Nature of the neutral Na(+)-Cl- coupled entry at the apical membrane of rabbit gallbladder epithelium: IV. Na+/H+, Cl-/HCO3- double exchange, hydrochlorothiazide-sensitive Na(+)-Cl- symport and Na(+)-K(+)-2Cl- cotransport are all involved. Transepithelial fluid transport was measured gravimetrically in rabbit gallbladder (and net Na+ transport was calculated from it), at 27 degrees C, in HCO(3-)-free bathing media containing 10(-4) M acetazolamide. Whereas luminal 10(-4) M bumetanide or 10(-4) M 4-acetamido-4'-iso-thiocyanostilbene-2,2'-disulfonate (SITS) did not affect fluid absorption, 25 mM SCN- abolished it; hydrochlorothiazide (HCTZ) in the luminal medium reduced fluid absorption from 28.3 +/- 1.6 (n = 21) to 8.6 +/- 1.6 microliters cm-2 hr-1 (n = 10), i.e., to about 30%. This maximum effect was already obtained at 10(-3) M concentration; the apparent IC50 was about 2 x 10(-4) M. The residual fluid absorption, again insensitive to SITS, was completely inhibited by SCN- or bumetanide. Cl- influx at the luminal border of the epithelium, measured under the same conditions and corrected for the extracellular space and paracellular influx, proved insensitive to 10(-4) M bumetanide, but was slowly inhibited by 10(-3) M HCTZ, with maximum inhibition (about 54%) reached after a 10-min treatment; it subsequently rose again, in spite of the presence of HCTZ. However, if the epithelium, treated with HCTZ, was exposed to 10(-4) M bumetanide during the measuring time (45 sec), inhibition was completed and the subsequent rise of Cl- influx eliminated. Intracellular Cl- accumulation with respect to the predicted activity value at equilibrium decreased significantly upon exposure to 10(-3) M HCTZ, reached a minimum within 15-30 min of treatment, then rose again significantly at 60 min. Simultaneous exposure to HCTZ and bumetanide decreased the accumulation to a significantly larger extent as compared to HCTZ alone, already in 15 min, and impeded the subsequent rise. Intracellular K+ activity rose significantly within 30 min treatment with HCTZ; the increase proved bumetanide dependent. The results obtained show that Na(+)-Cl- symport, previously detected under control conditions, is the HCTZ-sensitive type; its inhibition elicits bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport. Thus, the three forms of neutral Na(+)-Cl(-)-coupled transport so far evidenced in epithelia, Na+/H+, Cl-/HCO3- double exchange (in the presence of exogenous bicarbonate), HCTZ-sensitive Na(+)-Cl- symport and bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport, are all present in the apical membrane of rabbit gallbladder.


Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation. The regulation of intracellular pH (pHi) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2',7'-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO3(-)-containing medium (25 mM NaHCO3/5% CO2; pH 7.4) have a steady-state pHi of 7.25 +/- 0.02. Acid loading of acinar cells using the NH4+/NH3 prepulse technique resulted in a Na(+)-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, Ki approximately 0.42 microM) pHi recovery, the kinetics of which were not influenced by the absence of extracellular Cl-. The rate and magnitude of the pHi recovery were dependent on the extracellular Na+ concentration, indicating that Na+/H+ exchange plays a critical role in maintaining pHi above the pH predicted for electrochemical equilibrium. When the NH4+/NH3 concentration was varied, the rate of pHi recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na+/H+ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl(-)-free medium did not significantly alter resting pHi, suggesting the absence of Cl-/HCO3- exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t 1/2 < 30 sec; 0.16 +/- 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37 +/- 0.05 pH unit) or in the absence of extracellular Na+ (0.34 +/- 0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO3(-)-free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl-. The Ca2+ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid) to chelate intracellular Ca2+ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na+/H+ exchange activity; (ii) is most likely mediated by HCO3- efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca2+].


Nuclear magnetic resonance studies of the internal dynamics in Apo, (Cd2+)1 and (Ca2+)2 calbindin D9k. The rates of amide proton exchange with solvent. The backbone dynamics of the EF-hand Ca(2+)-binding protein, calbindin D9k, has been investigated in the apo, (Cd2+)1 and (Ca2+)2 states by measuring the rate constants for amide proton exchange with solvent. 15N-1H correlation spectroscopy was utilized to follow direct 1H-->2H exchange of the slowly exchanging amide protons and to follow indirect proton exchange via saturation transfer from water to the rapidly exchanging amide protons. Plots of experimental rate constants versus intrinsic rate constants have been analyzed to give qualitative insight into the opening modes of the protein that lead to exchange. These results have been interpreted within the context of a progressive unfolding model, wherein hydrophobic interactions and metal chelation serve to anchor portions of the protein, thereby damping fluctuations and retarding amide proton exchange. The addition of Ca2+ or Cd2+ was found to retard the exchange of many amide protons observed to be in hydrogen-bonding environments in the crystal structure of the (Ca2+)2 state, but not of those amide protons that were not involved in hydrogen bonds. The largest changes in rate constant occur for residues in the ion-binding loops, with substantial effects also found for the adjacent residues in helices I, II and III, but not helix IV. The results are consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops, accompanied by a change in the conformation of helix IV, as metal ions are chelated. Further analysis of the results obtained for the three states of metal occupancy provides insight into the nature of the changes in conformational fluctuations induced by ion binding.


Alpha-adrenergic modification of the Ca2+ transient and contraction in single rat cardiomyocytes. Intracellular Ca2+ transients and contraction were measured simultaneously in single rat cardiomyocytes loaded with the fluorescent Ca2+ indicator fura-2, using a recently described high-speed digital imaging method (O'Rourke et al., 1990, Am J Physiol 259: H230-H242). In cardiomyocytes electrically-stimulated at 1 Hertz, alpha-adrenoceptor activation in the presence of beta-adrenoceptor blockade resulted in enhanced cell shortening associated with an increase in the amplitude of the cytosolic Ca2+ transient. Both effects developed in parallel over a 10-min time period and occurred without a change in the half-times for decay of Ca2+ or relaxation of the cell. To determine if the increase in contractility was proportional to the increase in peak cytosolic Ca2+, the effect of raising extracellular Ca2+ ([Ca2+]o) from 0.5 to 3 mM was examined in the absence and presence of alpha-adrenoceptor activation. At [Ca2+]o concentrations up to 1 mM, alpha-adrenoceptor-mediated effects on contraction were directly correlated with changes in peak cytosolic Ca2+ and resembled the effect of raising [Ca2+]o alone. In 2 and 3 mM [Ca2+]o, peak cytosolic Ca2+ approached a maximal level and alpha-adrenoceptor activation induced a slight enhancement in the extent of shortening in the absence of a detectable alteration of the Ca2+ transient. In contrast, under similar conditions, beta-adrenergic effects on shortening never exceeded those of alpha-adrenoceptor activation, although much higher peak cytosolic Ca2+ concentrations were achieved at high [Ca2+]o. The results suggest that the mechanism underlying the positive inotropic effect of alpha-adrenergic stimulation in rat ventricular cells is primarily dependent on an enhancement of the cytosolic Ca2+ transient, although there is also an increase in the myofibrillar response to intracellular Ca2+ under the condition of high extracellular Ca2+.


Effects of the protein phosphatase inhibitors okadaic acid and calyculin A on metabolically inhibited and ischaemic isolated myocytes. Isolated adult rat myocytes were subjected to 180 min of metabolic inhibition or incubated in ischaemic pellets, in the presence and absence of 10 microM okadaic acid (OA) or calyculin A (CL-A). Contracture and viability was determined by light microscopic analysis of trypan blue-stained preparations and ATP levels by HPLC. Osmotic fragility was assessed by brief hypotonic swelling of cells in 170 or 85 mOsm media prior to determination of viability. Neither drug significantly affected the relatively rapid rates of contracture of myocytes during metabolic inhibition, and both afforded significant protection from development of trypan blue permeability and osmotic fragility. Both OA and CL-A significantly accelerated the rates of contracture and ATP depletion of myocytes during ischaemic incubations. Despite an enhanced rate of ATP depletion, which would be expected to accelerate development of injury, neither drug accelerated development of loss of viability or development of osmotic fragility as measured by 170 mOsm swelling. Mathematical compensation for different rates of ATP depletion confirmed that a protective effect of the drugs, during ischaemic incubation, was masked by their enhancement of the rate of injury, following swelling at 170 mOsm. When the effects of CL-A on ischaemic cells were examined at 85 mOsm, a more stringent test for osmotic fragility, protection was found without compensation for differing rates of ATP depletion. A dose/response curve for CL-A showed some effect at 100 nM and a nearly full effect during metabolic inhibition at 1 microM concentrations. It is concluded that protein phosphatase inhibitors reduce the rates of development of osmotic fragility of metabolically inhibited cells and reduces the rate of injury relative to the rate of ATP depletion of ischaemic cardiomyocytes. Phosphorylation mechanisms may be important to development of irreversible myocardial cell injury.


Density of 1,4-dihydropyridine receptors decreases in the hearts of aging hamsters. To determine whether density and properties of myocardial voltage-sensitive calcium channels may change in the hearts of aged animals, we have compared specific binding of [3H]-nitrendipine ([3H]-NTP) to 1,4-dihydropyridine (DHP) receptors in ventricular homogenate and membrane fractions prepared from the hearts of adult hamsters of different ages. In crude homogenate preparations from young adult animals (70 days) the density (Bmax) of [3H]-NTP binding was 82 +/- 6 fmol/mg protein (n = 5 hearts). By contrast, Bmax values for 400-, 500- and 600-day-old animals were 54 +/- 5, 59 +/- 11 and 50 +/- 2 fmol/mg protein, respectively (significantly lower than control, P = 0.004, n = 4-5 hearts/group). The affinity (Kd) of [3H]-NTP binding was not affected by increasing age. Similar results were obtained in a partially purified membrane fraction. Allosteric modulation of [3H]-NTP binding by the phenylalkylamine, verapamil and the benzothiazepine, diltiazem were preserved in aging heart. These studies demonstrate that the density of myocardial DHP receptors declines in the aged hamster, although the properties of these receptors are unchanged. This suggests that a decrease in voltage-sensitive calcium channels may be associated with normal myocardial aging.


ACE-inhibition induces NO-formation in cultured bovine endothelial cells and protects isolated ischemic rat hearts. The role of NO-formation induced by accumulated endogenous bradykinin (BK) via local ACE-inhibition with ramiprilat (RT) or by adding BK exogenously was evaluated in cultured bovine aortic endothelial cells (BAEC) and in isolated rat hearts with post-ischaemic reperfusion injuries. Furthermore we used the n-octyl-ester of ramipril (RA-octil) which was shown to have no ACE-inhibitory action. In BAEC, ACE-inhibition by RT (1 x 10(-8)-1 x 10(-6) mol/l) or addition of BK (1 x 10(-8)-1 x 10(-6) mol/l) stimulated the formation of NO and prostacyclin (PGI2) as assessed by endothelial cyclic GMP- and 6-keto-PGF1a formation. Cyclic GMP and PGI2 synthesis was completely suppressed by the NO synthase inhibitor NG-nitro-L-arginine (L-NNA, 1 x 10(-5) mol/l) and by the B2 kinin receptor antagonist HOE 140 (1 x 10(-7) mol/l). RA-octil (1 x 10(-8)-1 x 10(-4) mol/l) did not affect endothelial cyclic GMP production in BAEC. In isolated working rat hearts subjected to local ischemia with reperfusion both RT (1 x 10(-8) mol/l) and BK (1 x 10(-9) mol/l) reduced the incidence and duration of ventricular fibrillation. In parallel myocardial function (left ventricular pressure, coronary flow) and metabolism (high energy rich phosphates) were improved showing a comparable fingerprint for RT and BK. Addition of L-NNA (1 x 10(-6) mol/l) or HOE 140 (1 x 10(-9) mol/l) abolished these protective effects of RT and BK. As in the BAEC studies RA-octil was without beneficial effects on the isolated ischaemic rat heart. The findings on BAEC show that inhibition of ACE localized on the luminal side of the vascular endothelium results in increased synthesis of NO and prostacyclin by local accumulation of endothelium-derived BK. Similar mechanisms may occur in the ischaemic rat heart leading to cardioprotection.


Polyphosphoinositide metabolism in hypertrophic rat heart. The accumulations of inositol-1,4,5-trisphosphate (IP3) and inositol-1,3,4,5-tetrakisphosphate (IP4) after hormonal stimulation may have a physiological role, possibly by alteration of Ca2+ levels in cardiac tissue. But the accumulation of inositol polyphosphate in a pathophysiological condition has not been studied. We investigated phosphatidylinositol-4,5-bisphosphate (PIP2) metabolism in hypertrophic cardiac myocytes, and clarified that the accumulations of IP3, IP4 and diacylglyceride after stimulation with norepinephrine were significantly enhanced in isolated myocytes from spontaneously hypertensive rat heart. Phospholipase C activity increased with age in SHRSP heart cells. These data suggest that PI turnover pathways, which can be mediated by both phosphatidylinositol-4,5-bisphosphate and diacylglyceride, may play an important role in development of hypertrophy in the hearts of rats with spontaneous hypertension.


Arrhythmogenic effects of isoproterenol-activated Cl- current in guinea-pig ventricular myocytes. Possible arrhythmogenic effects of the isoproterenol-activated Cl- current were examined in isolated guinea-pig ventricular myocytes under various intra- and extracellular Cl- concentrations. Experiments were carried out with external K+ concentration ([K+]o) decreased to 2 or 3 mM. Under symmetrical concentrations of Cl- in intra- and extra-cellular solutions (ECl = 0 mV), 1 microM isoproterenol (ISP) depolarized resting membrane potential (RMP) by 6.2 +/- 1.1 mV and slowed repolarization with induction of early afterdepolarizations (EADs) in 9 out of 9 cells. EADs appeared at voltages positive to -40 mV, where L-type Ca2+ current is assumed to be activated. When Cl- concentrations were settled near physiological conditions (ECl = -40 - -50 mV), ISP depolarized RMP by 2.8 +/- 0.4 mV and elicited abnormal repolarization with occasional EADs in 6 out of 19 cells. When ECl was set to -80 mV, however, ISP depolarized RMP by only 0.5 +/- 0.5 mV without induction of abnormal activities. Thus, depolarizing effects of ISP and incidence of repolarization abnormalities including EADs were increased as ECl shifted to more positive potential levels. At [K+]o = 4 mM, no abnormal activities were observed when ECl was around -50 mV (0/8), and 6 out of 6 cells showed abnormal activities when ECl was set to 0 mV. ISP-elicited abnormal activities were abolished by 1 mM DNDS (4,4'-dinitrostilbene-2,2'-disulphonic acid), a blocker for Cl- channels.(ABSTRACT TRUNCATED AT 250 WORDS)


The effects of blocking the Na-Ca exchange at intervals throughout the physiological contraction-relaxation cycle of single cardiac myocyte. The method of rapid superfusion of the single isolated ventricular myocytes of guinea-pig heart was used in order to inhibit the Na-Ca exchange throughout the physiological contraction-relaxation cycle. Superfusion of the cell at selected intervals during the contraction with the Na,Ca-free solution resulted in increase in its amplitude, increase in time to peak shortening and in delay of relaxation, albeit the cells relaxed before reperfusion of normal Tyrode solution. The largest increase in amplitude of contraction (to 134 +/- 16%) was observed when the effective exchange of the cell's environment was attained approximately 50 ms after the pulse stimulating contraction. The effects declined promptly when the delay was increased beyond 100 ms. In the cells treated with 10 mM caffeine superfusion with the Na,Ca-free solution after the delay of 50-100 ms resulted in decrease in extent of shortening. Increase in delay resulted in slight increase in extent of shortening with respect to control and strong inhibition of relaxation. The strongest effects were observed when the delay was approximately 200 ms. Superfusion of the normal cells and of the cells treated with caffeine between contractions resulted in slight potentiation of the next beat. It is concluded that Na-Ca exchange provides an important mechanism of relaxation and outward Ca2+ transport in the physiological contraction of the ventricular cardiomyocyte.


High-dose chemotherapy for resistant germ cell tumors: recent advances and future directions. A review of the published literature shows that treatment with high-dose carboplatin and etoposide with autologous bone marrow transplantation results in durable complete responses (3-42 months, continuous at time of report) in about 10%-20% of heavily pretreated patients with cisplatin-resistant germ cell tumors and represents a curative therapy in patients who would otherwise die of their disease. In a recent follow-up study of 40 patients with refractory germ cell tumors, 15% (six) were alive and free of disease more than 24 months after treatment with high-dose carboplatin and etoposide with autologous bone marrow transplantation. The several studies that incorporated an oxazaphosphorine (usually cyclophosphamide) in the regimen strongly suggest that the addition of this third drug to high doses of carboplatin and etoposide results in a higher proportion of complete responses (35%, 23% durable) than carboplatin and etoposide alone (26%, 12% durable). Early intervention with high-dose chemotherapy and autologous bone marrow transplantation appears to reduce hematologic toxicity. Its role in the treatment of less heavily pretreated patients will depend on the definition of "cisplatin-resistant," which may encompass those who failed to respond to the drug or those who are judged not likely to respond. Studies to improve treatment efficacy and to lessen the burden of supportive care during hematopoietic reconstitution are ongoing and include the use of peripheral blood-derived stem cells.


Characterization of a topoisomerase II gene rearrangement in a human small-cell lung cancer cell line. Small-cell lung cancer (SCLC) is a highly chemosensitive tumor, but the recurrent disease that is common after initial response is often unresponsive to further chemotherapy. Although the mechanisms of drug resistance in SCLC have not been established, studies suggest that alterations of the nuclear enzyme DNA topoisomerase II may reduce the sensitivity of the cell to drug action. This enzyme is recognized as a primary target for cytotoxic activity of important antitumor agents. In this study, we attempted to determine if altered forms of DNA topoisomerase II are responsible for reduced drug sensitivity. We characterized a rearrangement of the topoisomerase II p170 gene (also known as TOP2) in a relatively chemoresistant SCLC cell line, NCI-H69, and compared topoisomerase II expression and activity in this line with those in the chemosensitive NCI-H187 cell line. Fragments of complementary DNA from the topoisomerase II gene were generated by polymerase chain reaction. Immunodetection was accomplished by using the monoclonal antibody 7E6 against the human topoisomerase II p170 isoform. Using DNA probes corresponding to different complementary DNA regions, we showed that the rearrangement was localized at the 3' terminus of one allele of the topoisomerase II gene. In addition to the normal 6.2-kilobase (kb) topoisomerase II messenger RNA (mRNA), the NCI-H69 line expressed a 7.4-kb topoisomerase II transcript, presumably encoded by the rearranged allele. Moreover, this transcript, although longer than the normal mRNA, lacked a substantial portion of the 3'-terminal p170 gene coding sequence. Topoisomerase II activity in nuclear extracts, as determined by the P4 phage DNA-unknotting assay, was more easily detected and measured at lower NaCl concentrations in NCI-H69 than in NCI-H187 cells. These results are consistent with the hypothesis that the chemoresistant NCI-H69 cell line may express, in addition to the normal enzyme, an altered topoisomerase II enzyme possibly encoded by the 7.4-kb mRNA, which in turn may be transcribed from the rearranged gene allele. These observations emphasize the role of topoisomerase II in determining drug sensitivity and suggest that such gene rearrangements may contribute to resistance of SCLC cells to topoisomerase II inhibitors.


Comparison of prognostic factors and survival among black patients and white patients treated with irradiation for non-small-cell lung cancer. Many studies have reported differences in cancer incidence and survival between populations of Blacks and Whites. A 45% higher death rate from lung cancer for Black men and a survival duration for Black patients with lung cancer that is generally shorter than that for White patients have also been reported. The purpose of this study was to evaluate whether race affects known prognostic factors for non-small-cell lung cancer in Black versus White patients. This analysis attempts to determine which prognostic factors may contribute to the reported differences in disease outcome. We used data from 1565 patients with non-small-cell lung cancer treated in four randomized prospective trials conducted by the Radiation Therapy Oncology Group (RTOG). The data were pooled for a retrospective analysis of survival and prognostic factors by race. Univariate analysis showed significant differences between Blacks and Whites with regard to sex, weight loss, histology, and RTOG T stage (P < .05), but the only clinically significant difference (P < or = .01) was weight loss. Despite these findings, overall survival for Blacks and Whites did not differ significantly (P = .67). Median survival for Blacks and Whites with a Karnofsky performance status (KPS) of 90 or more was 12.1 and 11.3 months, respectively (P = .45). Survival for Blacks and Whites with a KPS of less than 90 was 7.8 and 6.8 months, respectively. Cause of death did not differ between the two races. For both races, KPS, age, sex, weight loss, and RTOG T and N stages were significant prognostic factors for survival (P < .01), but race was not a significant prognostic factor. Further studies of the differential in cancer survival for Blacks and Whites may be indicated, but greater impact may be achieved by addressing socioeconomic factors, lifestyle and occupational risk factors, health education, and access to adequate health care.


Activity of topotecan, a new topoisomerase I inhibitor, against human tumor colony-forming units in vitro. Topotecan [(S)-9-dimethylaminomethyl(10-hydroxy-camptothecin), NSC 609699, SK&F 104864A], a semisynthetic analogue of the natural product camptothecin, is a cell cycle-specific drug that exerts antineoplastic activity through inhibition of topoisomerase I. Currently, topotecan is undergoing phase I and early phase II clinical trials. The dose-limiting toxicity for topotecan is myelosuppression. Our purpose was to determine plasma concentrations and exposure times necessary for optimal clinical activity and tumor types that may be responsive in phase II clinical studies of topotecan. A soft-agar cloning system assay was used to determine the in vitro effects of topotecan against cells from biopsy specimens of colorectal, breast, lung, ovarian, renal cell, and gastric cancers and cancers of unknown primary origin. We studied 141 freshly explanted tumor specimens, using 1-hour exposure to topotecan, and 80 were studied using continuous exposure. A decrease in tumor colony formation resulting from drug exposure was considered an in vitro response if survival of colonies was up to 50% of that in controls. With 1-hour exposure, in vitro responses were seen in 10% and 25% of assessable tumor specimens at final topotecan concentrations of 1.0 and 10.0 micrograms/mL, respectively. With continuous exposures at concentrations of 0.1 and 1.0 micrograms/mL, in vitro response rates were 34% and 76%, respectively. Specific activity was seen against colorectal, breast, non-small-cell lung, ovarian, and renal cell cancers, with responses observed in 27%, 25%, 32%, 39%, and 83%, respectively, of assessable tumor specimens after continuous exposure to 0.1 micrograms/mL topotecan. A subset of tumor specimens resistant to doxorubicin or fluorouracil was sensitive to topotecan, and the difference in sensitivity was statistically significant. In addition, some of the tumor specimens resistant to cyclophosphamide and etoposide were also sensitive to topotecan. Topotecan appears to be active in vitro against a variety of human tumors, including a subgroup resistant in vitro to standard antineoplastic agents. If plasma levels of 0.1 micrograms/mL can be achieved for prolonged periods of time in ongoing clinical trials, topotecan should have substantial clinical activity. Further clinical development of topotecan is warranted.


Silica-induced pulmonary inflammation and fibrosis in mice is altered by acute exposure to nitrogen dioxide. The biologic impact of consecutive exposures to two environmental pollutants was examined in mice exposed to silica crystals (SI) by intratracheal (IT) injection followed by an inhalation exposure to nitrogen dioxide (NO2). C57Bl/6 mice received an IT injection of 2 mg SI or sterile saline (SAL) followed by a 2-h inhalation exposure to NO2 at 20 ppm either within 2 h of or 24 h after SI instillation. During acute inflammation (d 3 postsilica), mice exposed to NO2 at either time showed a dramatic and significant reduction in the number of lavaged alveolar neutrophils (PMN) when compared to silica/air-exposed mice. Animals exposed to NO2 24 h after silica also evidenced significant decreases in levels of lavage albumin and lactate dehydrogenase (LDH) 3 d after silica, as well as significant decreases in hydroxyproline content of the lung 30 and 60 d postsilica injection when compared to silica/air-exposed animals. NO2 administration 24 h after silica appeared to shift the appearance of PMN in the lung from d 3 to d 14, but did not otherwise alter chronic cellular inflammation. These data suggest that the marked neutrophil response and collagen deposition induced by SI can be modulated by NO2 exposure and that the time of oxidant gas exposure after silica administration is critical to this modulation.


Experimental studies on the changes of adrenergic receptors in rat liver with acute injury and in endotoxemia. Previous studies suggested that hepatic alpha and beta adrenergic receptors is stable and it is in dynamic equilibrium under normal conditions. However, at a pathological condition these receptors may be changed. We studied the changes of adrenergic receptors in the rat liver with acute injury and in endotoxemia by light microscopic autoradiography. Our experimental results reveal that in the liver of rats with acute injury and in endotoxemia there were changes in varying degrees of alpha and beta adrenoceptors. Moreover, the changes were not uniform in various structures of the rat liver. Glucagon can keep adrenergic receptors stable on hepatic cells and muscular cells of blood vessels in the rat liver. Our experiment provides evidence for clinical use of adrenoceptor antagonists in the treatment of acute and chronic liver disease.


A comparison of radiographic interpretation of silica exposed workers using the 1963 and the 1986 Chinese roentgenodiagnostic criteria of pneumoconioses. As part of a larger study relating to silica exposure, silicosis, and lung cancer mortality in Chinese mine and factory workers, 1936 old posterior-anterior chest X-rays were re-interpreted according to the 1986 Chinese Roentgenodiagnostic Criteria of pneumoconioses. Each film was independently read by three individuals from a panel of eleven radiologists, and this reading was compared to the original one. Subsequent to the independent readings, a groups of three readers interpreted the films together, called the consensus readings. Comparisons were made by Chinese stage of pneumoconiosis. For the entire cohort, there was a crude agreement of 57.4% between the old and the new interpretations. Agreement within one step of full agreement was 92.5%. The interpretations done by median reading and by consensus were very similar. In general, there was a tendency for the old readings to be slightly higher compared to the new interpretations. This tendency was most marked in the tin mines, followed in decreasing order by the iron/copper mines, the potteries, and the tungsten mines. The agreement between the old and new interpretations is felt to be satisfactory.


Computer simulation of metoclopramide block of cardiac sodium channels. Based on the gate-related receptor hypothesis, a quantitative study on kinetics and state-dependency of metoclopramide (Met) blockade of cardiac sodium channels was performed by computer simulation. The characteristics of Met, a sodium channel blocker modulated simultaneously by both activation and inactivation gates were described and analyzed for the first time. The time constant of recovery from blocking for Met at resting potential -85 mV was 2.82 s, which was increased during depolarization or hyperpolarization. Its apparent rate of onset of blocking was 0.49 AP-1 at a stimulation frequency of 2.0 Hz. A concentration-dependent shift of h infinity curve was found at doses higher than 100 mumol/L Met. This study provides a new test and verification for our gate-related receptor hypothesis.


Human studies with the chelating agents, DMPS and DMSA. Meso-2,3-dimercaptosuccinic acid (DMSA) is bound to plasma albumin in humans and appears to be excreted in the urine as the DMSA-cysteine mixed disulfide. The pharmacokinetics of DMSA have been determined after its administration to humans po. For the blood, the tmax and t1/2 were 3.0 h + 0.45 SE and 3.2 h + 0.56 SE, respectively. The Cmax was 26.2 microM + 4.7 SE. To determine whether dental amalgams influence the human body burden of mercury, we gave volunteers the sodium salt of 2,3-dimercaptopropane-1-sulfonic acid (DMPS). The diameters of dental amalgams of the subjects were determined to obtain the amalgam score. Administration of 300 mg DMPS by mouth increased the mean urinary mercury excretion of subjects over a 9 h period. There was a positive correlation between the amount of mercury excreted and the amalgam score. DMPS might be useful for increasing the urinary excretion of mercury and thus increasing the significance and reliability of this measure of mercury exposure. DMSA analogs have been designed and synthesized in attempts to increase the uptake by cell membranes of the DMSA prototype chelating agents. The i.v. administration of the monomethyl ester of DMSA, the dimethyl ester of DMSA or the zinc chelate of dimethyl DMSA increases the biliary excretion of platinum and cadmium in rats.


Bicarbonate ingestion: effects of dosage on 60 s cycle ergometry. Nine healthy male subjects who were all participating in athletic events volunteered to take part in this study, the aim of which was to determine whether there are specific dosages of sodium bicarbonate (HCO3-) that are useful as an ergogenic aid as far as anaerobic performance times are concerned. A control, placebo (CaCO3 500 mg kg-1) and five dosages of bicarbonate (100, 200, 300, 400 and 500 mg kg-1) were used. The anaerobic test consisted of pedalling a Repco Exertech cycle ergometer for 1 min during which total work (kJ) and peak power (W) were measured. The subjects completed more work in the 200 (P < 0.05), 300, 400 and 500 mg kg-1 (P < 0.005) trials with most work being undertaken in the 300 mg kg-1 trial (41.9 kJ min-1). Peak power was not significantly different from the control until the 300 mg kg-1 dose, and there were no further changes from this with increasing doses of HCO3-. The highest level of peak power achieved was 1295 +/- 72.8 W at the 300 mg kg-1 dosage. Blood pH indicated that after ingestion of all but the 100 mg kg-1 dose, a state of alkalosis was achieved (P < 0.005), and this was also indicated by changes in base excess. Bicarbonate levels increased post-ingestion in all but the 100 mg kg-1 dose, with these changes reflecting the changes that occurred in the work output. Blood lactate (BLa) levels increased post-exercise (P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)


Sodium bicarbonate ingestion and its effects on anaerobic exercise of various durations. Four groups of male subjects participated in anaerobic testing on a Repco EX10 cycle ergometer to determine the effectiveness of sodium bicarbonate (0.3 g kg-1 body mass) as an ergogenic aid during exercise of 10, 30, 120 and 240 s duration. Blood was collected 90 min prior to ingestion of sodium bicarbonate (NaHCO3), after ingestion of NaHCO3 and immediately post-exercise from a heated (43-46 degrees C) fingertip and analysed immediately post-collection for pH, base excess, bicarbonate and lactate. The total work undertaken (kJ) and peak power achieved during the tests were also obtained via a Repco Work Monitor Unit. Blood bicarbonate levels were again increased above the control and placebo conditions (P < 0.001) and blood lactate levels were also increased following the bicarbonate trials. The pH levels fell significantly (P < 0.05) below the control and placebo conditions in all trials. The results indicate that NaHCO3 at this dosage has no ergogenic benefit for work of either 10 or 30 s duration, even though blood bicarbonate levels were significantly increased (P < 0.05) following ingestion of NaHCO3. For work periods of 120 and 240 s, performance was significantly increased (P < 0.05) above the control and placebo conditions following NaHCO3 ingestion.


Percutaneous ethanol injection under sonographic guidance of hepatocellular carcinoma in compensated and decompensated cirrhotic patients. Forty-six patients with cirrhosis and 75 biopsy-proved hepatocellular carcinoma (HCC) nodules underwent percutaneous ethanol injection (PEI) regardless of number (up to five) and size (mean diameter, 3.6 cm) of tumoral lesions and clinical severity of cirrhosis (11 patients in Child's class C were included). Ethanol was injected under sonographic guidance through 20 to 22 gauge needles so as to obtain homogeneous hyperechogenicity of lesions. A total of 271 PEI sessions were carried out, delivering 2 to 14 ml per session. All nodules but one decreased in size, and seven were no longer appreciable on sonography. Recurrence was detected in two patients. The 3 year survival rate of all cases was 86%. Child's classes A and B patients fared better (3 yr survival 100%); 2 year survival of subjects with HCC < or = 3 cm was 92%. Multifocality did not affect survival. Most patients experienced mild pain at the site of injection, but only two major complications were encountered: partial chemical thrombosis of the left portal vein and cholangitis. Both cases were managed conservatively. In conclusion, PEI seems to offer a safe and valuable tool for therapy of HCC, especially in patients with good functional liver reserve and small (< or = 3 cm) tumors.


New technique using intraductal ultrasonography for the diagnosis of diseases of the pancreatobiliary system. Intraductal ultrasonography (IDUS), a new technique for visualizing arterial structures, operates at an ultrasound frequency of 30 MHz to produce high resolution, cross-sectional images in real time. The purpose of this study was to provide a basis for interpreting IDUS images in vitro. We also attempted to determine the clinical usefulness of the IDUS system in diagnosing pancreatobiliary diseases in vivo. IDUS echograms of both the bile duct (BD) and main pancreatic duct (MPD) from autopsy specimens of 15 patients demonstrated three distinct layers with a fine reticular pattern in the pancreas in vitro. In clinical cases, the MPD and BD of four patients could be scanned by inserting the IDUS catheter via the major papilla without requiring endoscopic sphincterotomy. We hope that IDUS will become routine in scanning the BD and MPD to achieve early and accurate diagnoses of pancreatobiliary diseases.


Myristate-protein interactions in poliovirus: interactions of VP4 threonine 28 contribute to the structural conformation of assembly intermediates and the stability of assembled virions. The VP4 capsid protein of poliovirus is N-terminally modified with myristic acid. Within the poliovirus structure, a hydrogen bond is observed between the myristate carbonyl and the hydroxyl side chain of threonine 28 of VP4. This interaction is between two fivefold symmetry-related copies of VP4 and is one of several myristoyl-mediated interactions that appears to structurally link the promoters within the pentamer subunit of the virus particle. Site-specific substitutions of the threonine residue were constructed to investigate the biological relevance of these myristate-protein interactions. Replacement of the threonine with glycine or lysine is lethal, generating nonviable viruses. Substitution with serine or valine led to viable viruses, but these mutants displayed anomalies during virus assembly. In addition, both assembled serine- and valine-substituted virion particles showed reduced infectivity and were more sensitive to thermal inactivation and antibody neutralization. Thus the threonine residue provides interactions necessary for efficient assembly of the virus and for virion stability.


Multiple amino acids in the capsid structure of canine parvovirus coordinately determine the canine host range and specific antigenic and hemagglutination properties. Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are over 98% similar in DNA sequence but have specific host range, antigenic, and hemagglutination (HA) properties which were located within the capsid protein gene. In vitro mutagenesis and recombination were used to prepare 16 different recombinant genomic clones, and viruses derived from those clones were analyzed for their in vitro host range, antigenic, and HA properties. The region of CPV from 59 to 91 map units determined the ability to replicate in canine cells. A complex series of interactions was observed among the individual sequence differences between 59 and 73 map units. The canine host range required that VP2 amino acids (aa) 93 and 323 both be the CPV sequence, and those two CPV sequences introduced alone into FPV greatly increased viral replication in canine cells. Changing any one of aa 93, 103, or 323 of CPV to the FPV sequence either greatly decreased replication in canine cells or resulted in an inviable plasmid. The Asn-Lys difference of aa 93 alone was responsible for the CPV-specific epitope recognized by monoclonal antibodies. An FPV-specific epitope was affected by aa 323. Amino acids 323 and 375 together determined the pH dependence of HA. Amino acids involved in the various specific properties were all around the threefold spikes of the viral particle.


Immunological characterization of the gag gene products of bovine immunodeficiency virus. The bovine immunodeficiency virus (BIV) gag gene encodes a 53-kDa precursor (Pr53gag) that is involved in virus particle assembly and is further processed into the putative matrix (MA), capsid (CA), and nucleocapsid (NC) functional domains in the mature virus. Gag determinants are also found in the Gag-Pol polyprotein precursor. To immunologically identify the major precursors and processed products of the BIV gag gene, monospecific rabbit sera to recombinant BIV MA protein and Pr53gag and peptides predicted to correspond to the CA and NC proteins and the MA-CA cleavage site were developed and used in immunoprecipitations and immunoblots of BIV antigens. Monospecific antisera to native and recombinant human immunodeficiency virus type 1 proteins were also used to identify analogous BIV Gag proteins and to determine whether cross-reactive epitopes were present in the BIV Gag precursors or processed products. The BIV MA, CA, and NC Gag proteins were identified as p16, p26, and p13, respectively. In addition to BIV Pr53gag, the major Gag precursor, two other Gag-related precursors of 170 and 49 kDa were identified that have been designated pPr170gag-pol and Pr49gag, respectively; pPr170gag-pol is the Gag-Pol polyprotein precursor, and Pr49gag is the transframe Gag precursor present in pPr170gag-pol. Several alternative Gag cleavage products were also observed, including p23, which contains CA and NC determinants, and p10, which contains a peptide sequence conserved in the CA proteins of most lentiviruses. The monospecific antisera to human immunodeficiency virus type 1 CA (p24) and NC (p7) proteins showed cross-reactivity to and aided in the identification of analogous BIV proteins. Based on the present data, a scheme for the processing of BIV Gag precursors is proposed.


A novel sequence-specific DNA-binding protein, LCP-1, interacts with single-stranded DNA and differentially regulates early gene expression of the human neurotropic JC virus. We have identified a novel brain-derived single-stranded-DNA-binding protein that interacts with a region of the human neurotropic JC virus enhancer designated the lytic control element (LCE). This nuclear factor, LCP-1 (for lytic control element-binding protein 1), specifically recognizes the LCE, as determined by gel retardation assays. Alkylation interference showed that specific nucleotides within the LCE were contacted by LCP-1. Subsequent experiments revealed that point mutations within the LCE differentially affected LCP-1 binding. UV cross-linking and competition analysis suggested that the LCP-1 DNA-protein complexes were 50 to 52 and 100 to 120 kDa in size. Promoter mutations that affected LCP-1 binding reduced early mRNA transcription during the early phase of the lytic cycle. However, upon DNA replication in the presence of JC virus T antigen, when early mRNA initiation shifts to new locations indicative of the late phase, the LCP-1 mutations had no effect. We suggest that the JC virus early transcription unit is differentially regulated by LCP-1 prior to but not after DNA replication, suggesting a novel mechanism by which DNA structure regulates eukaryotic gene expression.


Homologous sequences in adenovirus E1A and human papillomavirus E7 proteins mediate interaction with the same set of cellular proteins. Studies of adenovirus E1A oncoprotein mutants suggest that the association of E1A with the retinoblastoma protein (pRB) is necessary for E1A-mediated transformation. Mutational analysis of E1A indicates that two regions of pRB are required for E1A to form stable complexes with the retinoblastoma protein. In addition to pRB binding, these regions are necessary for E1A association with several other cellular proteins, including p130, p107, cyclin A, and p33cdk2. Here we show that short synthetic peptides containing the pRB-binding sequences of E1A are sufficient for interaction with p107, cyclin A, and p130. The E7 protein of human papillomavirus type 16 contains an element that binds to pRB and appears to be functionally homologous to the E1A sequences. Peptides containing this region of the E7 protein were able to interact with p107, cyclin A, and p130 in addition to pRB. These findings suggest that the common mechanism of transformation used by these viral oncogenes involves their association with a set of polypeptides.


Regulation of transcription from the hepatitis B virus major surface antigen promoter by the Sp1 transcription factor. The DNA-binding proteins which recognize the regulatory sequence elements of the hepatitis B virus (HBV) major surface antigen promoter were examined by gel retardation analysis, using nuclear extracts from the human hepatoma cell line Huh7. Using this assay, we identified four regions (B, D, E, and F) of the promoter that interact with the same or similar transcription factor(s). In addition, the recognition sequence for the Sp1 transcription factor bound the same or similar transcription factor(s) present in Huh7 cell nuclear extracts, and this binding was inhibited by the four major surface antigen promoter elements, B, D, E, and F. Purified Sp1 transcription factor was shown to bind to three (B, D, and F) of the major surface antigen promoter regulatory sequence elements by DNase I footprinting. Using transient transfection assays with Drosophila Schneider line 2 cells, we found that transcription from the major surface antigen promoter was transactivated by exogenously expressed Sp1, whereas transcription from the other three HBV promoters was not. Deletion analysis of the major surface antigen promoter demonstrated that the promoter region between -35 and +157 was sufficient to confer Sp1 responsiveness. This promoter region includes one of the regulatory elements footprinted by the purified Sp1 transcription factor. The function of the B, D, E, and F promoter elements was further examined by using these binding sites cloned into a minimal promoter element. Each of these regulatory regions transactivated transcription from the minimal promoter element in response to exogenously expressed Sp1. This finding demonstrates that the HBV major surface antigen promoter contains four functional Sp1 binding sites which probably contribute to the level of expression from this promoter during viral infection.


Assembly of viruslike particles by recombinant structural proteins of adeno-associated virus type 2 in insect cells. The three capsid proteins VP1, VP2, and VP3 of the adeno-associated virus type 2 (AAV-2) are encoded by overlapping sequences of the same open reading frame. Separate expression of these proteins by recombinant baculoviruses in insect cells was achieved by mutation of the internal translation initiation codons. Coexpression of VP1 and VP2, VP2 and VP3, and all three capsid proteins and the expression of VP2 alone in Sf9 cells resulted in the production of viruslike particles resembling empty capsids generated during infection of HeLa cells with AAV-2 and adenovirus. These results suggest a requirement for VP2 in the formation of empty capsids. Individual expression of the AAV capsid proteins in HeLa cells showed that VP1 and VP2 accumulate in the cell nucleus and VP3 is distributed between nucleus and cytoplasm. Coexpression of VP3 with the other structural proteins also led to nuclear localization of VP3, indicating that the formation of a complex with VP1 or VP2 is required for accumulation of VP3 in the nucleus.


Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing. We have shown previously that a novel herpes simplex virus-induced activity, LPF, selectively increases RNA 3'-end processing at the poly(A) site of a late virus gene (J. McLauchlan, S. Simpson, and J. B. Clements, Cell 59:1093-1105, 1989). Here, our in vivo and in vitro analyses both demonstrate that LPF is induced during early stages of virus infection. Studies of virus mutants indicate that expression of the immediate-early IE63 gene is required for induction of this activity. The selective effects on 3' processing displayed in the presence of IE63 provide direct evidence that IE63 can influence this posttranscription process. This extends previous studies which reported increases in reporter gene activity with certain poly(A) sites by IE63 (R. M. Sandri-Goldin and G. E. Mendoza, Genes Dev. 6:848-863, 1992).


Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines. Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.


In vivo model for pseudo-templated transcription in Sendai virus. A synthetic Sendai virus-like recombinant RNA was used to develop a model system for pseudo-templated transcription of the P/C gene. The synthetic RNA molecule contains a 42-base stretch of nucleotide sequence derived from the RNA editing site of the P/C gene embedded into the chloramphenicol acetyltransferase gene. When this construct was rescued into Sendai virus, it was found that this 42-base sequence was sufficient to allow the Sendai virus polymerase to transcribe mRNAs with G-nucleotide insertions. Edited mRNA species containing a single nontemplated G insertion were found at a frequency of 6.5%, while rare messages had two G residues inserted. Edited viral RNA was not apparent, suggesting that this event is appropriately excluded during replication of the model genome. By progressively deleting from the 3' end, we found that a 24-nucleotide sequence spanning the G-insertion site was sufficient for pseudo-templated transcription in our system.


The 5' end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease. The presence of a papainlike cysteine protease (PCP) domain in the N-terminal region of the equine arteritis virus (EAV) replicase, which had been postulated on the basis of limited sequence similarities with cellular and viral thiol proteases, was confirmed by in vitro translation and mutagenesis studies. The EAV protease was found to direct an autoproteolytic cleavage at its C terminus which leads to the production of an approximately 30-kDa N-terminal replicase product (nsp1) containing the PCP domain. Amino acid residues Cys-164 and His-230 of the EAV replicase polyprotein were identified as the most likely candidates for the role of PCP catalytic residues. By means of N-terminal sequence analysis of a PCP cleavage product, derived from a bacterial expression system, it was shown that cleavage occurs between Gly-260 and Gly-261. No evidence for PCP-directed cleavages at other positions in the EAV replicase was obtained. In cotranslational and posttranslational trans-cleavage assays, neither EAV nsp1 nor its precursor was able to process the PCP cleavage site in trans.


A second gene for the African green monkey poliovirus receptor that has no putative N-glycosylation site in the functional N-terminal immunoglobulin-like domain. Using cDNA of the human poliovirus receptor (PVR) as a probe, two types of cDNA clones of the monkey homologs were isolated from a cDNA library prepared from an African green monkey kidney cell line. Either type of cDNA clone rendered mouse L cells permissive for poliovirus infection. Homologies of the amino acid sequences deduced from these cDNA sequences with that of human PVR were 90.2 and 86.4%, respectively. These two monkey PVRs were found to be encoded in two different loci of the genome. Evolutionary analysis suggested that duplication of the PVR gene in the monkey genome had occurred after the species differentiation between humans and monkeys. The NH2-terminal immunoglobulin-like domain, domain 1, of the second monkey PVR, which lacks a putative N-glycosylation site, mediated poliovirus infection. In addition, a human PVR mutant without N-glycosylation sites in domain 1 also promoted viral infection. These results suggest that domain 1 of the monkey receptor also harbors the binding site for poliovirus and that sugar moieties possibly attached to this domain of human PVR are dispensable for the virus-receptor interaction.


Replication-defective mutants of herpes simplex virus (HSV) induce cellular immunity and protect against lethal HSV infection. Live viruses and live virus vaccines induce cellular immunity more readily than do inactivated viruses or purified proteins, but the mechanism by which this process occurs is unknown. A trivial explanation would relate to the ability of live viruses to spread and infect more cells than can inactivated virus. We have used live but replication-defective mutants to investigate this question. Our studies indicate that the immune responses of mice to live virus differ greatly from the responses to inactivated virus even when the virus does not complete a replicative cycle. Further, these studies indicate that herpes simplex virus-specific T-cell responses can be generated by infection with replication-defective mutant viruses. These data indicate that the magnitude of the cellular immunity to herpes simplex virus may be proportional to the number or quantity of different viral gene products expressed by an immunizing virus.


Contributions to transcriptional activity and to viral leukemogenicity made by sequences within and downstream of the MCF13 murine leukemia virus enhancer. We have identified nucleotide sequences that regulate transcription in both a cell-type-specific and general manner in the long terminal repeat of the MCF13 murine leukemia virus. Besides the enhancer element, we have observed that the region between the enhancer and promoter (DEN) has a profound effect on transcription in different cell types. This effect, however, was dependent on the copy number of enhancer repeats and was detectable in the presence of a single repeat. When two enhancer repeats were present, the effect of DEN on transcription was abrogated except in T cells. DEN also makes a significant contribution to the leukemogenic property of the MCF13 retrovirus. Its deletion from the MCF13 virus dramatically reduced the incidence of thymic lymphoma and increased the latency of disease in comparison with the wild-type virus. This effect was most marked when one rather than two enhancer repeats was present in the mutant viruses. We also observed that the removal of one repeat alone remarkably reduced leukemogenicity by the MCF13 virus. A newly identified protein-binding site (MLPal) located within DEN affects transcription only in T cells, and its deletion attenuates the ability of an MCF13 virus with a single enhancer repeat to induce thymic lymphoma. This observation suggests that the MLPal protein-binding site contributes to the effect of the DEN region on T-cell-specific transcription and viral leukemogenicity. This study identifies the importance of nonenhancer sequences in the long terminal repeat for the oncogenesis of the MCF13 retrovirus.


Role of cell surface spikes in alphavirus budding. Alphaviruses mature by budding at cell surfaces. According to a prevailing hypothesis, the viral membrane protein, which is a heterodimeric protein unit, is transported to the plasma membrane (PM), where it awaits binding to the viral nucleocapsid (NC). This hypothesis predicts that the viral membrane protein heterodimers accumulate at the cell surface when expressed in the absence of NCs. We have tested this prediction by analyzing the spike protein expression phenotype of a Semliki Forest virus (SFV) variant which contains a capsid gene deletion. We found that viral membrane protein heterodimers were formed and transported to the cell surface normally. However, instead of accumulating at the PM as expected, the membrane proteins were rapidly degraded. In the case of the E1 subunit, degradation resulted in the release of a soluble E1 fragment into the medium. The fact that this pathway of protein degradation is mostly inhibited during wild-type virus infection suggests that viral membrane proteins are very efficiently captured by NCs into budding complexes and that normally no sizeable pool of free membrane protein complexes exists at the PM.


The pseudorabies virus homology of the herpes simplex virus UL21 gene product is a capsid protein which is involved in capsid maturation. We mutagenized, mapped, and sequenced the pseudorabies virus (PRV) homology of gene UL21 of herpes simplex virus type 1. A polyclonal mouse antiserum against the protein encoded by the UL21 homolog was generated and used to monitor the expression and subcellular localization of the UL21-encoded protein. We found that the protein is identical to a previously detected PRV capsid protein. We analyzed viable PRV strains encoding mutant UL21 homologys, truncated by insertion of an oligonucleotide that contains stop codons in all reading frames. In two PRV mutants carrying the oligonucleotide at two sites within the gene, processing of newly replicated viral DNA was impaired. In addition, we show that one of the UL21 mutants has strongly reduced virulence for mice.


Multiple glycoproteins synthesized by the smallest RNA segment (S10) of bluetongue virus. The genome of bluetongue virus, an orbivirus, consists of 10 double-stranded RNAs, each encoding at least one polypeptide. The smallest RNA segment (S10) encodes two minor nonstructural proteins, NS3 and NS3A, the structures and functions of which are not understood. We have expressed these two proteins in mammalian cells by using the T7 cytoplasmic transient expression system. Using a deletion mutant (lacking the first AUG initiation codon), we have demonstrated that the second initiation codon is used to initiate the synthesis of NS3A protein and that the two initiation codons are responsible for the synthesis not only of NS3 and NS3A but also of high-molecular-weight forms of both proteins. These higher-molecular-weight forms (GNS3 and GNS3A) are glycosylated. We have also demonstrated that the carbohydrate chains of GNS3 and GNS3A could be further modified by heterogeneous extension to polylactosaminoglycan forms. The glycosylated and nonglycosylated forms are found in similar intracellular locations in the Golgi complex. In the presence of cycloheximide, NS3 and NS3A immunofluorescence staining was pronounced in the Golgi complex, confirming that NS3 and NS3A are competent for transport to the Golgi apparatus after synthesis. We conclude that S10 gene products are integral membrane glycoproteins.


Glycosylation requirements for intracellular transport and function of the hemagglutinin of influenza virus. The contribution of each of the seven asparagine-linked oligosaccharide side chains on the hemagglutinin of the A/Aichi/68 (X31) strain of influenza virus was assessed with respect to its effect on the folding, intracellular transport, and biological activities of the molecule. Twenty mutant influenza virus hemagglutinins were constructed and expressed, each of which had one or more of the seven glycosylation sites removed. Investigations of these mutant hemagglutinins indicated that (i) no individual oligosaccharide side chain is necessary or sufficient for the folding, intracellular transport, or function of the molecule, (ii) at least five oligosaccharide side chains are required for the X31 hemagglutinin molecule to move along the exocytic pathway to the plasma membrane, and (iii) mutant hemagglutinins having less than five oligosaccharide side chains form intracellular aggregates and are retained in the endoplasmic reticulum.


Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents. Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.


Mutations in the VP1 coding region of polyomavirus determine differentiating stage specificity. Polyomavirus mutants capable of replicating in undifferentiated murine C2 myoblasts were selected and characterized. These mutants grow normally in 3T6 mouse fibroblast cells, and they do not complement the wild-type virus in coinfection experiments of C2 myoblasts. Of 12 isolates, 10 possess duplications of the regulatory region including the enhancer A domain. On the bases of the regulatory region structure and the presence and length of the enhancer duplication, the mutant viruses could be grouped into three classes. One mutant class (e.g., PyMB3) possesses an enhancer duplication of 91 bp identical to that of a previously characterized polyomavirus mutant, PyNB11/1. We have demonstrated that this enhancer duplication gives rise at its junction to a novel recognition motif for the transcriptional factor NF-1 (M. Caruso, C. Iacobini, C. Passananti, A. Felsani, and P. Amati, EMBO J. 9:947-955, 1990). The regulatory region PyMB3 virus recombined in a wild-type genome context maintains the mutant phenotype. The other two types of mutants, one with a 30-bp enhancer duplication (e.g., PyMB40) and one with a wild-type enhancer structure (e.g., PyMB27), possess two similar but distinct 6-bp deletions in the same region of the VP1 coding gene. In both cases, the ability to replicate in undifferentiated C2 myoblasts is strictly correlated to the mutation in the VP1 coding region.


Antiviral effects of a thiol protease inhibitor on foot-and-mouth disease virus. The thiol protease inhibitor E-64 specifically blocks autocatalytic activity of the leader protease of foot-and-mouth disease virus (FMDV) and interferes with cleavage of the structural protein precursor in an in vitro translation assay programmed with virion RNA. Experiments with FMDV-infected cells and E-64 or a membrane-permeable analog, E-64d, have confirmed these results and demonstrated interference in virus assembly, causing a reduction in virus yield. In addition, there is a lag in the appearance of virus-induced cellular morphologic alterations, a delay in cleavage of host cell protein p220 and in shutoff of host protein synthesis, and a decrease in viral protein and RNA synthesis. The implications of using E-64-based compounds as potential antiviral agents for FMDV are discussed.


Tryptase Clara, an activating protease for Sendai virus in rat lungs, is involved in pneumopathogenicity. Tryptase Clara is an arginine-specific serine protease localized exclusively in and secreted from Clara cells of the bronchial epithelium of rats (H. Kido, Y. Yokogoshi, K. Sakai, M. Tashiro, Y. Kishino, A. Fukutomi, and N. Katunuma, J. Biol. Chem. 267:13573-13579, 1992). The purified protease was shown in vitro to behave similarly to trypsin, cleaving the precursor glycoprotein F of Sendai virus at residue Arg-116 and activating viral infectivity in a dose-dependent manner. Anti-tryptase Clara antibody inhibited viral activation by the protease in vitro in lung block cultures and in vivo in infected rats. When the enzyme-specific antibody was administered intranasally to rats that were also infected intranasally with Sendai virus, activation of progeny virus in the lungs was significantly inhibited. Thus, multiple cycles of viral replication were suppressed, resulting in a reduction in lung lesions and in the mortality rate. These findings indicate that tryptase Clara is an activating protease for Sendai virus in rat lungs and is therefore involved in pulmonary pathogenicity of the virus in rats.


Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro. The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein.


Characterization of lymphocytic choriomeningitis virus-binding protein(s): a candidate cellular receptor for the virus. The attachment of lymphocytic choriomeningitis virus (LCMV) to murine and primate cell lines was quantitated by a fluorescence-activated cell sorter assay in which binding of biotinylated virus was detected with streptavidin-fluorescein isothiocyanate. Cell lines that were readily infected by LCMV (e.g., MC57, Rin, BHK, Vero, and HeLa) bound virus in a dose-dependent manner, whereas no significant binding was observed to lymphocytic cell lines (e.g., RMA and WIL 2) that were not readily infected. Binding was specific and competitively blocked by nonbiotinylated LCMV. It was also blocked by LCMV-specific antiserum and a neutralizing monoclonal antibody to the virus glycoprotein GP-1 but not by antibodies specific for GP-2, indicating that attachment was likely mediated by GP-1. Treatment of cells with any of several proteases abolished LCMV binding, whereas phospholipases including phosphatidylinositol-specific phospholipase C had no effect, indicating that one or more membrane proteins were involved in virus attachment. These proteins were characterized with a virus overlay protein blot assay. Virus bound to protein(s) with a molecular mass of 120 to 140 kDa in membranes from cell lines permissive for LCMV but not from nonpermissive cell lines. Binding was specific, since unlabeled LCMV, but not the unrelated enveloped virus herpes simplex virus type 1, competed with 125I-labeled LCMV for binding to the 120- to 140-kDa band. The proteinaceous nature of the LCMV-binding substance was confirmed by the lack of virus binding to proteinase K-treated membrane components. By contrast, glycosidase treatment of membranes did not abolish virus binding. However, in membranes treated with endoglycosidase F/N-glycosidase F, and/or neuraminidase and in membranes from cells grown in tunicamycin, the molecular mass of the LCMV-binding entity was reduced. Hence, LCMV attachment to rodent fibroblastic cell lines is mediated by a glycoprotein(s) with a molecular mass of 120 to 140 kDa, with complex N-linked sugars that are not involved in virus binding.


The Epstein-Barr virus immediate-early promoter BRLF1 can be activated by the cellular Sp1 transcription factor. Disruption of viral latency in Epstein-Barr virus-infected cells is mediated through the activation of the BZLF1 (Z) immediate-early gene product. The Z protein can be derived from either of two promoters: the BZLF1 promoter, which directs transcription of a 1.0-kb mRNA encoding the Z gene product alone, or the upstream BRLF1 promoter, which directs transcription of a 2.8-kb bicistronic mRNA encoding the BRLF1 and BZLF1 immediate-early proteins. In this study we have examined the regulation of the BRLF1 promoter by viral and cellular factors. We found that the BRLF1 promoter is autoregulated by the BRLF1 transactivator through a nonbinding mechanism. We show that the BRLF1 (but not the BZLF1) promoter is highly responsive to the Sp1 transcription factor. Sp1 activation of the BRLF1 promoter is mediated through a consensus Sp1-binding site located from -39 to -44 (relative to the mRNA start site). We demonstrate that the BRLF1 promoter has high constitutive activity in C-33 cells (an epithelial cell line) and that the proximal Sp1-binding site is required for this activity. Despite the ubiquitous presence of Sp1 in many cell types, we found that the BRLF1 promoter has essentially no activity in lymphoid cell lines, suggesting that factors other than Sp1 may negatively regulate the BRLF1 promoter in these cells. Our findings demonstrate that the two potential promoters directing BZLF1 transcription are differentially regulated and that Sp1 can activate the BRLF1 promoter but not the BZLF1 promoter.


A constitutive enhancer in the bovine papillomavirus upstream regulatory region shares genetic elements with the viral P1 promoter. The bovine papillomavirus upstream regulatory region represents a common element in the regulation of transcription from the five early viral promoters. We have determined the sequences required for transcription from the viral P1 promoter, which is located at the 5' end of the upstream regulatory region. In vitro transcription from P1 requires a 123-bp fragment (nucleotides 7153 to 7275; -33 to +90) consisting of an upstream TATA-like sequence as well as an unidentified protein which binds to sequences immediately downstream of the initiation site. In vivo, this promoter requires additional downstream sequences (to position +160; nucleotide 7345) for maximal activity but does not require any additional DNA sequence upstream of a putative TATA box. Four regions within the downstream sequence from +9 to +160 are protected from DNase I digestion by proteins present in a HeLa cell extract. The presence of these sites correlates with the level of P1 activity. A constitutive enhancer maps to this same region, and mutations in this enhancer have been shown to affect downstream promoters. Deletion analysis indicates that the same sequences are required by both the P1 promoter and the constitutive enhancer, suggesting that the same proteins function in both activities.


Herpes simplex virus type 1 mutant strain in1814 establishes a unique, slowly progressing infection in SCID mice. Ocular infection of immunocompetent (BALB/c) mice with wild-type herpes simplex virus type 1 (HSV-1) 17+ may lead to acute fatal encephalitis; however, in surviving animals, a latent (nonproductive) infection of the nervous system is established. In contrast, 17+ infection invariably kills mice with severe combined immunodeficiency (SCID mice) within 2 weeks. Ocular infection of immunocompetent mice with a mutant HSV-1 strain, in1814, which does not produce a functional alpha-transinducing protein, results in no detectable viral replication in the nervous system during the time corresponding to the acute phase of infection, no mortality, and the establishment of latency. In SCID mice, however, the in1814 virus establishes a unique, slowly progressing infection. In studying the courses of in1814 infection in SCID and BALB/c mice, we found that although intact B- and/or T-lymphocytic functions were required for the control of viral replication in the nervous system, some of the infected neurons of SCID mice seemed to be able to restrict in1814 replication and harbor the virus in a latent state.


Strong trans activation of the human cytomegalovirus major immediate-early enhancer by p40tax of human T-cell leukemia virus type I via two repetitive tax-responsive sequence elements. The immediate-early 1 and 2 gene locus of human cytomegalovirus (HCMV) that encodes trans-activator proteins with effects on both homologous and heterologous promoters is expressed under control of a complex enhancer/promoter regulatory region. This enhancer contains four types of repetitive sequence elements with 17, 18, 19, and 21 bp that bind cellular transcription factors. Although the HCMV enhancer acts as a powerful stimulator of transcription in most cell types examined, human T cells do not support strong activity. The present study demonstrates that the tax gene product of human T-cell leukemia virus type I trans activates the major enhancer of HCMV more than 60-fold in the T-cell line Jurkat. When a series of chloramphenicol acetyltransferase expression plasmids containing synthetic oligonucleotides with the 17-, 18-, 19-, or 21-bp motif upstream of a minimal immediate-early 1 and 2 gene promoter was tested, two of the four repeat motifs could be identified as Tax-responsive elements. Both the 18- and the 19-bp motifs were able to act as strong Tax-responsive elements even when they were present as single copies. Thus, in addition to interacting with human immunodeficiency virus, HCMV is able to interact with a second retrovirus of clinical importance.


Evidence that a sequence similar to TAR is important for induction of the JC virus late promoter by human immunodeficiency virus type 1 Tat. A specific RNA sequence located in the leader of all human immunodeficiency virus type 1 (HIV-1) mRNAs termed the transactivation response element, or TAR, is a primary target for induction of HIV-1 long terminal repeat activity by the HIV-1-derived trans-regulatory protein, Tat. Human neurotropic virus, JC virus (JCV), a causative agent of the degenerative demyelinating disease progressive multifocal leukoencephalopathy, contains sequences in the 5' end of the late RNA species with an extensive homology to HIV-1 TAR. In this study, we examined the possible role of the JCV-derived TAR-homologous sequence in Tat-mediated activation of the JCV late promoter (Tada et al., Proc. Natl. Acad. Sci. USA 87:3479-3483, 1990). Results from site-directed mutagenesis revealed that critical G residues required for the function of HIV-1 TAR that are conserved in the JCV TAR homolog play an important role in Tat activation of the JCV promoter. In addition, in vivo competition studies suggest that shared regulatory components mediate Tat activation of the JCV late and HIV-1 long terminal repeat promoters. Furthermore, we showed that the JCV-derived TAR sequence behaves in the same way as HIV-1 TAR in response to two distinct Tat mutants, one of which that has no ability to bind to HIV-1 TAR and another that lacks transcriptional activity on a responsive promoter. These results suggest that the TAR homolog of the JCV late promoter is responsive to HIV-1 Tat induction and thus may participate in the overall activation of the JCV late promoter mediated by this transactivation.


Herpes simplex virus type 1 protease expressed in Escherichia coli exhibits autoprocessing and specific cleavage of the ICP35 assembly protein. The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a protease which is responsible for the C-terminal cleavage of the nucleocapsid-associated proteins, ICP35 c and d, to their posttranslationally modified counterparts, ICP35 e and f. To further characterize the HSV-1 protease, the UL26 gene product was expressed in Escherichia coli. The expressed protease underwent autoproteolytic processing at two independent sites. The first site is shared with ICP35 and results in removal of 25 amino acids from the C terminus of the protease. The second unique site gives rise to protein species consistent with deletion of a 28-kDa fragment at the N terminus. A mutant protease, which showed no activity in a mammalian cell cotransfection assay (F. Liu and B. Roizman, Proc. Natl. Acad. Sci. USA 89:2076-2080, 1992), failed to exhibit autoproteolytic processing at either site when expressed in bacteria. The inactive mutant was able to serve as a substrate in a trans assay in which the substrate and protease were coexpressed in bacteria. This experiment demonstrated that the unique N-terminal processing was mediated exclusively by the HSV-1 protease. ICP35 c,d also served as a substrate in this assay and was correctly processed by HSV-1 protease in E. coli. This trans-cleavage assay will aid in the characterization of HSV-1 protease and assist in investigation of the role of proteolytic processing in the virus.


N glycosylation of the virus binding domain is not essential for function of the human poliovirus receptor. The human poliovirus receptor (hPVR) is a glycoprotein with three immunoglobulin-like extracellular domains, of which the N-terminal domain (V-type domain) is necessary and sufficient for virus binding and uptake. The effect of N glycosylation of the V domain of hPVR on binding and entry of poliovirus was studied. Stable mouse L-cell lines were generated that express PVR-specific cDNA. One of the cell lines expressed a mutant of hPVR, in which both asparagine residues of the two N-glycosylation sites of the V domain were changed to aspartate (N105D) and serine (N120S), respectively. In the second mutant cell line, the portion of the cDNA encoding the V domain of hPVR was substituted by the homologous sequence of the recently isolated PVR cDNA from monkey cells. This V domain naturally lacks both N glycosylation sites and encodes D105 and S120 at the respective positions of the open reading frame. Absence of N glycosylation at these sites was demonstrated by in vitro translation of the two mutant coding sequences in the presence of microsomal membranes. Both PVR mutant cell lines were capable of poliovirus binding and replication. However, binding of anti-PVR monoclonal antibody D171 and protection from viral replication by this antibody were observed only with the glycosylation mutant carrying the human V domain. In contrast, infection of the cell line expressing the monkey-human hybrid receptor was not blocked even though monkey cells are fully protected by monoclonal antibody D171. The data suggest that N glycosylation of the V domain of hPVR is not essential for viral replication in human tissues and that differential glycosylation of hPVR at these sites is likely not a determinant of viral tissue tropism. Furthermore, the virus binding site and the epitope recognized by monoclonal antibody D171 do not appear to overlap.


A chimeric avian retrovirus containing the influenza virus hemagglutinin gene has an expanded host range. We have investigated what protein sequences are necessary for glycoprotein incorporation into Rous sarcoma virus (RSV) virions by utilizing the hemagglutinin (HA) protein of influenza virus. Two chimeric HA genes were constructed. In the first the coding sequence for the signal peptide of the RSV env gene product was fused in frame to the entire HA structural gene, and in the second the hydrophobic anchor and cytoplasmic domain sequences of the HA gene were also replaced with those from the RSV env gene. Both chimeric genes, expressed from a simian virus 40 expression vector in CV-1 cells, yielded functional HA proteins that were transported to the cell surface and were able to bind to erythrocytes. When the genes were expressed in combination with the RSV gag-pol gene region in QT6 cells by using a vaccinia virus-T7 expression/complementation system, virions that efficiently incorporated either chimeric protein were assembled. This result indicated that the presence of the RSV env membrane anchor and cytoplasmic sequences did not facilitate HA glycoprotein incorporation into virions. The presence of the RSV env signal sequence allowed the chimeric HA genes to be substituted into the RSV-derived BH-RCAN.HiSV viral genome in place of the RSV env gene. Both chimeric genomes yielded infectious virus that could infect human and avian cells with equal efficiency. These experiments demonstrate that a foreign glycoprotein, efficiently incorporated into virions lacking a native glycoprotein, can confer a broadened host range on the virus. Moreover, because the HA of influenza virus requires the acidic pH of the endosome in order to be activated, these results imply that foreign proteins can modify the normal route of entry of this avian retrovirus.


Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and of cell lines after in vitro infection. After infection of 1-day-old chickens, chicken anemia virus (CAV) causes a complete depletion of the thymic cortex by day 14. Since cell death can be caused either by necrosis or by apoptosis, we investigated which type of cell death occurs after in vivo and in vitro infections with CAV. Using electron microscopy and biochemical methods, we demonstrated that CAV induces apoptosis of cortical thymocytes after in vivo infection and of lymphoblastoid cell lines after in vitro infection. At day 13 after in vivo infection, virus-like particles were detected in apoptotic bodies that were absorbed by epithelial cells. These results show that apoptosis, a phenomenon that has been observed for a few other viruses, is also an important phenomenon during the pathogenesis of CAV.


Repression of liver-specific hepatitis B virus enhancer 2 activity by adenovirus E1A proteins. Two regions of the hepatitis B virus (HBV) genome have been shown to display properties of a transcriptional enhancer. Enhancer 1 is active in most hepatoma lines examined as well as in some non-hepatocyte-derived cell lines. In contrast, enhancer 2 activity is strictly liver specific. In this study, we show that adenovirus E1A expression in the highly differentiated human hepatoma line Huh6 strongly inhibits HBV enhancer 2-stimulated transcription while having no effect on HBV enhancer 1 activity. A sequence motif in HBV enhancer 2 which is essential for its enhancer function is the target for E1A-mediated repression. The repression of HBV enhancer 2 activity is mediated through the N-terminal region of the E1A proteins known to bind a 300-kDa cellular protein. Our results suggest that HBV enhancer function may be modulated by a cellular mechanism similar to E1A-mediated transcriptional repression.


The Epstein-Barr virus (EBV) nuclear antigen 1 BamHI F promoter is activated on entry of EBV-transformed B cells into the lytic cycle. In Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cell lines exhibiting the latency I form of infection (i.e., EBV nuclear antigen 1 [EBNA1] positive in the absence of other latent proteins), the EBNA1 mRNA has a unique BamHI Q/U/K splice structure and is expressed from a novel promoter, Fp, located near the BamHI FQ boundary. This contrasts with the situation in EBV-transformed lymphoblastoid cell lines (LCLs) exhibiting the latency III form of infection (i.e., positive for all latent proteins), in which transcription from the upstream Cp or Wp promoters is the principal source of EBNA mRNAs. We carried out cDNA amplifications with oligonucleotide primer-probe combinations to determine whether Fp is ever active in an LCL environment. The results clearly showed that some LCLs express a Q/U/K-spliced EBNA1 mRNA in addition to the expected Cp/Wp-initiated transcripts; this seemed inconsistent with the concept of Cp/Wp and Fp as mutually exclusive promoters. Here we show that Fp is indeed silent in latency III cells but is activated at an early stage following the switch from latency III into the virus lytic cycle. Four pieces of evidence support this conclusion: (i) examples of coincident Cp/Wp and Fp usage in LCLs are restricted to those lines in which a small subpopulation of cells have spontaneously entered the lytic cycle; (ii) transcripts initiating from Fp can readily be demonstrated in spontaneously productive lines by S1 nuclease protection; (iii) the presence of Fp-initiated transcripts is not affected by acyclovir blockade of the late lytic cycle; and (iv) infection of latently infected LCLs with a recombinant vaccinia virus encoding the EBV immediate-early protein BZLF1, a transcriptional transactivator which normally initiates the lytic cycle, results in the appearance of the diagnostic Q/U/K-spliced transcripts.


Purification and characterization of poliovirus polypeptide 3CD, a proteinase and a precursor for RNA polymerase. A cDNA clone encoding the 3CD proteinase (3CDpro) of poliovirus type 2 (Sabin), the precursor to proteinase 3Cpro and RNA polymerase 3Dpol, was expressed in bacteria by using a T7 expression system. Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3Cpro and 3Dpol. Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CDpro T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing. This mutant was purified to 86% homogeneity and used for subsequent proteolytic studies. Purified 3CDproM (M designates the cleavage site mutant 3CDpro T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide. Purified 3CDproM demonstrated the same detergent sensitivity in processing experiments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates. Purified 3CDproM did not have any detectable RNA polymerase activity, whereas 3Dpol, separated from 3CDproM by gel filtration in the last step of purification, did. We conclude that 3CDproM can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate. Moreover, cleavage of 3CD to 3Dpol is needed to activate the 3D RNA polymerase.


Antibodies to varicella-zoster virus modulate antigen distribution but fail to induce viral persistence in vitro. Varicella-zoster virus (VZV) persists in human sensory ganglia. One of the hypotheses to explain the induction or the maintenance of VZV latency is that it could be promoted by the immune response itself. It is known that in the case of viruses which bud off the infected cell membrane, virus-specific antibodies can induce antigenic modulation, i.e., spatial redistribution of viral antigens and modulation of their synthesis. To determine whether antigenic modulation occurs during VZV infection in vitro and could possibly be involved in viral persistence, we have grown infected cells in the presence of anti-VZV antibodies either transiently or permanently. The distribution of immune complexes and viral proteins was then analyzed. In transient immunomodulation experiments, the distribution of one or more viral antigens was modified not only in the cytoplasmic membranes but also in the cytoplasm and nucleoplasm of infected cells. When infected cells were kept permanently in the presence of antibodies, the same pattern of redistribution of immune complexes was observed and the localization of internal viral glycoproteins was significantly modified. However, antibodies did not prevent the lytic effect of infection; they altered neither the infectious virus yield nor the Western immunoblot pattern of viral proteins, suggesting that immunomodulation is not the primary effector of viral persistence.


The kappa B enhancer motifs in human immunodeficiency virus type 1 and simian virus 40 recognize different binding activities in human Jurkat and H9 T cells: evidence for NF-kappa B-independent activation of the kappa B motif. The kappa B transcriptional enhancer motif, present in many viruses, is broadly active in many cell types. It is recognized by c-Rel/HIVEN86A in DNA affinity precipitation (DNAP) assays and by the Rel-related p50 and p65 subunits of the nuclear factor NF-kappa B in electrophoretic mobility shift assays (EMSA). We have analyzed activities that bind the human immunodeficiency virus type 1 and simian virus 40 kappa B motifs in two human leukemia cell lines, Jurkat and H9. In both DNAP and EMSA analyses of Jurkat cell extracts, we detected multiple kappa B motif-binding activities in addition to c-Rel/HIVEN86A and p50-p65 NF-kappa B. In Jurkat cell nuclear extracts, EMSA analysis revealed at least six specific DNA-protein complexes, of which one comigrated with the p50-p65 NF-kappa B complex. Formation of all six complexes was enhanced by stimulation of the cells with phorbol 12-myristate-13-acetate and phytohemagglutinin but was differentially affected by the salt concentration in the binding reaction and by the conditions of Jurkat cell growth. Nuclear extracts from both unstimulated and stimulated H9 cells revealed similar levels of five kappa B motif-specific complexes, all of which displayed mobilities distinct from those of the Jurkat cell complexes. Indeed, a complex corresponding to p50-p65 NF-kappa B was not detectable in nuclear extracts from unstimulated H9 cells although such a complex was apparent in nuclear extracts from stimulated H9 cells. In contrast to the inducibility of a p50-p65 NF-kappa B-like complex, transcriptional enhancers composed of multimerized kappa B motifs displayed similar high levels of activity in both the unstimulated and stimulated H9 cells. Thus, the activity of the kappa B motif in H9 cells corresponded to the abundance of the H9 cell-specific kappa B motif complexes and not to the levels of p50-p65 NF-kappa B complex. These results suggest that the broad activity of the kappa B enhancer element is not only due to the broadly distributed NF-kappa B activator but also to cell type-specific kappa B motif-binding activities.


The herpes simplex virus type 1 (HSV-1) a sequence serves as a cleavage/packaging signal but does not drive recombinational genome isomerization when it is inserted into the HSV-2 genome. We inserted the terminal repeat (a sequence) of herpes simplex virus type 1 (HSV-1) strain KOS into the tk gene of HSV-2 strain HG52 in order to assess the ability of the HSV-1 a sequence to provoke genome isomerization events in an HSV-2 background. We found that the HSV-1 a sequence was cleaved by the HSV-2 cleavage/packaging machinery to give rise to novel genomic termini. However, the HSV-1 a sequence did not detectably recombine with the HSV-2 a sequence. These results demonstrate that the viral DNA cleavage/packaging system contributes to a subset of genome isomerization events and indicate that the additional recombinational inversion events that occur during infection require sequence homology between the recombination partners.


An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit. We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1). R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus. When the N terminus was separately expressed in E. coli and partially purified, a similar autophosphorylation reaction was observed. Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted. Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E. coli contaminant protein kinase. We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication.


A mouse mammary tumor virus mammary gland enhancer confers tissue-specific but not lactation-dependent expression in transgenic mice. The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) is known to contain a number of transcriptional regulatory elements, including glucocorticoid response elements. In this study, we showed that a mammary gland/salivary gland enhancer found in the LTR of this virus directs expression of a heterologous promoter to both virgin and lactating mammary glands in transgenic mice. Using transgenic mice containing hybrid gene constructs with various deletions of the LTR sequences linked to marker genes, we also showed that the dramatic increase in MMTV expression that occurs during lactation is due to the glucocorticoid response elements. Thus, the MMTV LTR encodes two distinct elements, both of which are required for a high level of expression in lactating mammary glands.


Epstein-Barr virus nuclear protein 2 is a critical determinant for tumor growth in SCID mice and for transformation in vitro. Injection of Epstein-Barr virus (EBV)-transformed human lymphoblastoid B cells into immunodeficient SCID mice results in the appearance of rapidly growing, fatal human B-cell tumors. To evaluate the role of EBV nuclear protein 2 (EBNA-2) in this process, we generated lymphoblastoid cell lines transformed by several EBV mutants which were identical except for deletions in the EBNA-2 gene (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991). These cell lines were injected intraperitoneally into SCID mice, and the interval until tumor detection was determined. Cell lines transformed with EBV type 1 (strain W91) or with EBV type 2 (strain P3HR-1) with an inserted type 1 EBNA-2 gene grew at the same rapid rate, indicating the potential importance of EBNA-2 for tumor formation in vivo. Cell lines derived from three different EBV mutants with deletions in the amino half of EBNA-2 produced tumors more slowly than cell lines transformed by wild-type W91 virus. In contrast, a cell line transformed with an EBV mutant with a deletion in the carboxy terminus of EBNA-2 grew more rapidly than cell lines transformed by wild-type virus. EBV mutants with deletions in the amino half of EBNA-2 had had reduced transforming activity in vitro, while the carboxy-terminal EBNA-2 mutant had had transforming activity greater than or equal to that of the wild type. These data indicate that EBNA-2 plays a critical role both for B-cell tumor growth in SCID mice and for B-lymphocyte transformation in vitro.


Alphavirus assembly and entry: role of the cytoplasmic tail of the E1 spike subunit. The alphavirus Semliki Forest virus (SFV) matures by budding at the cell surface. This process is driven by interactions of its membrane protein heterodimer E2-E1 and the nucleocapsid. The virus penetrates into new cells by an E1-mediated membrane fusion event. The E1 subunit has a short, strongly positively charged cytoplasmic tail peptide (Arg-Arg) which is very conserved among different alphavirus E1 proteins. In this work, we have used in vitro mutagenesis of a full-size cDNA clone of SFV to study the role of the tail peptide of the E1 subunit in virus budding and fusion processes in baby hamster kidney cell culture. Our results suggest that the E1 tail plays no major role in SFV multiplication in animal cell culture.