:321-330, 1977). The objective of the experiments described in this communication was to determine the location within the map of the EBV genome (D. Given and E. Kieff, J. Virol. 28:524-542, 1978) of the DNA which encodes the viral RNA in the poly(A)+ and non-polyadenylated [poly(A)-] RNA fractions of Namalwa cells. Hybridization of labeled DNA homologous to Namalwa poly(A)+ or poly(A)- RNA to blots containing EcoRI, Hsu I, or Hsu I/EcoRI double-cut fragments of EBV (B95-8) or (W91) DNA indicated that these RNAs are encoded by DNA contained primarily in the Hsu I A/EcoRI A and Hsu I B/EcoRI A fragments and, to a lesser extent, in other fragments of the EBV genome. Hybridizations of Namalwa poly(A)+ and poly(A)- RNA in solution to denatured labeled EcoRI A or B fragments, Hsu I A, B, or D fragments, and Hsu I A/EcoRI A or Bam I S fragments and of Raji polyribosomal poly(A)+ RNA to the EcoRI A fragment indicated that (i) Namalwa poly(A)+ RNA is encoded primarily by 6 x 10(5) daltons of a 2 x 10(6)-dalton segment of DNA, Bam I S, which is tandemly reiterated, approximately 10 times, in the Hsu I A/EcoRI A fragment and is encoded to a lesser extent by DNA in the Hsu I B, EcoRI B, and Hsu I D fragments. Raji polyribosomal poly(A)+ RNA is encoded by a similar fraction of the EcoRI A fragment as that which encodes Namalwa poly(A)+ RNA. (ii) The fraction of the Bam I S fragment homologous to Namalwa poly(A)- RNA is similar to the fraction homologous to Namalwa poly(A)+ RNA. However, Namalwa poly(A)- RNA is homologous to a larger fraction of the DNA in the Hsu I B, Hsu I D, and EcoRI B fragments.


Control of herpes simplex virus type 1 mRNA synthesis in cells infected with wild-type virus or the temperature-sensitive mutant tsK. This paper deals with control of mRNA levels, assayed by in vitro translation, in cells infected with herpes simplex virus type 1 (HSV-1). A particularly useful marker has been pyrimidine deoxyribonucleoside kinase (dPyK) mRNA, for which the enzymatically active product can be assayed quantitatively. Cells infected with the HSV-1 temperature-sensitive mutant tsK at the nonpermissive temperature (38.5 degrees C) or with wild-type HSV-1 in the continuous presence of cycloheximide contained no detectable dPyK mRNA. Upon temperature shift-down of tsK-infected cells to 31 degrees C, dPyK mRNA was produced, and this event was inhibited by actinomycin D but not cycloheximide. This result demonstrated that the defective polypeptide in tsK-infected cells was involved in transcription of the dPyK gene and could regain activity at 31 degrees C. Because tsK-infected cells synthesized mainly immediate early polypeptides at 38.5 degrees C, the involvement of this polypeptide class in synthesis of dPyK mRNA was investigated. Analysis of the kinetics of inductions of dPyK mRNA indicated that the temperature-sensitive lesion in tsK lies in an immediate early polypeptide which is directly responsible for activation of the dPyK gene at the transcriptional level.


Extracellular cleavage of the glycoprotein precursor of Rous sarcoma virus. The kinetics of cleavage of pr92gp, the precursor of the two glycoproteins of Rous sarcoma virus gp85 and gp35, were followed. Viral glycoproteins were detected by immunoprecipitation with anti-gp85 and anti-gp35 serum. It could be shown in pulse-chase experiments that little or no intracellular cleavage of the precursor took place during the time in which the majority of newly synthesized viral glycoprotein was exported from the cells. Soon after its synthesis, however, pr92gp underwent some modification that made it migrate slightly faster on sodium dodecyl sulfate-polyacrylamide gels. Under steady state conditions the precursor was shown to be the predominant form of intracellular viral glycoprotein. Virus which was harvested every 2 min from infected cells prelabeled for 90 min with [3H]mannose contained mostly uncleaved and only a little mature glycoprotein. By incubation of this freshly released virus in serum-free buffer, the majority of the glycoprotein precursor could be cleaved into mature gp85 and gp35. Virus harvested every 10 min contained only mature glycoproteins.


Specific protein phosphorylation in interferon-treated uninfected and virus-infected mouse L929 cells: enhancement by double-stranded RNA. The enhanced phosphorylation of specific protein(s) observed in extracts from interferon-treated cells (in the presence of ATP and double-stranded [ds] RNA) was also seen in intact mouse L929 cells upon treatment with dsRNA, polyriboinosinic.polyribocytidylic acid [poly(rI.rC)] or reovirus dsRNA, using 32Pi as radiolabel. Labeling of a 65,000-dalton protein(s) with 32P was greatly increased in interferon-treated cells in the presence of added dsRNA, suggesting that the expression in vivo of the kinase activity involved is regulated by dsRNA. This was used as a test system to investigate whether the activity of interferon-induced enzyme(s) is stimulated following virus infection, possibly owing to the accumulation of dsRNA. No obvious increase in 32P-labeling of 65,000-dalton protein(s) was observed upon infection of interferon-treated cells with mengovirus or vesicular stomatitis virus. A basal level of 32P-labeling of the 65,000-dalton protein(s) was detected in interferon-treated cells in the absence of added dsRNA, indicating a basal level of expression of the kinase activity involved. The possible implications of these results are discussed.


Growth and maturation of a vesicular stomatitis virus temperature-sensitive mutant and its central nervous system isolate. A temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), tsG31, produces a prolonged central nervous system disease in mice with pathological features similar to those of slow viral diseases. tsG31 and the subsequent virus recovered from the central nervous system (tsG31BP) of mice infected with tsG31 were compared with the parental wild-type (WT) VSV for plaque morphology, growth kinetics, thermal sensitivity of the virions, and viral protein synthesis and maturation. Several properties of the central nervous system isolate distinguished this virus from the original tsG31 and the WT VSV. The WT VSV produced clear plaques with complete cell lysis, and the tsG31 produced diffuse plaques and incomplete cell lysis, whereas the tsG31BP had clear plaques similar to those of the WT VSV. Although plaque morphology suggested that tsG31BP virus was a revertant to the WT, growth kinetics in either BHK-21 or neuroblastoma (N-18) cells indicated that this virus was similar to tsG31, with a productive cycle at 31 degrees C and no infectious virus at 39 degrees C. At 37 degrees C, however, the tsG31BP matured much slower than did the original tsG31 (and produced only 1% of the yield measured at 31 degrees C). WT VSV produced similar quantities of infectious virions at 31, 37, and 39 degrees C. The lack of infectious virions at 39 degrees C for the ts mutants was presumably not due to a greater rate of inactivation at 39 degrees C. Unlike WT VSV, which synthesized viral proteins equally well at all three temperatures, tsG31 had a reduced synthesis of all the structural proteins at 37 and 39 degrees C, compared with that at 31 degrees C; the formation of the M protein was most temperature sensitive. In addition, fractionation of the infected cells indicated that the incorporation of the M and N proteins into the cellular membranes was also disrupted at the higher, nonpermissive temperatures. Several characteristics of protein synthesis during tsG31BP infection at 39 degrees C distinguished this virus from tsG31: (i) no mature viral proteins were detected at 39 degrees C; (ii) several host proteins were [ill], suggesting that the virus was incapable of completely depressing host macromolecular synthesis; and (iii) a great proportion of the incorporated radioactivity was found in unusually high-molecular-weight proteins. In addition, at 37 degrees C, the tsG31BP virus showed a decreased synthesis of viral proteins and reduced assembly of the viral structural proteins.


Gene D5 product of bacteriophage T5: DNA-binding protein affecting DNA replication and late gene expression. Gene D5 is not only necessary for replication of bacteriophage T5 DNA and for shutoff of expression of some early genes, but has been found to be necessary also for the expression of late T5 genes. The polypeptide product of gene D5 has been identified, an intragenic map of gene D5 has been constructed, and the direction of transcription of gene D5 has been established. The polypeptide coded by gene D5 has been shown to be a DNA-binding protein with affinity for both double- and single-stranded DNA.


Selective packaging of host tRNA's by murine leukemia virus particles does not require genomic RNA. The 4S RNA contained in RNA tumor virus particles consists of a selected population of host tRNA's. However, the mechanism by which virions select host tRNA's has not been elucidated. We have considered a model which specifies that 35S genomic RNA determines which tRNA's are to be encapsidated as well as the relative amounts of these tRNA's within the virion. The model was tested by comparing the free 4S RNA composition of normal murine leukemia virus (MuLV) particles and noninfectious virions from actinomycin D (ActD)-treated cells, which are deficient in genomic RNA (ActD virions). Viral 4S RNA was analyzed by two-dimensional polyacrylamide gel electrophoresis. Surprisingly, the patterns obtained for control and ActD 4S RNA were identical to each other and were clearly distinct from the cell 4S RNA pattern. The viral patterns had three prominent areas of radioactivity. One of the spots was identified on the basis of its oligonucleotide fingerprint as tRNA (Pro), the primer for MuLV RNA-directed DNA synthesis. These results were obtained with two different MuLV strains, AKR and Moloney, each grown in SC-1 cells. The demonstration that ActD virions contain primer tRNA and in general exhibit the characteristic MuLV tRNA pattern rather than the complete representation of cell 4S RNA leads to the conclusion that genomic RNA is not the major determinant in selective packaging of host tRNA's. A possible role for one or more viral proteins, including reverse transcriptase, is suggested.


Oligosaccharide chains of avian RNA tumor virus glycoproteins contain heterogeneous oligomannosyl cores. Chicken embryo fibroblasts (C/E phenotype) infected with subgroups B and C of the Prague strain of Rous sarcoma virus were radiolabeled with either [6-(3)H]-glucosamine or [2-(3)H]mannose, and virus was purified from the growth medium. The large envelope glycoprotein, gp85, was the only major radiolabeled component of purified virus. Pronase-digested glycopeptides from purified virus were analyzed by a combination of (i) gel filtration with columns of Sephadex G15/G50 and Bio-Gel P4 and (ii) enzymatic digestion of the oligosaccharide chains with specific exoglycosidases and endo-beta-N-acetylglucosaminidases. The rather broad molecular weight distribution (approximately 2,000 to 4,000) for glycopeptides in these studies and previous studies in other laboratories was shown to represent actual heterogeneity in the carbohydrate moieties: (i) the glycopeptides contained both mannose-rich, neutral chains and complex, acidic chains with terminal sialic acid; and (ii) both classes of asparagine-linked carbohydrate structures exhibited heterogeneity in the size of the oligomannosyl core (a mixture of approximately 5 to 9 mannose units for the neutral structures, and 3 or 5 mannose units for the acidic structures). With the [2-(3)H]mannose-labeled glycopeptides from Rous sarcoma virus, Prague strain subgroup C, most of the oligosaccharide chains were high-molecular-weight, acidic structures, with similar numbers of 3-mannose and 5-mannose core structures.


Restricted replication of two alphaviruses in ricin-resistant mouse L cells with altered glycosyltransferase activities. Two mouse L cell variant lines (CL 3 and CL 6) selected for resistance to the toxic plant lectin ricin were restricted in their ability to replicate the two alphaviruses Sindbis virus and Semliki Forest virus. CL 3 cells have been shown to exhibit increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, whereas CL 6 cells have been shown to contain decreased UDPgalactose:glycoprotein galactosyltransferase and UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activities. The adsorption of Sindbis virus to CL 6 cells was considerably reduced, suggesting that the loss or inaccessibility of the receptors for Sindbis virus accounted for a major defect in virus production in these cells. In contrast, CL 3 synthesized Sindbis viral RNA and proteins but were unable to convert the precursor glycoprotein PE2 to the structural protein E2. The cleavage of PE2 to E2 was also blocked in both CL 3 and CL 6 cells infected with Semliki Forest virus.


Poliovirus polyuridylic acid polymerase and RNA replicase have the same viral polypeptide. A poliovirus-specific polyuridylic acid [poly(U)] polymerase that copies a polyadenylic acid template complexed to an oligouridylic acid primer was isolated from the membrane fraction of infected HeLa cells and was found to sediment at 4 to 5S on a linear 5 to 20% glycerol gradient. When the poly(U) polymerase was isolated from cells labeled with [(35)S]methionine and was analyzed by glycerol gradient centrifugation and polyacrylamide gel electrophoresis, the position of only one viral protein was found to correlate with the location of enzyme activity. This protein had an apparent molecular weight of 62,500 based on its electrophoretic mobility relative to that of several molecular weight standards and was designated p63. When the poly(U) polymerase was isolated from the soluble fraction of a cytoplasmic extract, the activity was found to sediment at about 7S. In this case, however, both p63 and NCVP2 (77,000-dalton precursor of p63) cosedimented with the 7S activity peak. When the 7S polymerase activity was purified by phosphocellulose chromatography, both p63 and NCVP2 were found to co-chromatograph with poly(U) polymerase activity. The poliovirus replicase complexed with its endogenous RNA template was isolated from infected cells labeled with [(35)S]methionine and was centrifuged through a linear 15 to 30% glycerol gradient. The major viral polypeptide component in a 26S peak of replicase activity was p63, but small amounts of other poliovirus proteins were also present. When the replicase-template complex was treated with RNase T1 before centrifugation, a single peak of activity was found that sedimented at 20S and contained only labeled p63. Thus, p63 was found to be the only viral polypeptide in the replicase bound to its endogenous RNA template, and appears to be active as a poly(U) polymerase either as a monomer protein or as a 7S complex.


Structural relationship between the 100,000- and 17,000- molecular-weight T antigens of simian virus 40 (SV40) as deduced by comparison with the SV40-specific proteins coded by the nondefective adenovirus type 2-SV40 hybrid viruses. The two-dimensional peptide maps of the methionine-containing tryptic peptides of the 100,000-molecular-weight (100K) and 17K T antigens of simian virus 40 (SV40) have been compared. The two proteins share nine methionine-containing tryptic peptides in common. The 17K T antigen has two peptides not found in the 100K T antigen, and the 100K T antigen has 14 unique peptides. The peptide maps of the 100 K and 17K T antigens were also compared with those of the SV40-specific proteins found in cells infected by the nondefective adenovirus type 2-SV40 hybrid viruses, which we have previously shown are encoded by defined sequences within the early region of SV40 (K. Mann, T. Hunter, G. Walter, and H.K. Linke, J. Virol. 24:151-169, 1977). This comparison shows that the 100K and 17K T antigens share common N-terminal sequences coded for between 0.65 and 0.59 map units on the SV40 genome. Furthermore, none of the sequences in the 17K T antigen arises from the region between 0.54 and 0.18 map units. We deduce that the sequences unique to the 17K T antigen originate between 0.59 and 0.54 map units. This type of structural relationship between the 100K and 17K T antigens fits well with the proposed model (L.V. Crawford, C.N. Cole, A. E. Smith, E. Paucha, P. Tegtmeyer, K. Rundell, and P. Berg, Proc. Natl. Acad. Sci. U.S.A. 75:117-121, 1978) for the expression of the early region of SV40.


Gibbon ape leukemia virus-Hall's Island: new strain of gibbon ape leukemia virus. Gibbon ape leukemia virus-Hall's Island (GaLV-H), a type C virus related to previous isolates of GaLV and simian sarcoma virus, was isolated from a gibbon ape with lymphocytic leukemia from a small colony of free-ranging gibbon apes on Hall's Island near Bermuda. We show here by molecular hybridization experiments that GaLV-H is approximately 60% related to three previous isolates of GaLV (GaLV-SF, GaLV-SEATO, and GaLV-Br) and is less closely related to simian sarcoma virus. The oligopyrimidine pattern of a transcript of the terminal 135 +/- 5 nucleotides of the viral RNA of GaLV-H is similar to that of GALV-Br but distinct from that of GaLV-SF and simian sarcoma virus. GaLV-H thus represents a fifth distinct strain of the infectious primate type C viruses, which among the previously described isolates of GaLV is most closely related to GaLV-Br.


Integration of Rous-associated virus type O provirus in susceptible chicken cells. The number of viral genome equivalents per haploid cell genome was determined in normal chicken embryos from three selected chicken lines and in cultured fibroblasts (CEF) from these embryos. The cellular concentration of endogenous proviral DNA is similar in embryos from chickens of lines SPAFAS, 7, 15, 7 x 15, and 100. The concentration of proviral DNA is not affected by in vitro cultivation in CEF from lines that do not spontaneously produce virus, nor in CEF from line 7, which lacks receptors for Rous-associated virus type 0 (RAV-0). There is, however, a restricted increase in the number of integrated proviral genome equivalents in CEF from line 7 x 15, which produces RAV-0 and can support replication of this virus, and in CEF from line 15 experimentally infected with RAV-0.


Persistence of herpes simplex virus type 1 in rat neurotumor cells. Herpes simplex virus type 1 (HSV-1) infection of a rat central nervous system tumor cell line led to almost complete destruction of the cells. Cells that survived the infection could be isolated and shown to produce infectious HSV particles for variable lengths of time in culture ranging from 20 to 57 passages. Even though infectious virus production eventually ceased, the cell lines continued to produce herpes-specified proteins as measured by immunological techniques. These cells also showed herpesvirus-like structures in the electron microscope. The persistently infected cells that produced HSV antigens and bore HSV sequences were resistant to superinfection by HSV-1. The resistance was not due to failure of adsorption of the virus or to the production of interferon by the cells. The nature of the block in HSV replication in these neurotumor cells, which contain and partially express the HSV genome, is unknown, but may offer an interesting parallel to the known latency of HSV in neural tissues.


Relationship between T-antigen and tumor-specific transplantation antigen in simian virus 40-transformed cells. The simian virus 40 (sv40) tumor antigen (T-antigen) and tumor-specific transplantation antigen (TSTA) have been partially purified and studied to clarify their relationship. The T-antigen and the TSTA were partially purified from nuclei of SV AL/N cells, and SV40-transformed mouse embryo fibroblast line, by precipitation with ammonium sulfate and chromatography on DEAE- and DNA-cellulose. The T-antigen was assayed by complement fixation, and the TSTA was assayed by its ability to immunize mice against SV40-containing ascites tumor cells. When T-antigen- and TSTA-containing preparations were sedimented through sucrose gradients, each antigen had a major peak of activity at a sedimentation coefficient of 6.7 and minor peaks in other regions. Antiserum against T-antigen (from tumor-bearing hamsters) immunoprecipitated the TSTA activity. A preparation of T-antigen from human SV80 cells, which exhibited only one protein band after sodium dodecylsulfate-polyacrylamide gel electrophoresis, had TSTA activity when as little as 0.6 microgram of protein per mouse was used for immunization. These experiments demonstrate that the T-antigen, the product of the SV40 early A gene is capable of inducing specific immunity against transplantation of SV40-transformed tumor cells in mice.


Herpes simplex virus type 2 functions expressed during stimulation of human cell DNA synthesis. Experiments were designed to identify herpes simplex virus type 2 (HSV-2)-specific functions expressed during stimulation of human embryo fibroblast DNA synthesis. Cultures were partially arrested in DNA synthesis by pretreatment with 5-fluorouracil and maintenance in low-serum (0.2%) medium during virus infection. Results showed that continuous [methyl-(3)H]thymidine uptake into cellular DNA was ninefold greater in HSV-2-infected than in mock-infected cultures measured after 24 h of incubation at 42 degrees C. Shifting mock-infected cultures from low- to high-serum (10%) medium also caused some stimulation, but [methyl-(3)H]thymidine uptake was only twofold greater than in cells maintained with low serum. Plating efficiencies of both HSV-2-infected and mock-infected cells at 42 degrees C were essentially the same and ranged from 37 to 76% between zero time and 72 h of incubation. De novo RNA and protein syntheses were continuously required for HSV-2 stimulation of cellular DNA synthesis. HSV-2 infection markedly enhanced transport, phosphorylation, and rate of incorporation of [methyl-(3)H]thymidine into cellular DNA, starting at 3 h and reaching a maximum by 12 h; after 12 h, these processes gradually declined to low levels. In mock-infected cells these processes remained at low levels throughout the observation period. Pretreatment of cells with interferon or addition of arabinofuranosylthymine at the time of virus infection inhibited stimulation caused by HSV-2. 5-Bromodeoxyuridine density-labeled experiments revealed that HSV-2 stimulates predominantly semiconservative DNA replication and some DNA repair. Stimulation of [methyl-(3)H]thymidine into cellular DNA correlated with detection of virus-specific thymidine kinase activity. In conclusion, HSV-2 stimulation of cellular DNA synthesis appeared to involve at least four virus-specific functions: induction of thymidine transport, HSV-2 thymidine kinase activity, semiconservative replication, and repair of cellular DNA.


Characterization of some isolates of newly recovered avian sarcoma virus. We previously reported the isolation of a newly recovered avian sarcoma virus (rASV) from tumors of chickens injected with transformation-defective (td) mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV). In this paper, we present further biological and biochemical characterization of the recovered sarcoma viruses. High titers of rASV's were generally obtained by cocultivation of tumor cells with normal chicken embryo fibroblasts or by homogenization of tumor tissues. Most rASV isolates were similar to SR-RSV, subgroup A (SR-RSV-A), in their growth characteristics and were nondefective in replication. The subgroup specificity of rASV's and the electrophoretic mobilities of their structural proteins were the same as those parental td viruses. The nondefectiveness of rASV's was further substantiated by the size of their genomic RNA, which was indistinguishable from that of SR-RSV-A and substantially larger than that of parental td RNA. Molecular hybridization using complementary DNA specific to the src gene of SR-RSV (cDNAsrc) showed that the RNAs of td mutants used in this study contained extensive deletions within the src gene (7 to 30% hybridization with cDNAsrc); the same probe hybridized up to 90% with RNA from two isolates of rASV. These data indicate that rASV has regained genetic information which had been deleted in the td mutants and strongly suggest that the generation of rASV involves a genetic interaction between td virus and host cell genetic information.


Herpes simplex virus type 1 Angelotti and a defective viral genotype: analysis of genome structures and genetic relatedness by DNA-DNA reassociation kinetics. DNA-DNA reassociation kinetics of herpes simplex virus type 1 Angelotti DNA and a class of defective viral DNA revealed that the viral standard genome has a total sequence complexity of about 93 X 10(6) daltons and that a portion of 11 X 10(6) daltons occurs twice on the viral genome. These results agree with structural features of herpes simplex virus type 1 DNA derived from electron microscopic studies and restriction enzyme analyses by several investigators. The defective viral DNA (molecular weight, about 97 X 10(6)) displays a sequence complexity of about 11 X 10(6) daltons, suggesting that the molecule is built up by repetitions of standard DNA sequences comprising about 15,000 base pairs. A 2 X 10(6)-dalton portion of these sequences maps in the redundant region and a 9 X 10(6)-dalton portion maps in the unique part of the standard herpes simplex virus type 1 Angelotti DNA, as could be shown by reassociation of viral standard DNA in the presence of defective DNA and vice versa. No cellular DNA sequences could be detected in defective DNA. A 12% molar fraction of the defective DNA consists of highly repetitive sequences of about 350 to 500 base pairs in length.


Noninfectious vesicular stomatitis virus particles deficient in the viral nucleocapsid. Several temperature-sensitive mutants of vesicular stomatitis virus in complementation group III produce, at nonpermissive temperature, noninfectious particles which contain the viral M (matrix) and G (glycoprotein) proteins but less than 10% of the normal proportion of N protein or RNA. Since group III mutants are thought to be defective in the structural gene for the virus M protein, these findings demonstrate that an interaction between M and the nucleocapsid is of importance in virus budding. Taken together with earlier results, they suggest that M is the key protein in bud formation.


Anatomy of herpes simplex virus DNA. XII. Accumulation of head-to-tail concatemers in nuclei of infected cells and their role in the generation of the four isomeric arrangements of viral DNA. Previous reports (H. Delius and J. B. Clements, J. Gen. Virol. 33:125-134, 1976; G. S. Hayward, R. J. Jacob, S. C. Wadsworth, and B. Roizman, Proc. Natl. Acad. Sci. U.S.A. 72:4243-4247, 1975; B. Roizman, G. S. Hayward, R. Jacob, S. W. Wadsworth, and R. W. Honess, Excerpta Med. Int. Congr. Ser. 2:188-198, 1974) have shown that herpes simplex virus DNA extracted from virions accumulating in the cytoplasm of infected cells consists of four populations of linear molecules differing in the orientation of the covalently linked large (L) and small (S) components relative to each other. Together, these four isomeric arrangements of viral DNA display four different termini and four different L-S component junctions. In the studies reported in this paper, we analyzed with restriction endonucleases the newly replicated viral DNA shortly after the onset of viral DNA synthesis, the progeny DNA accumulating in the nuclei late in infection, and rapidly sedimenting DNA present in nuclei of infected cells at 8 h after infection. In each instance the nuclear viral DNA contained a decreased concentration of all four terminal fragments and an increase in the concentration of fragments spanning the junction of L and S components relative to the concentration of other DNA fragments. The results are consistent with the hypothesis that the viral DNA accumulating in the nuclei consists of head-to-tail concatemers arising from the replication of DNA by a rolling-circle mechanism. A model is presented for generation of all four isomeric arrangements of herpes simplex virus DNA from one arrangement based on excision and repair of unit length DNA from head-to-tail concatemers and known features of the sequence arrangement of viral DNA.


Evidence that the RNA polymerase usually does not make a complete transcript of the late strand of simian virus 40 DNA. An important model for the transcription of the late (L) strand of simian virus 40 DNA is that, late after infection of permissive monkey cells, the RNA polymerase makes a complete transcript of the L DNA strand before terminating transcription. The purpose of the current work was to test a prediction of this model, namely, that the rate of synthesis of all RNA sequences from the L DNA strand should be equal. About one-half of the L DNA strand is transcribed into late mRNA sequences and the other half into late dRNA sequences, which do not leave the nucleus. Using glucosamine to reduce the size of the intracellular UTP pool before and after a pulse-label with radioactive uridine, a pulse-chase experiment was performed to determine the half-lives of these sequences. The half-life of the late dRNA sequences was determined to be 4 min. The late mRNA sequences were degraded more slowly, on the average, than the late dRNA sequences. In a parallel experiment with similarly treated cells, it was shown that after a 2-min label with radioactive uridine there was only 0.2 times as much radioactivity in the late dRNA sequences as in the late mRNA sequences in the total cellular RNA population. The results could be combined to calculate that the rate of synthesis of the late dRNA sequences was at most 0.3 times that of the late mRNA sequences. Consequently they provide strong evidence that when the RNA polymerase transcribes the late mRNA sequences, it usually terminates transcription before all the late dRNA sequences are transcribed.


Effect of 5-methylcytidine on virus production in avian sarcoma virus-infected chicken embryo cells. 5-Methylcytidine (5mC) is a minor constituent of RNA in procaryotes as well as eucaryotes. The function of this modified nucleoside is not known. We studied the effect of this compound on virus production in avian sarcoma virus-infected chicken embryo fibroblasts. We found, surprisingly, that virus release into the medium was severely reduced in cultures treated with 5mC. In contrast to the effect of 5mC on virus release, intracellular levels of virus-specific RNA transcripts as well as the proteins were slightly elevated. Analysis of intracellular RNA transcripts on velocity gradients and virus-specific proteins in polyacrylamide gels did not reveal any qualitative differences in 5mC-treated cells compared to cytidine-treated cells. From these results we conclude that the effect of 5mC is probably at the level of virus maturation or packaging.


Murine mammary tumor virus expression during mammary tumorigenesis in BALB/c mice. Steady-state levels of murine mammary tumor virus (MuMTV) RNA were quantitated during mammary tumorigenesis in BALB/c mice by molecular hybridization with a representative MuMTV complementary DNA (cDNA) probe. Hyperplastic alveolar nodule (HAN) lines are preneoplastic mammary lesions that were induced in BALB/c mice by hormones alone or in combination with 7,12-dimethylbenz(a)anthracene and give rise to mammary tumors. The hormone-induced HAN lines D1 and D2 contained detectable amounts of hybridizable MuMTV sequences. MuMTV RNA sequences were also observed in five of the six transplanted BALB/c mammary tumors that were examined. Similar levels of hybridizable MuMTV RNA were observed between the D1 or D2 HAN line and mammary tumors derived from each HAN line. The D2 HAN line as well as D2, C4, and CD8 mammary tumors accumulated RNA that was apparently homologous to most of the MuMTV genome. Thermal denaturation of hybrids indicated extensive sequence homology between the MuMTV cDNA and hybridizable RNA in the BALB/c HAN lines and mammary tumors. A low level of type C viral RNA was observed in the BALB/c HAN lines and most mammary tumors by molecular hybridization with a cDNA to Moloney murine leukemia virus. These data demonstrate that MuMTV sequences are frequently expressed in hormone-induced BALB/c HAN lines and mammary tumors derived from HAN lines or ductal hyperplasias induced in BALB/c mice by hormones and/or a chemical carcinogen. The transition from the preneoplastic to the neoplastic state in BALB/c mice does not appear to be due to a change in the steady-state levels of MuMTV RNA since the hormone-induced HAN lines and mammary tumors had similar levels of hybridizable MuMTV RNA.


Rescue and transmission of a replication-defective variant of moloney murine leukemia virus. We have described a clone of mouse cells, termed "8A," which appears to be infected with a replication-defective variant of Moloney murine leukemia virus (MuLV) (Rein et al., J. Virol. 25:146-156, 1978). Clone 8A cells release virus particles which do not form plaques in the standard XC test. However, approximately 10(2) particles per ml of clone 8A supernatant do form plaques in a modified XC test (the "complementation plaque assay"), in which the assay cells are coinfected with the XC-negative, nondefective amphotropic MuLV as well as the test virus. Superinfection of clone 8A cells themselves with amphotropic MuLV results in the production of approximately 10(5), rather than approximately 10(2), particles per ml which register in the complementation plaque assay. This increase is due to the rescue of replication-defective ecotropic MuLV from clone 8A cells by amphotropic MuLV since (i) this ecotropic MuLV can only form XC plaques in cells which are coinfected with amphotropic MuLV; and (ii) it is possible to transmit this defective variant, rescued from superinfected clone 8A cells, to a fresh clone of normal mouse cells. The time course of production of the rescued MuLV particles by superinfected clone 8A cells is virtually identical to that of rescue from these cells of murine sarcoma virus. Amphotropic MuLV superinfection of "NP-N" cells, which contain a "non-plaque-forming" variant of N-tropic MuLV (Hopkins and Jolicoeur, J. Virol. 16:991-999, 1975), also increases the titer of particles registering in the complementation plaque assay; thus, NP-N cells, like clone 8A cells, contain a rescuable defective variant of ecotropic MuLV.


Genes controlling receptors for ecotropic and xenotropic type C virus in Mus cervicolor and Mus musculus. Gene loci controlling cell surface receptors for murine leukemia virus were studied by using murine X Chinese hamster hybrid cells. Hybrids which exclusively segregate murine chromosomes were made by fusing Mus cervicolor and Mus musculus lymphocytes to hamster fibroblasts. Sensitivity to Moloney murine leukemia virus infecotion and specific binding of the envelope glycoprotein of Rauscher murine leukemia virus (gp70) cosegregate and isozyme analysis show an association with chromosome 5 in both species. With the possible exception of one clone, no evidence was found for a proviral integration site independent of chromosome 5. Evidence is presented for additional unlinked ectropic and xenotropic receptors independent of chromosome 5.


Nature of Rous sarcoma virus-specific RNA in transformed and revertant field vole cells. Cytoplasmic and polyribosomal RNAs from Rous sarcoma virus-transformed and phenotypically reverted field vole cells were fractionated by rate-zonal sedimentation and hybridized with a (3)H-labeled complementary DNA viral probe to determine the size classes of virus-specific RNA present in these cell types. In contrast to Rous sarcoma virus-infected permissive avian cells, only two of three discrete species of virus-specific RNA were detected in the cytoplasm of these vole cells. These included genome-length 35S RNA and a 21S RNA. However, viral 28S RNA, routinely detected in the cytoplasm of productively infected avian cells, could not be found in cytoplasmic RNA from vole cells. In addition, a low-molecular-weight viral RNA sedimenting less than 16S was detected in both infected avian and vole cells. Because of its heterogeneity this latter species is most likely generated from the intracellular degradation of the larger viral RNAs. Both the viral 35S and 21S RNA were also found to be associated with total polyribosomes from these vole cells. Studies were also performed to determine the distribution of both total viral genomic and sarcoma-specific RNA sequences among the size classes of fractionated total polyribosomes. In both vole cell types the majority of cytoplasmic viral RNA sequences were also associated with polyribosomes and were similarly distributed among the size classes of total polyribosomes. Sarcoma-specific sequences were present on both the 35S and 21S RNA species. These data suggest that the expression of the viral transforming gene in revertant field vole cells may be controlled at some stage subsequent to translation of the viral RNA.


Precipitation of visna viral proteins by immune sera of rabbits and sheep. Eighteen polypeptides equivalent to 1.2 x 10(6) daltons of visna virus were specifically precipitated by immune sera from rabbits and sheep. The hyperimmunized rabbit antisera contained high concentrations of antibodies against p25 and p14, whereas the sera from sheep actively infected with visna virus showed a large quantity of anti-gp115 antibody. The results indicate that almost all the polypeptides reported previously (F. H. Lin, J. Virol. 25:207--214, 1978) are virus-specific components of visna. The presence of anti-gp115 antibody in sera of infected sheep may offer a simple and sensitive diagnostic procedure for visna.


Effect of alkylation on the physical properties of simian virus 40 T-antigen species. We analyzed large and small species of T-antigen by immunoprecipitation and two-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis either directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by two-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on two dimensional gels. Large-T did not form a discrete protein spot, but rather formed a streak from approximately pH 6.5 to 6.9 on isoelectric focusing gels. Small-t formed a sharp protein spot at approximately pH 7.2 when subjected to electrophoresis under non-equilibrium conditions which extended the pH gradient to include proteins with basic isoelectric points. Treatment with N-ethylmaleimide decreased the mobility of the T-antigen species during sodium dodecyl sulfate gel electrophoresis. We suggest that the apparent increase in molecular weight was due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of simian virus 40 which do not induce the synthesis of small-t but product small-t-related polypeptides were used to localize the cysteine-rich region of small-t to between 0.54 and 0.59 on the genetic map of simian virus 40.


In vitro, the major ribosome binding site on Rous sarcoma virus RNA does not contain the nucleotide sequence coding for the N-terminal amino acids of the gag gene product. Ribosomes from the reticulocyte lysate bind strongly and mainly to a region located in the 5' end of the Rous sarcoma virus RNA molecule between residues 9 and 53. This binding involves the participation of initiator tRNA and is sensitive to inhibitors of initiation of protein synthesis such as 7-methyl-GMP and aurintricarboxylic acid. The nucleotide sequence of this ribosome binding site has been determined: it conatains a GUG codon centered at position 26 that is not in phase with any termination codon within the 5' end nucleotide sequence of the RNA that we have analyzed (101 residues). However, the predicted N-terminal amino acid sequence starting from this GUG codon (or even from any AUG or GUG codon in the 5' end of the RNA) does not coincide with that of the in vitro-synthesized product of the 5' end proximal gag gene. Nevertheless, inhibition of ribosome binding to this site is accompanied by an inhibition of the in vitro translation of the gag gene.


Intracellular forms of simian virus 40 nucleoprotein complexes. I. Methods of isolation and characterization in CV-1 cells. A new method was developed for isolation of intracellular forms of simian virus 40 (SV40) nucleoprotein complexes from SV40-infected CV-1 cells late in the infectious cycle. In contrast to the Triton extraction method, which yields only a 60-70S complex, this new procedure yielded three forms of SV40 nucleoprotein complexes: complex I, complex II, and the nature virion (V). The three nucleoprotein complexes differed in physical as well as biochemical properties. Complex I, which is only a small portion of the total SV42 nucleoprotein complexes late during infection, was active in synthesizing both SV40-specific DNA and RNA. Pulse-labeling experiments suggest the following metabolic pathway: I leads to II leads to V. Conversion of complex I to II occurred shortly after the completion of SV40 DNA replication and resulted in the inactivation of the biosynthetic activities of I.


State and organization of polyoma virus DNA sequences in transformed rat cell lines. Polyoma virus-transformed rat cell lines were isolated as colonies growing in agar after infection of F2408 cells with low multiplicities of wild-type virus. Viral DNA present in the transformed cells was analyzed by fractionating the cellular DNA on agarose gels before and after digestion with various restriction endonucleases, followed by detection of the DNA fragments containing viral sequences using the procedure described by Southern (E. Southern, J. Mol. Biol., 98:503--515, 1975). Five lines, independently derived, were studied in detail. All five lines, when examined after a minimum number of passages in culture, contained both free and apparently integrated viral DNA. The free polyoma DNA in three of the lines was indistinguishable, by restriction enzyme analysis, from wild-type viral DNA, whereas the two other lines also contained smaller free DNA molecules which lacked parts of the wild-type genome. The integrated DNA in the five lines studies existed as head-to-tail tandem repeats of unit-length polyoma DNA covalently attached to nonviral DNA. The same five polyoma-transformed rat lines were examined after further passage in culture. Free viral DNA was then either undetectable or greatly reduced in amounts, whereas the high-molecular-weight, integrated units persisted after passage of the cells. The subclones, derived from one of the five lines selected for detailed analysis, showed some variations in the quantity and size of the free viral DNA as well as minor alterations in the pattern of the apparently integrated sequences.


Feline sarcoma virus-coded polyprotein: enzymatic cleavage by a type C virus-coded structural protein. A purified 15,000-molecular-weight (Mr) Prague strain Rous sarcoma virus gag gene-coded structural protein, p15, was shown to enzymatically cleave the previously described 130,000 Mr feline sarcoma virus-coded polyprotein, Pr130. Cleavage products included proteins ranging in molecular weight from 12,000 to 110,000. The specificity of this cleavage reactivity was indicated by the fact that, under similar conditions, neither purified type C viral structural proteins nor nonviral proteins such as bovine serum albumin were cleaved to significant extents. Moreover, feline leukemia virus Pr65gag was efficiently cleaved, resulting in the generations of proteins of 30,000 (p30), 15,000 (p15), 12,000 (p12), and 10,000 (p10) Mr. Using enzymatically (p15) treated feline sarcoma virus Pr130 as starting material, we were able to purify a major 72,000 Mr cleavage product and to show it to contain the previously described feline sarcoma virus-coded nonstructural component.


Nucleosomal structure of Epstein-Barr virus DNA in transformed cell lines. Micrococcal nuclease digestion was used to analyze Epstein-Barr virus (EBV) DNA structure in nuclei of transformed cells. Digests of virus-producing (P3HR-1), non-virus-producing (Raji), and superinfected Rajii cell nuclei were fractionated by electrophoresis on agarose gels, transferred to nitrocellulose, and hybridized to 32P-labeled EBV DNA. The viral DNA of Raji nuclei produced a series of bands on electrophoresis whose lengths were integral multiples of a unit size, which was the same as the repeat length of host DNA. Viral DNA in nuclei of P3HR-1 and superinfected Raji cells produced faintly visible bands superimposed on a smear of viral DNA which dominated the hybridization pattern. No differences were detected in the patterns when total DNA digests from Raji, P3HR-1, and an EBV DNA-negative cell line (U-698M) were analyzed by ethidium bromide staining or by hybridization with the use of 32P-labeled lymphoblastoid cell DNA as probe. We conclude that the EBV episomal DNA of Raji cells is folded into nucleosomes, whereas most of the viral DNA of P3HR-1 and superinfected Raji cells is not. This pattern of DNA organization differs signficantly from that in papova group viruses.


Molecular genetics of herpes simplex virus. II. Mapping of the major viral glycoproteins and of the genetic loci specifying the social behavior of infected cells. We have mapped the location in herpes simplex virus (HSV) DNA of (i) three mutations at different loci (syn loci) which alter the social behavior of infected cells from clumping of rounded cells to polykaryocytosis, (ii) a mutation which determines the accumulation of one major glycoprotein [VP8.0(C(2))], and (iii) the sequences encoding four major virus glycoproteins [VP8.0(C(2)), VP7(B(2)), VP8.5(A), and VP19E(D(2))]. The experimental design and results were as follows. (i) Analysis of HSV-1 x HSV-2 recombinants showed that the sequences encoding the VP19E(D(2)) glycoprotein map in the S component, whereas the sequences encoding the other three major glycoproteins are in two locations in the L component of HSV DNA. The templates specifying the HSV-1 and HSV-2 glycoprotein VP8.0(C(2)) appear not to be colinear; we isolated recombinants specifying glycoproteins comigrating in sodium dodecyl sulfate-polyacrylamide gels with VP8.0(C(2)) of both HSV-1 and HSV-2. (ii) Marker rescue of a ts mutant defective in accumulation of glycoprotein VP7(B(2)) showed that the mutation maps within a region containing the sequences encoding that glycoprotein. (iii) Marker transfer experiments involving transfection of rabbit skin cells with donor HSV-1(F) DNA and fragments from several donor strains causing fusion of Vero or both Vero and HEp-2 cells revealed the existence of three syn loci specifying the social behavior of cells and one locus (Cr) determining the accumulation of glycoprotein VP8.0(C(2)). The Cr locus maps to the right of the template specifying VP8.0(C(2)) glycoprotein. Loci syn 1 and syn 2 map at or near the Cr locus but can be segregated from it. Locus syn 3 maps at or near the template specifying glycoproteins VP7(B(2)) and VP8.5(A). The expression of mutations in the syn 1 and syn 3 loci appear to be cell type dependent, in that recombinants with these mutations fuse Vero cells but not HEp-2 cells. Recipients of the syn 2 locus or of both syn 2 and syn 1 loci fuse both Vero and HEp-2 cells.


Molecular genetics of herpes simplex virus. III. Fine mapping of a genetic locus determining resistance to phosphonoacetate by two methods of marker transfer. We have transferred a genetic locus determining resistance to phosphonoacetic acid (PAAr) from one herpes simplex viral genome to another by two methods of marker transfer. One method requires recombination between an intact DNA molecule and a restriction endonuclease DNA fragment, whereas the other requires repair of a partial heteroduplex formed between the two DNA molecules. These two methods mapped the PAAr locus between positions 0.45 and 0.53 map units on the physical map of the viral DNA. Fine mapping of the PAAr locus showed that it maps at or near an EcoRI restriction endonuclease site at either 0.46 or 0.49 map units. We also describe and compare the two methods of marker transfer.


Phosphorylation of T-antigen and control T-antigen expression in cells transformed by wild-type and tsA mutants of simian virus 40. Chinese hamster lung (CHL) cells transformed by wild-type simian virus 40 (cell line CHLWT15) or transformed by the simian virus 40 mutants tsA30 (cell lines CHLA30L1 and CHLA30L2) or tsA239 (cell line CHLA239L1) were used to determine the rates of turnover and synthesis of the T-antigen protein and the rate of turnover of the phosphate group(s) attached to the T-antigen at both the permissive and restrictive temperatures. The phosphate group turned over several times within the lifetime of the protein to which it was attached, with the exception of the phosphate group in the tsA transformants at 40 degrees C, which turned over at the same rate as the T-antigen protein. The steady-state levels of the T-antigens (molecular weights, 92,000 [92K] and 17K) and the amount of simian virus 40-specific RNA was also determined in each of the lines. The CHLA30L1 line contained two to three times more early simian virus 40 RNA than the CHLA30L2 line; although neither line formed colonies in agar at 40 degrees C, CHLA30L1 overgrew a normal monolayer at 40 degrees C. The rate of 92K-T-antigen synthesis was 1.5 times faster in CHLA30L1 than in CHLA30L2 at 33 degrees C and 4 times faster at 40 degrees C. The different phenotype of these two presumably isogenic cell lines seem to be related to the levels of the T-antigens. The ratios of the 92K T-antigen to the 17K T-antigens were similar in the two lines. Transformed CHL cell lines, unlike transformed mouse 3T3 cell lines, were found to contain very small amounts of the 56K T-antigen.


Transformation of human embryonic kidney cells by human papovarirus BK. Infection of secondary human embryonic kidney (HEK) cells with human papovavirus BK (BKV) resulted in cellular lysis and degeneration within 7 days. After 30 days, multilayered colonies of transformed cells were found and subcultured for analyses. These BK-HEK cells uniformly expressed the BKV T-antigen but were only 1% V-antigen positive. They produced infectious virus and were resistant to superinfection by BKV. They reached a saturation density of 1.3 x 10(5) cells per cm2 in medium with 5% fetal calf serum, were able to grow in medium containing 2% serum, and did not form colonies in soft agar or tumors in nude mice. Nonintegrated, superhelical BKV DNA was detected in the noncloned cells as expected because they were persistently infected and contained RNA transcripts complementary to both early and late regions of the BKV genome. Analysis of T-antigen-positive clonal isolates of these BK-HEK cells by the Southern technique revealed an absence of free viral DNA and the presencce of integrated BKV DNA sequences corresponding to the early region of the BKV genome. These studies demonstrate the stable transformation of human cells by BKV. However, the transformed human cells which retain and express part of the BKV genome do not fully manifest the growth properties of other papovarirus-transformed cells.


Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation. The avian sarcoma virus (ASV) protein responsible for cellular transformation in vitro and sarcomagenesis in animals was studied structurally with special reference to the sites of phosphorylation on the polypeptide. The product of the ASV src gene, pp60src, is a phosphoprotein of 60,000 daltons. We found that pp60src contained two major sites of phosphorylation, one involving phosphoserine and the other involving phosphothreonine and possible addtional minor sites of phosphorylation. By using N-formyl[35S]methionyl-tRNAf as a radiolabeled precursor in the cell-free synthesis of the src protein in conjunction with partial proteolysis mapping, we determined that the major phosphoserine residue was located on the amino-terminal two-thirds of the molecule and that the phosphothreonine was located on the carboxy-terminal third. We further determined that the phosphorylation of pp60src in cell extracts involved at least two protein kinases, the one that phosphorylated the major serine site being cyclic AMP dependent and the other, acting on the threonine residue, being a cyclic nucleotide-independnet phosphotransferase. Finally, analysis of the pp60src isolated from cells infected with a temperature-sensitive src gene mutant of ASV revealed that phosphorylation of the major threonine residue was severely reduced when infected cells were grown at the nonpermissive temperature, whereas a phosphorylation pattern characteristic of the wild-type pp60src was observed at the permissive temperature. As pp60src has an associated protein kinase activity, the possible involvement of phosphorylation-dephosphorylation reactions in the functional regulation of ASV transforming protein enzymatic activity is discussed.


Nucleotide sequence analysis of two simian virus 40 mutants with deletions in the late region of the genome. Two mutants of simian virus 40, dl-1261 and dl-1262, have deletions that map between coordinated 0.90 and 0.95 (Cole et al., J. Virol 24:277--294, 1977). Both affect the structure of the two minor proteins VP2 and VP3. The precise location and size of the deletions have now been determined by nucleotide sequence analysis. Mutant dl-1261 is deleted of 54 base pairs, is temperature sensitive for the protein defined by the D complementation group, and promotes the synthesis of shorter VP2 and VP3 polypeptides. Mutant dl-1262 is viable irrespective of temperature and has a deletion of 36 base pairs, 23 of which overlap the deletion in dl-1261. Since these mutants produce normal VP1, the deleted regions probably have no function in the splicing of precursor RNA to the VP1 mRNA.


Monomer and multimer covalently closed circular forms of Rous sarcoma virus DNA. Covalently closed circular molecules of viral DNA synthesized in virus-infected cells are composed mainly of monomers sedimenting at 22 to 27S in neutral sucrose gradients. These monomers are detected by annealing with complementary DNA or transfection assay. However, 11% of the infectious circles sediment faster than monomers. There is a peak at 32S which may correspond to dimer molecules. Traces of infectivity (about 3%) found between 32S and 65S suggest the presence of higher oligomers. In alkaline sucrose gradients, covalently closed monomers are found at 64 to 71S. Infectivity of these monomers is reduced by alkali treatment to less than one-tenth, and, perhaps for this reason, no infectious dimers or higher oligomers are observed. It has been shown that upon resedimentation the dimers of 95 can be separated from monomers and detected by hybridization.