pseudotyping envelopes of viral vectors are heterologous glycoproteins with the key role of mediating vector entry into target cells .
thus , their nature , function , and density on the vector surface may deeply influence the transduction ability of the vectors . a powerful strategy to increase
the expression of heterologous proteins in eukaryotic cells is codon optimization ( co ) , which is an artificial process through which dna sequences are modified by the introduction of silent mutations , generating synonymous codons . by degeneracy of the genetic code , all amino acids ( aa ) except met and trp are encoded by more than one codon ; i.e. , synonymous codons .
genetic code redundancy makes dna triplets tolerant for point mutations , which do not result in corresponding aa mutations ( silent mutations ) .
codon optimization is exploited to overcome species - specific codon usage bias and ultimately improve heterologous protein production .
the frequency of codon distribution within the genome ( codon usage bias ) is variable and differs depending on species .
it follows that trnas corresponding to synonymous codons are not equally abundant in different cell types and species .
therefore , for a certain aa , there are synonymous codons more often used , influencing the timing and efficiency of protein translation.2 , 3 , 4 the codon adaptation index ( cai ) technique measures synonymous codon usage bias in a given species .
the cai uses a range ( between 0 and 1 , where 1 is the maximum translational efficiency ) of high - rate expression genes ( i.e. , ribosomal proteins and elongation factors ) to assess the relative contribution of each codon in a specific organism , allowing comparison with the nucleotide sequence of interest .
thus , it is possible to increase the expression of a certain gene in a specific organism / cell type by simply changing rare codons with more frequent ones , resulting in modification of the cai .
codon optimization has been extensively used to increase the production of either recombinant proteins or viral vectors.6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 rd114-tr is a chimeric mutant deriving from the feline endogenous retrovirus rd114 envelope , in which the tr domain of the gamma retroviral vector ( -rv ) moloney leukemia virus ( mlv ) amphotropic 4070-a envelope , fused at the c - terminal end of rd114 , increases envelope incorporation into lentiviral vector ( lv ) particles .
rd114-tr is first translated in a non - functional precursor ( pr ) that is then processed by the membrane - associated endoprotease furin in the surface ( su ) and transmembrane ( tm ) active subunits .
rd114-tr processing occurs either in furin - rich compartments of the trans - golgi network , where the pr accumulates during its way to the plasma membrane or in the recycling endosomes close to the plasma membrane .
the cleavage and post - translational glycosylation of rd114-tr are crucial for trafficking to the plasma membrane and for incorporation into nascent virion coats .
the tm subunit mediates plasma membrane anchoring of the su subunit . upon recognition and engagement of functional subunits to specific receptors , fusion between viral and cell membranes mediates the entry of the vector into target cells .
rd114-tr - pseudotyped retroviral vectors are suitable for both ex  vivo and in  vivo gene therapy applications because they can be concentrated by centrifugation and are resistant to human serum complement inactivation.20 , 21 , 22 , 23 to improve and simplify the expression of the rd114-tr envelope during development of the rd - molpack packaging technology for stable and constitutive manufacturing of lvs,21 , 23 we codon - optimized the entire rd114-tr open reading frame ( orf ) .
this idea stemmed from our previous observation that rd114-tr expression is achieved only when the -globin intron ( bgi ) is inserted between the promoter and the rd114-tr cdna of the expression cassette of  many different expression plasmids tested . to explain this constraint
, we hypothesized that bgi may attenuate the negative effect of interfering sequences existing in the rd114-tr cdna . to eliminate these sequences and to simplify the vector design , we decided to codon - optimize the entire rd114-tr orf .
in fact , the elimination of the interfering sequences would have avoided using the bgi , therefore reducing the size of the vector .
unexpectedly , we  found that , despite the high level of transcription / translation and cytosol export , rd114-trco is functionally dead .
our data strengthen the conclusion , also supported by other studies , that codon optimization may not always lead to functional improvement of the gene of interest .
we initially analyzed the expression of the rd114-trwt envelope in rd3-molpack - gfp producer cells and in their derived lvs to confirm previous studies describing proper processing and trafficking to the plasma membrane of the wild - type ( wt ) envelope .
rd3-molpack - gfp cells contain 12 copies of the integrated self inactivating ( sin)-rd114-trwt - in - rev - responsive element ( rre ) transfer vector ( tv ) ( figure  1a , scheme 2 ) , and the originated rd114-trwt pseudo - typed lvs are proficient in cell transduction , as reported previously .
we used two specific antibodies ( abs ) , each recognizing either the pr and su ( anti - su ) subunits or the pr and tm ( anti - tm ) subunits , respectively ( figure  1b ) . to visualize the expression of rd114-trwt at the rd3-molpack - gfp plasma membrane
, we carried out pull - down ( pd ) of biotinylated and de - glycosylated total cell extracts . because , in sds - page , glycosylated pr and su molecules co - migrate , we first pulled down membrane proteins , which were first biotinylated in  vivo and then deglycosylated by  peptide n - glycosidase f ( pngasef ) treatment in  vitro .
pngasef cleaves the link between asparagine and n - acetylglucosamine residues ( complex oligosaccharides ) that are added in the endoplasmic reticulum ( er ) and the golgi stack .
we here confirmed the results previously reported by sandrin et  al . , showing that both tm and su subunits are correctly localized at the plasma membrane , whereas the pr does not reach and/or accumulate on it ( figure  2a , lane 8) .
the very low level of pr detected in the pngasef - treated sample likely derives from contamination of the endoplasmic reticulum or other membranes ( figure  2a , anti - su , top right panel , lane 8) . to further characterize rd114-tr glycosylation and trafficking to the plasma membrane , we treated in  vitro producer cellular and derived vector extracts not only with pngasef but also with endoglycosidase h ( endoh ) enzyme .
the latter is active on n - linked high - mannose oligosaccharides ( simplex oligosaccharides ) , added in the er compartment , but not on high - glucose residues attached later during glycosylation in the golgi apparatus .
it follows that glycoproteins carrying complex oligosaccharides become resistant to the attack of endoh ( endoh - resistant proteins ) . of note
, we observed that , in both cells and derived lvs , pr and tm subunits are endoh - sensitive ( figure  2b , lanes 3 and 6 , anti - tm ) .
on the contrary , the su subunit is endoh - resistant because it carries complex oligosaccharides ( figure  2b , lanes 3 and 6 , anti - su ) .
the tm contains one putative n - linked glycosylation site ( nxs and nxt , where x is any aa ) , whereas su contains 11 sites ( figure  s1 ) .
it is possible that this unique n - linked site in tm is glycosylated with simplex and not complex oligosaccharides and that the tm subunit is transported to the plasma membrane linked to the su .
furthermore , the average titer of rd3-molpack - gfp lvs tested in this study is 1.6   10   4.7   10 sem transducing units ( tu)/ml ( n  = 5 ) , in line with our previous collective data.21 , 23 overall , these findings demonstrate that expression of rd114-trwt in rd3-molpack - gfp producer cells and stemmed lvs is correctly achieved . in an attempt to enhance the transduction efficiency of rd3-molpack - derived lvs by increasing the expression and stability of rd114-tr glycoprotein , we codon - optimized its complete cdna .
after recoding , the cai of the rd114-tr orf shifted from 0.64 to 0.98 , and the average gc content increased from 48% ( wt ) to 61% ( co ) , resulting in 73% identity between the wt and co sequences ( figures s2 and s3 ) . to test the function of rd114-trco ,
the new orf , cloned into the pires - puro3 expression vector , was transiently co - transfected in pk-7 cells together with the sin - gfp tv to produce rd114-trco - expressing lvs .
we analyzed the expression of rd114-tr proteins by western blot , treating cell and virion extracts with or without pngasef and endoh ( figure  3 ) .
surprisingly , the pattern of rd114-trco subunits greatly differed from that of the wt counterparts .
in fact , both cell and lv protein extracts showed very high levels of prco and very low levels or even absence of processed suco and tmco subunits ( figures 3a and 3b ) .
in contrast , the expression profile of rd114-trwt in cell and vector extracts was identical to that of rd3-molpack - gfp producer cells and the lvs shown in figure  2 .
in agreement with these data , the viral titer of rd114-trwt pseudotyped lvs calculated on cem a3.01 cells was 3.9   10   7.1   10 sem tu / ml ( n  = 3 ) , whereas that of rd114-trco - pseudotyped lvs was consistently undetectable .
to better understand the difference between prwt and prco processing , we tested whether codon optimization might have somehow compromised furin - mediated cleavage of rd114-trco . to this purpose , we treated cell extracts derived from pk-7 cells transfected with either the rd114-trwt or rd114-trco plasmid with recombinant furin overnight at 16c .
untreated and treated extracts were then analyzed by western blot using the anti - tm ab ( figure  4a ) .
we observed that , after furin treatment in  vitro , the level of tmco subunit clearly increases ( figure  4a , lane 4 ) , even though it is difficult to appreciate the corresponding decrement of prco because of its high level of expression . on the contrary ,
the amount of prwt is clearly decreased , although it is difficult to appreciate the corresponding increase of tmwt because the wild - type protein is already abundantly cleaved before cell protein extraction .
overall , these results support the idea that codon optimization does not compromise furin - mediated cleavage of the envelope , at least in  vitro . based on this notion
, we then tried to understand why the prco is not correctly processed in  vivo .
one possible explanation was that a large amount of prco could trigger the phenomenon known as excess substrate inhibition . to exclude this possibility , we transfected hek293 t cells with a scalar amount of rd114-trco plasmid and tested the corresponding cell extracts in a western blot to find the lowest possible dose of prco substrate not inhibiting endogenous furin action ( figure  4b ) .
we observed that , even at the lowest amount of plasmid generating detectable prco , the tmco subunit was not visible , indicating that  in  vivo prco is not processed ( figure  4b , lane 3 ) .
we next evaluated whether partial recoding of the orf could restore the function of the rd114-trco envelope . to this aim
, we generated two cdnas recoded only in the 5 or 3 half of the cdna sequence .
we transiently transfected either the rd114-tr5co or rd114-tr3co chimera , cloned into the pires - puro3 plasmid , into pk-7 cells together with the sin - egfp tv .
we then tested cellular and lv extracts in a western blot and lv titer in cem a3.01 cells .
immunoblot analysis demonstrated that , for both chimeric rd114-tr glycoproteins , prco processing was impaired ( figure  s4 ) .
furthermore , although we transfected equal amounts of rd114-tr , rd114-tr5co , and rd114-tr3co plasmid dna , the expression of rd114-tr3co was lower than that of rd114-tr5co and rd114-trwt ( figures s4a and s4c , lanes 3 and 4 ) .
the tm3co and tm5co subunits were not detectable in the respective lvs , whereas , after pngasef treatment , su3co and su5co were barely visible and visible , respectively .
we explain the difference between anti - tm and anti - su staining with an intrinsic difference in the specific affinity of the two abs . in agreement ,
to see whether rd114-trco differs from rd114-trwt in its subcellular localization , we carried out confocal microscope imaging in cos-7 cells transfected with pires - rd114-tr plasmids .
forty - eight hours after transfection , rd114-tr expression was visualized together with that of calnexin and vamp8/endobrevin , which are er and early and late endosomal markers , respectively . as shown previously by sandrin et  al .
, rd114-trwt is expressed in the cytosol and perinuclear region and is co - localized mostly with calnexin and very poorly with endobrevin / vamp8 .
a similar staining pattern and subcellular localization was observed for rd114-trco in either the cos-7 ( figure  5 ) or pk-7 cell experimental setting ( figure  s5 ) , indicating that er and early and late endosome trafficking of rd114-tr is not affected by codon optimization . because many groups have demonstrated that silent mutations affect correct pre - mrna splicing by introducing cryptic splice sites or altering splicing control elements ( i.e. , exonic splicing enhancers and silencers),3 , 25 , 26 we and two service provider companies analyzed rd114-trco mrna both in  silico and in  vitro for the presence of potential cryptic splicing sites .
the first in  silico service - provided analysis identified one consensus ( cryptic ) splice donor site that was nullified by codon optimization , whereas the second service - provided analysis recognized no cryptic sites ( figures s2 , s3 , s6 , and s7 ) .
we also examined the rd114-trwt and rd114-trco orfs in  silico using the netgene2 server , which calculates the probability of cryptic splicing sites in pre - mrna sequences .
we did not pinpoint any differences between wild - type and codon - optimized orfs . to further confirm these results , we assessed rd114-trwt and rd114-trco mrna transcripts derived from pk-7 cells transiently transfected with the sin - rd114-trwt / co - in - rre constructs ( figure  1a , schemes 2 and 3 ) by northern blot ( figure  6a ) .
two sequence - specific probes targeting rd114-trwt and rd114-trco , respectively , recognized qualitatively comparable rd114-tr mrna transcript patterns ( figure  6a ) .
similar results were obtained by using a probe directed against the packaging signal (  ) , which is a sequence common to both constructs .
the overall steady - state level of rd114-trco rna detected by the  probe was only slightly reduced compared with the wild - type counterpart , but no extra spliced bands were observed .
these results indicate that the two lentiviral vector plasmids were equally transfected and correctly expressed from the 5 long terminal repeat ( ltr)-cytomegalovirus ( cmv ) vector promoter .
these findings indicate that no cryptic splicing sites are present either in the orf or in the vector backbone ( figure  6a ) .
to assess whether mrna metabolism differs between rd114-trwt and rd114-trco , we studied mrna nuclear - cytoplasm export in the pk-7 cell setting using the pires - puro3-based expression vectors , which generate only one mrna transcript .
northern blot analysis of total , nuclear ( nucl ) , and cytoplasmic ( cyt ) mrnas and quantification by typhoon phosphorimager of the band intensity normalized by cellular equivalents loaded revealed that the unique codon - optimized mrna is exported 1.4-fold more ( wt cyt / nucl band intensity  = 1.1 and co cyt / nucl band intensity  = 1.6 ; 1.6/1.1  = 1.4 ) than wild - type mrna ( figure  6b ) .
qrt - pcr analysis , using the expression of nuclear u6 and cytosolic / total gapdh genes as an internal normalizer , revealed that rd114-trco mrna is exported 3.6-fold more than rd114-trwt mrna ( figure  6c ) .
overall , these data establish that recoding affects nuclear export but not transcription and splicing processes .
we then investigated whether codon optimization could influence mrna secondary structure and , thereafter , protein translation , as reported recently by several groups.3 , 4 , 27 thus , we examined rd114-trwt and rd114-trco mrna sequences by mfold software ( figures 7a and 7b ) . this computational analysis predicts the most thermodynamically stable rna configurations ( up to 50 ) based on the free energy value ( g ) of the molecules , where a lower g indicates a higher stability .
we retrieved 33 different configurations for rd114-trwt and 37 for rd114-trco ( figure  7e ) .
as expected , wild - type structures are very different from codon - optimized ones ; the average g for rd114-trwt mrna is 462.25 ( where g  =  468.70 is the most stable configuration ) , whereas the average g for rd114-trco mrna is 679.39 ( where g  = 687.60 is the most stable configuration ) .
this finding indicates that recoded mrna molecules are more stable than wild - type counterparts ( p  < 0.0001 ) ( figure  7e ) . because 5 end and 3 end substructures are fundamentally important for translational dynamics and protein folding , we scanned the 5 and 3 ends of all wild - type and recoded mrnas to identify any possible structural conserved domains . over the 33 conformations of rd114-trwt mrna
, we identified a conserved domain at both the 5 end ( nucleotides [ nt ] 1320 ) and 3 end ( nt 1,3081,677 ) ( figure  7c ) .
these 5 end and 3 end domains are also conserved in the corresponding region of the rd114-trco mrna structure . above the 37 structures calculated by mfold for rd114-trco mrna , we identified nt 1330 at the 5 end and nt 1,3901,677 at the 3 end ( figure  7d ) .
to evaluate the similarity between identified domains , we studied rd114-trwt and rd114-trco mrna 5 end and 3 end substructures with simtree software .
this software compares each node complexity ( branch - loop ) of two structures that eventually grades in a similarity score ( between 01 , where 1 is the maximum ) .
the score is normalized by the number of nucleotides of the substructures from two rna structures showing the lowest g in mfold ( table s1 ) . at the 5 end of the rd114-trwt mrna substructure ,
26 complexities were identified , which corresponded to only ten complexities in rd114-trco ( normalized symmetric similarity [ nss ]  = 0.5213 ) ( figure  7a ) . at the 3 end
mrna substructures , 42 complexities were found in rd114-trwt and only 18 in rd114-trco ( nss  = 0.6178 ) ( figure  7b ) .
these results point out that codon optimization of the rd114-tr gene introduced significant alterations at both the 5 end and 3 end of the rd114-trco mrna secondary structure .
codon optimization and de - optimization have been used extensively for a lot of different biotechnological practices , primarily in heterologous systems to increase recombinant protein yield and as an adaptive response to environmental conditions and natural host selection in bacteria , yeasts and viruses.29 , 30 , 31 , 32 , 33 , 34 in some eukaryotes ( i.e. , c.  elegans and drosophila melanogaster ) , it has been exploited to control intracellular trna to modulate translational efficiency.35 , 36 , 37 , 38 in autologous hosts , such as the mammalian chinese hamster ovary ( cho ) or hek293 cell lines , which are the most widespread systems for manufacturing pharmaceuticals , codon optimization is a valuable strategy to prevent transcriptional silencing , mrna destabilization , or inefficient translation other than being a powerful tool to increase immunogenicity in dna vaccinology applications.9 , 10 , 16 , 17 , 39 finally , hiv - derived lv production has also benefited from codon optimization by enhancing production of structural and functional viral proteins ( i.e. , gag and pol);9 , 11 , 12 neutralizing cis - repressive sequences present in gag / pol genes , thereby making the expression of these genes rev - independent;13 , 14 and eliminating homology between packaging gag - pol genes and the cis - regulatory packaging (  ) sequence contained in the packaging construct and tv , respectively , therefore reducing the risk of generating a replication - competent lentivirus ( rcl ) .
based on these premises , we analyzed dna codon optimization of the rd114-tr gene with the aim of improving envelope translation in rd3-molpack producer cells .
in fact , codon optimization would have neutralized the interfering sequences contained in the rd114-tr orf , thereby sparing the use of the bgi in vector design and , at the same time , increasing the production and density of rd114-tr on the cellular plasma membrane and , consequently , on virion coats .
we asked for two recoding analyses by two independent companies , which provided similar results ( figure  s8 ) .
therefore , we believe that the quality of the analysis could not have affected the final output . the consistently negative results obtained with the chimeric rd114-tr3co and 5co support this idea . in this study , we show that rd114-trwt expressed either stably ( rd3-molpack-24 cells ) or transiently ( pk-7 cells ) naturally traffics from the er through the golgi network to reach the plasma membrane .
prwt is processed in suwt and tmwt subunits , which are eventually embedded into nascent lvs .
in contrast , rd114-trco reaches the plasma membrane mainly as unprocessed prco , and maturation into suco and tmco functional subunits is drastically reduced or even absent . as a consequence , a high level of prco is erroneously incorporated into budded viral particles , which become defective vectors . because n - linked glycosylation is crucial for maturation of different envelope ( env ) proteins , such as the hepatitis c virus glycoprotein e2 and human t  cell leukemia virus type i ( htlv - i ) envelopes,39 , 40 , 41 we studied the glycosylation status of rd114-trwt and rd114-trco . here
we confirm the conclusions reached by sandrin et  al . , showing that prwt , suwt , and tmwt are deglycosylated by pngasef .
furthermore , we also expanded glycosylation studies showing that , in contrast to suwt , tmwt is endoh - sensitive .
this result suggests that tmwt either reaches the plasma membrane anchored to su or loses complex oligosaccharides when on the membrane , or , alternatively , does not need complex oligosaccharides for its function .
the analysis of prco glycosylation demonstrates sensitivity to both pngasef and endoh enzymes , suggesting defective glycosylation of the recoded protein .
further studies will clarify whether a correlation between the observed defective glycosylation and maturation of rd114-trco does exist .
although prco is cleaved in  vitro by recombinant furin both under reducing and non - reducing conditions ( data not shown ) , it is not cleaved in  vivo . to explain this result
, we reasoned that cleavage in  vivo could be prevented by an excess of substrate : prco is , in fact , much more abundant compared with prwt .
alternatively , the deficit of prco processing could be secondary to a deficit of retrograde transport of prco from the cell membrane to the endosomes , where the active form of furin is accumulated .
we ruled out the first hypothesis because furin is not active in  vivo , even with a very low amount of prco substrate , whereas the second hypothesis requires further analysis to be formally accepted .
synonymous mutations have been considered ineffective for a long time , and , for this reason , they are also named silent mutations .
however , their nature has been recently re - evaluated because evidence has shown that these mutations have a great effect on pre - mrna splicing and mrna secondary structure formation , therefore affecting protein translational efficiency and folding.4 , 43 even a single synonymous codon substitution can have a significant effect on protein folding and function .
protein dysfunction can be caused either by disruption ( or introduction ) of splicing enhancers , by altering mrna stability at the  global and local level , or by altering the kinetic of protein production , the ribosomal pausing sites , and co - translational folding.27 , 44 our results exclude that codon optimization has introduced aberrant pre - mrna splicing sites . rather , they establish that rd114-trco mrna is exported more efficiently into the cytosol than rd114-trwt mrna and support the theory that some alterations occurred at the mrna secondary structure , thereby influencing protein translation .
we focused our study on the mrna 5 end and 3 end because previous findings from others demonstrated that these two domains crucially influence translation dynamics , such as translation initiation and rna global and local stability.45 , 46 , 47 the in  silico mfold and sintree software analyses highlighted that the secondary structures of rd114-trwt and rd114-trco mrna are significantly different .
especially some conserved domains at the 5 end and the 3 end of  rd114-trwt mrna are lost in the rd114-trco isoform .
interestingly , the generation of chimeric rd114-tr5co and rd114-tr3co led to even worse functional impairment .
these findings suggest that rd114-trco inactivity is not due to single mutations clustered at the 5 end or 3 end but , more likely , due to conformational modifications distributed along the mrna molecule that affect global mrna stability and , thereby , protein folding and processing .
demonstrated that modifications in the cytoplasmic tail of rd114 and rd114-tr alter pr subunit transport from the cell membrane to the trans - golgi network .
in particular , transport of  the envelopes associated with core protein ( i.e. , gag ) to the endosomal compartment , where active furin accumulates , is important because it affects cleavage efficiency . we observed , by confocal microscope imaging , that both rd114-trwt and rd114-trco are  localized mostly in the er compartment when assessed either in the presence ( pk-7 setting ) or in the absence ( cos-7 ) of gag protein . to this extent , secondary structure modifications identified  in  rd114-trco mrna might result in alteration of  protein folding , which , in turn , is responsible for protein dysfunction
altogether , this study suggests that rd114-tr is not suitable for codon optimization and that this strategy can not be applied to improve its performance .
cosset ( inserm ) , encodes the chimeric rd114-tr envelope that derives from fusion of the extracellular and transmembrane domains of the feline endogenous retrovirus rd114 envelope and the cytoplasmic tail ( tr ) of the amphotropic ( a)-mlv 4070 envelope ( figure  1a , scheme 1 ) . the pires - rd114-trwt - internal ribosome entry site ( ires)-puro - woodchuck hepatitis post - transcriptional regulatory element ( wpre ) plasmid was obtained by excising the cmv - rd114-trwt cassette from the phcmv - rd114-tr plasmid and cloning it into the pires - puro 3 plasmid ( clontech laboratories , a takara bio company ) ( figure  1a , scheme 7 ) .
the generation of the sin - rd114-trwt and sin - rd114-trco vectors ( figure  1a , schemes 2 and 3 ) as well as the constructs encoding the hiv gag , pol , and rev genes ( figure  1a , schemes 4 and 5 , respectively ) have been described previously .
the sin - gfp tv encoding the egfp gene was kindly provided by l. naldini ( tiget , osr ) ( figure  1a , scheme 6 ) .
the rd114-tr orf was codon - optimized , synthesized , and cloned in either the pmk - rq or pms - rq plasmid by geneart .
we further cloned the rd114-trco orf into either the pires - puro3 or sin - lv plasmid .
four different molecules were generated : pires - cmv - rd114-tr - flco , in which full - length ( fl ) cdna was codon - optimized and cloned into the ecorv and nsii sites of the pires - puro3 plasmid by excising the orf from the pmk - rq - rd114-tr - flco plasmid ; pires - cmv - rd114-tr-5co , obtained by recoding only the 5-half sequence ( 789  bp ) of the rd114-trwt orf ( rd114-tr5co was modified by adding eco47iii and nsii restriction sites at the 5 end and 3 end of the gene sequence , respectively , and the orf was then cloned into the eco47iii and nsii sites of pires - puro3 plasmid ) ; pires - cmv - rd114-tr-3co , obtained by recoding only the 3-half sequence ( 789  bp ) and cloning into the eco47iii and sphi restriction sites of the pires - puro3 plasmid by  excising the orf from pmk - rq - rd114-tr-3co ( figure  1a , scheme 7 ) ; and sin - rd114-trco fl ( figure  1a , scheme 3 ) , generated by inserting rd114-trco into a sin - lv through a three - step cloning strategy .
first , the rd114-trwt - ires - puro - wpre fragment was excised from the sin - rd114-trwt - in - rre vector ( figure  1a , scheme 2 ) and cloned into the ecori site of a pgem - t plasmid , generating the pgem - rd114-trwt - ires - puro - wpre plasmid .
then , rd114-trwt - ires - puro was excised from pgem - rd114-trwt - ires - puro - wpre ( obtaining the pgem - wpre intermediate ) using bamhi , and the rd114-trco - ires - puro orf was excised from the pires - cmv - rd114-tr - fl - co using ecorv / xbai enzymes .
rd114-trco - ires - puro was then cloned into the  pgem - wpre intermediate through blunt ligation , obtaining the  pgem - rd114-trco - ires - puro - wpre plasmid .
finally , the rd114-trco - ires - wpre orf was cut out from pgem - rd114-trco - ires - wpre and cloned into the ecori site of sin - rd114-trwt - in - rre , generating the sin - rd114-trco - in - rre vector .
t cells and their derivative pk-7 clone , which constitutively expresses the hiv gag - pol - rev genes , were propagated in iscove s modified dulbecco s medium ( imdm ) ( biowhittaker , lonza group )  supplemented with 10% australian fetal calf serum ( fcs ) ( biowhittaker ) and a combination of 1% penicillin - streptomycin and glutamine ( psg ) ( lonza ) .
the cem a3.01 t  cell line was  grown in rpmi 1640 medium ( biowhittaker ) supplemented with 10% fcs and 1% psg .
cos-7 cells were grown in dmem ( biowhittaker ) supplemented with 10% fcs and 1% psg .
briefly , 35   10 cells were plated on 100-mm tissue culture dishes ( becton dickinson ) . after 24  hr of culture ,
the egfp tv , rev , packaging , and envelope constructs were co - transfected at a 4:1:0.88:0.48 ratio using either profection mammalian transfection system calcium phosphate ( promega ) or fugene 6 transfection reagent ( roche diagnostics ) according to the manufacturer s instructions .
transfection efficiency was calculated 48  hr later by analyzing the percentage of egfp - positive cells by fluorescence - activated cell sorting ( facs ) analysis .
the cem a3.01 cell line was transduced by spinoculation at 1,024   g for 2  hr at 37c in the presence of polybrene ( 8  g / ml ) ( sigma - aldrich ) .
physical titer was evaluated by measuring the level of p24gag released in the culture supernatant with the alliance hiv-1 p24 antigen elisa kit ( perkinelmer ) according to the manufacturer s instructions .
pk-7 cells were transfected with the pires - rd114-tr or sin - rd114-tr tv plasmid encoding either rd114-trwt or rd114-trco .
forty - eight hours after transfection , total , nuclear , and cytoplasmic rnas were extracted by trizol reagent ( life technologies ) following the manufacturer s instructions and analyzed by northern blot assay .
five micrograms rna / sample was run on 0.8% agarose - formaldehyde gel , transferred onto a hybond - n membrane by capillary transfer , and finally probed with 1   10 dpm / ml of a p - labeled 550-bp rd114-trwt or rd114-trco probe in perfecthyb plus hybridization buffer ( sigma - aldrich ) .
membranes were extensively washed and then exposed to x - ray films at 80c or to a typhoon phosphorimager 9000 ( ge healthcare ) for direct quantification of the radioactive signal .
after stripping , membranes were re - hybridized with an internal control probe encompassing the packaging sequence (  ) to detect full - length mrnas .
total , nuclear , and cytoplasmic rnas , obtained as described above , were retrotranscribed with the superscript first - strand synthesis system kit for rt - pcr ( invitrogen ) .
the cdna ( 1.25  ng ) was quantified by qpcr sybr green technology with the following specific primers : rd114-trwt ( for 5 aac ggg tca gtc ttc ctc tg ; rev 5 atc aat ggc agg aat ggg ga ) , rd114-trco ( for 5 ccg tgc agt tca ttc ctc tg ; rev 5 ctc agc ttg gtg tac tgg gt ) , u6 ( for 5 ctc gct tcg gca gca ca ; rev aac gct tca cga att tgc gt 5 ) , and gapdh ( for 5 tgc acc aca act gct tag c ; rev 5 ggc atg gac tgt ggt cat gag ) . normalization was calculated using gapdh for total and cytosolic mrna and u6 for nuclear mrna .
cellular extracts and viral proteins derived from isolated cell - free virus - like particles ( vlps ) or lvs were prepared as described previously.21 , 49 briefly , lv supernatants were concentrated by centrifugation at 15,000   g for 90  min at 4c .
then , the liquid phase was gently removed , and pelleted virions were directly lysed by adding 5  l of pbs/0.5% np-40 ( calbiochem , merck - millipore , # 492016 ) .
proteins were size - fractionated on 8% , 12% , or 4%15% gradient sds - page ( mini - protean tgx gels , # 456 - 1084 , bio - rad ) .
then , proteins were  electroblotted on either hybond enhanced chemiluminescence ( ecl ) nitrocellulose membranes ( ge healthcare ) or transblot turbo transfer pack membranes ( bio - rad , # 170 - 4159 ) . membranes were blocked in 5% low - fat dry milk tris - buffered saline ( tbs ) , 1% tween 20 ( tbs - t ) and then incubated with the appropriate primary antibody diluted in 5% bsa and tbs - t . the anti - tm rd114-tr rabbit serum , kindly provided by f .-
the anti - su rd114-tr mab , generated by areta international , was diluted 1:50 .
the anti - extracellular signal - related kinase-1 ( erk ) rabbit ab was diluted 1:1,000 ( cell signaling technology , # 16 ) .
the anti - calnexin rabbit ab was diluted 1:2,000 ( santa cruz biotechnology , g1910 ) .
the extravidin horseradish peroxidase ( hrp ) ab was diluted 1:2,000 ( sigma - aldrich , # e2886 ) .
the anti - hiv human serum , obtained from an aids patient , was kindly donated by g. poli ( osr ) and diluted 1:1,000 .
the secondary hrp - linked abs anti - human ( # na933v ) and anti - rabbit ( na934v ) ( ge healthcare ) were diluted 1:5,000 .
the anti - mouse ( # a2066 ) ab ( sigma - aldrich ) was diluted 1:10,000 .
ecl western blotting detection reagent ( ge healthcare , rpmn2106 ) was used for the chemiluminescence reaction .
pk-7 and cos-7 cells were transfected with pires - rd114-tr plasmids encoding either rd114-trwt or rd114-trco , seeded on poly - l - lysine - coated glass slides ( thermo fisher scientific ) .
forty - eight hours after transfection , cells were fixed with pbs and 3% paraformaldehyde ( pfa)/0.1  mm cacl2/0.1  mm mgcl2 , permeabilized with pbs and 0.1% triton x-100 , and then stained with the following abs : rabbit anti - vamp8 ( endobrevin synaptic systems , catalog no .
1047 302 ) at 1:200 dilution , rabbit anti - calnexin ( santa cruz , h-70 , catalog no .
the secondary abs were alexa fluor goat anti - rabbit a488 ( invitrogen , catalog no .
a11034 ) and alexa fluor goat anti - mouse a568 ( invitrogen , catalog no .
slides were mounted with fluorescence mounting medium ( dako ) , and images were captured with a laser - scanning confocal microscope ( leica tcs sp5 ) with an hcx pl apo blue 63 ( na 1.4 ) objective in oil immersion .
images were acquired using the leica application suite ( las ) advanced fluorescence ( af ) software ( leica microsystems ) and processed by  the public domain imagej image processing and analysis software  ( http://rsb.info.nih.gov/ij/ ) ( image processing and analysis in java ) .
protein extracts from either cells or virions were treated with pngasef and endoh enzymes according to the manufacturer s instructions ( new england biolabs , # p0704s and # p0702s , respectively ) .
briefly , proteins were first denatured for 10  min at 99c and then digested for 1  hr at 37c with 250500  u of pngasef or endoh enzyme .
4 loading buffer containing -mercaptoethanol was added to samples that were then boiled for 5  min at 99c and finally loaded onto 4%15% sds - page precast gels ( mini - protean tgx gels , # 456 - 1084 , bio - rad ) for western blot analysis . in  vitro furin digestion
was carried out by treating 35  g cellular extracts with 4  u of recombinant furin ( neb , # p8077s ) for 16  hr at 16c following the manufacturer s instructions .
rd3-molpack - sin - gfp producer cells were plated in 60-mm culture dishes at 1   10 cells / cm density .
forty - eight hours after cell seeding , rd3-molpack - sin - gfp cells reached about 90% confluency .
cellular monolayers were gently washed in pbs supplemented with 1  mm mgcl2 and 0.1  mm cacl2 to keep epithelial junctions tight and impermeable to molecules .
cells were then incubated on ice for 30  min with 0.5  mg / ml ez - link sulfo - nhs - lc - biotin ( thermo scientific ) in pbs/1  mm mgcl2/0.1  mm cacl2 and gently shaken .
after biotinylation , cell monolayers were washed for 5  min with pbs/100  mm glycine/1  mm mgcl2/0.1  mm cacl2 for quenching the biotin excess .
cells were finally lysed as described previously,21 , 49 and protein extracts were quantified by protein assay ( bio - rad , # 500 - 0006 ) .
one milligram of proteins was incubated with 5060  l of biotin binder magnetic beads ( dynabeads myone streptavidin t1 , # 65602 , invitrogen ) for 1  hr at room temperature by gentle rocking .
beads were washed four to five times with 1  ml of pbs/0.1% bsa , and then protein / bead complexes were processed with pngasef .
after addition of 4 loading dye and boiling for 5  min at 99c , proteins were separated by sds - page on 4%15% precast gels ( mini - protean tgx gels , # 456 - 1084 , bio - rad ) and analyzed by western blot assay .
matched p24gag equivalents of vector particles were incubated with 5  l of anti - su ab ( 0.9  mg / ml ) for 3  hr at 4c under rocking conditions .
the pd was performed by washing three times 100  l of dynabeads ( sheep anti - mouse immunoglobulin g [ igg ] dynabeads , invitrogen , # 422.01 ) with pbs/0.5% bsa/2 mm edta and then rocking them for 30  min at room temperature in the presence of lv particles and the anti - su ab .
after virion pd , the dynabeads were washed several times , 4 loading dye was added , and proteins were separated by sds - page on 4%15% precast gels for western blot analysis .
prediction of rna splice sites was generated by the software made available by the netgene2 server ( http://www.cbs.dtu.dk/services/netgene2/ ) and prediction of mrna structure by the software mfold ( http://mfold.rit.albany.edu/?q=mfold/rna-folding-form ) using the following parameters : linear rna sequence ; 37c folding temperature ; 1  m nacl ionic condition : number of calculated folding ; differences between the calculated foldings  = default parameters ; maximum extension of the calculated loops  = 30 ; maximum asymmetry between the calculated loops  = 30 ; and no limit in base pairing distance .
statistical analysis was performed using jmp statistical software and by running the wilcoxon - mann - whitney ranked - sum non - parametric test .
contributed to the conception , acquisition , analysis , and interpretation of data and drafting the article .
contributed to the final approval of the version to be published . c. bovolenta contributed to the conception , design , analysis , and interpretation of the data , drafting and approving the final version to be published
 lentiviral vectors ( lvs ) are a highly valuable tool for gene transfer currently exploited in basic , applied , and clinical studies . 
 their optimization is therefore very important for the field of vectorology and gene therapy . 
 a key molecule for lv function is the envelope because it guides cell entry . 
 the most commonly used in transiently produced lvs is the vesicular stomatitis virus glycoprotein ( vsv - g ) envelope , whose continuous expression is , however , toxic for stable lv producer cells . in contrast , the feline endogenous retroviral rd114-tr envelope is suitable for stable lv manufacturing , being well tolerated by producer cells under constitutive expression . 
 we have previously reported successful , transient and stable production of lvs pseudotyped with rd114-tr for good transduction of t lymphocytes and cd34 + cells . to further improve rd114-tr - pseudotyped lv cell entry by increasing envelope expression , we codon - optimized the rd114-tr open reading frame ( orf ) . here 
 we show that , despite the rd114-trco precursor being produced at a higher level than the wild - type counterpart , it is unexpectedly not duly glycosylated , exported to the cytosol , and processed . 
 correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in  vivo , and , consequently , the unprocessed precursor is incorporated into lvs , making them inactive . 
Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest