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Laboratório de Virologia Molecular, Instituto de Biologia, Universidade Federal do Rio de Janeiro, CCS Bloco A2 sala 121, Cidade Universitária, Ilha do Fundão, 2194421944-970 Rio de Janeiro, Brazil
Correspondence
Amilcar Tanuri
atanuri{at}biologia.ufrj.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This process is selective, in that the vRNA constitutes <1 % of the RNA in the infected cell's cytoplasm, yet constitutes most of the RNA inside the virion (Berkowitz et al., 1995; Zhang & Barklis, 1995). The specificity of packaging is believed to depend largely on direct interactions between the viral Gag polyprotein and a cis-acting region located in the 5' leader region of the vRNA known as the packaging signal (
site) (Berkowitz & Goff, 1994; Berkowitz et al., 1996). These interactions are mediated by the nucleocapsid domain of Gag (Aldovini & Young, 1990; Dorfman et al., 1993; Gorelick et al., 1993; Poon et al., 1996; Provitera et al., 2001). Furthermore, in order to obtain the interactions required for assembly, Gag molecules first must be concentrated at a cellular site. Detergent-resistant domains at the plasma membrane have been proposed to play an important role in the concentration and alignment of Gag and Gag–Pol molecules (Halwani et al., 2003). Although Gag alone is sufficient to drive protein multimerization, vRNA incorporation and the assembly of virus particles, incorporation of Gag–Pol is clearly an important event during virus assembly and maturation.
Lentivirus full-length RNA is the template for protein synthesis and is also packaged as the viral genome. Production of infectious particles requires that both vRNA and a number of other viral molecules, including the Gag, Gag–Pol and Env polyproteins, are present concomitantly at budding sites on the plasma membrane. Several lines of evidence suggest that the Gag and Gag–Pol precursors and the vRNA form an intermediate viral-assembly complex in the cytoplasm, which is transported as a unit to the cell membrane for virus assembly and budding (Basyuk et al., 2003; Hill et al., 2001).
Elucidation of the relationships between viral protein translation and viral RNA packaging is necessary for complete understanding of the retroviral replicative cycle. Gag/Gag–Pol interactions with vRNA can be explained by two different models: either nascent Gag/Gag–Pol molecules in ribosomes interact specifically with the unspliced vRNA that is being used as the template, which then directs that RNA for encapsidation in cis, or the vRNA interacts with Gag/Gag–Pol molecules translated by other unspliced RNA in a random fashion in trans. Studies of Gag precursor packaging have shown that Pr55Gag translation contributes to incorporation of vRNA and suggested that full-length mRNA can be packaged in cis by the same Gag that it synthesizes (Liang et al., 2002; Poon et al., 2002). In this work, the relationship between translation of the Gag–Pol polyprotein and its association with cognate vRNA during a single cycle of virus replication has been explored. We used an anti-retroviral nevirapine (NVP)-resistance marker within the rt gene region to characterize the relationship between the encapsulation of Gag–Pol molecules and the vRNA used as template during the translation process. Co-transfection experiments with the wild-type genome and its counterpart carrying the NVP-resistant marker in the presence of NVP demonstrated that Gag–Pol molecules carrying the resistance mutation were responsible for a higher incorporation of its cognate vRNA into virus particles, despite an excess of the wild-type counterpart in transfected cells. These results were observed both directly in virus particles and also for the proviral DNA after one round of infection, suggesting that Gag–Pol has a role in selecting its cognate vRNA for packaging. Our data support a cis packaging model in which nascent Gag–Pol molecules are preferentially co-encapsulated with Gag multimers carrying the former cognate vRNA. The model proposed here places together resistant proteins and their viral genomes inside the same virus particle, increasing the chance of survival of viruses exposed to selective external influences, such as anti-retroviral drugs.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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nefgpt infectious clone (Tanuri et al., 2000). This mutant harbours the xanthine–guanine phosphoribosyl transferase (gpt) selective marker gene from Escherichia coli, in place of the Nef open reading frame. This vector can express the gpt product in infected cells, transducing a positive selective marker for mycophenolic acid (MPA) resistance, a potent inhibitor of de novo purine synthesis (Mulligan & Berg, 1981). A nucleotide (T) insertion was introduced into the env gene to prevent the expression of the envelope protein; this plasmid was called Z6gptRTwt. Pseudotyped virions were obtained through co-transfections with the plasmid SV-A-MLV-env encoding the murine leukemia virus (MLV) envelope protein (Page et al., 1990). A mutation that confers high levels of resistance to the reverse transcriptase (RT) inhibitor NVP (Y181C) was introduced into Z6gptRTwt to generate the Z6gptRTY181C mutant (Richman et al., 1994). The Z6gptRTY181C mutant was also modified to include a new BamHI restriction site, which acted as a silent marker to follow recombination events during reverse transcription (Fig. 1). Each mutation was introduced separately into the respective plasmid by using a QuikChange site-directed mutagenesis kit (Stratagene). The following primers were used: for env gene inactivation, 5'-ATTGATGACCTGTAGTTAATGCAGACAATCT-3' (F) and 5'-AGATTGTCTGCATTAACTACAGGTCATCAAT-3' (R); for the Y181C mutation, 5'-AGAAATAGTTATCTGTCAATACATGGATG-3' (F) and 5'-CATCCATGTATTGACAGATAACTATTTCT-3' (R); and for the 5' long terminal repeat (LTR) BamHI site, 5'-GTGGTAACTAGGGATCCCTCAGA-3' (F) and 5'-CACCATTGATCCCTAGGGAGTCT-3' (R). HIV-1-expressing vectors were designed with minimal modifications to avoid changes in RNA and protein export and stability.
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). The molecular ratios of Z6gptRTwt and Z6gptRTY181C for co-transfections varied from 1 : 1 to 5 : 1 in different experiments. COS-7 culture supernatants were harvested 72 h after transfection, centrifuged and filtered through a 0.45 µM pore-size filter. Virus production was quantified by measuring p24 antigen levels in the cell-culture supernatants (Vironostika HIV-1 Antigen; bioMériex Laboratories). Virus aliquots were stored at –70 °C.
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HIV genome incorporation in Z6gpt+ cell clones.
Total genomic DNA was extracted from MPA-resistant cell clones by using a Wizard DNA Genomic Extraction kit (Promega). Two different HIV-1 genomic regions were targeted for PCR amplification: the rt gene and the U5 region from the 3' LTR. Amplification of the rt gene was performed as described previously (Caride et al., 2000). Amplification products were purified by using QIAquick purification columns (Qiagen) and sequenced in an ABI model 3100 automated DNA sequencer (Applied Biosystems) to evaluate the presence of the NVP-resistance marker (Y181C) in the rt gene. The U5 region from the 3' LTR was amplified by using 5'-GTCCCGCCAATCTCCGGTCGCTAA-3' as the forward primer annealing at the 3' end of the gpt gene and 5'-GCTAGAGATTTTCCACTCTGACTA-3' as the reverse primer annealing at the 3' end of the HIV-1 genome. Amplification products of the U5 region were submitted to digestion reactions with BamHI (Promega) and separated on a 1.2 % agarose gel. The Z6gptRTwt-derived fragment was visualized as a 702 bp fragment (uncut) and the Z6gptRTY181C-derived fragment as two fragments (668 and 34 bp) after digestion.
Natural endogenous RT (NERT) reaction.
The NERT assay was performed by a modification of the technique of Hooker et al. (2001). Aliquots of intact virus particles were exposed or not to increasing concentrations of NVP 2 h prior to NERT reactions. After NVP treatment, virus aliquots were incubated with 20 U DNase I (Invitrogen) and 10 mM MgCl2 for 60 min at 37 °C in a final reaction volume of 25 µl. After incubation, 400 µM dNTPs were added to each tube (except to the negative controls) and further incubated at 37 °C for 2 h. Enzymic activity was terminated by the addition of 37.5 µl stop solution [10 mM Tris/HCl (pH 7.4), 10 mM EDTA, 20 mg sheared salmon sperm DNA ml–1, 50 mg proteinase K ml–1], followed by incubation for 10 min at 37 °C and a further 10 min at 96 °C. The Z6gptRTwt and Z6gptRTY181C viruses used for NERT reactions encoded HIV-1 wild-type Env proteins. Samples of each stopped reaction mixture were assayed for minus-strand strong-stop DNA (–ssDNA) by quantitative real-time PCR using combinations of the following oligonucleotides and probe: SSF1 (5'-GCTAACTAGGGAACCCACTGCTT-3'), SSR1 (5'-CAACAGACGGGCACACACTACT-3') and single-stranded DNA probe (5'-AGCCTCAATAAAGCTTGCCTTGAGTGCTTC-3'). Reaction mixtures (final volume 25 µl) contained 1x Taqman Universal PCR mixture (Applied Biosystems), 0.25 pmol each primer, 0.05 pmol probe and 4 µl NERT stopped reaction. Amplification was performed with an ABI Prism 7000 sequence detection system (Applied Biosystems). Cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, and 50 cycles of 95 °C for 15 s and 60 °C for 1 min. Real-time PCR standard curves used for the quantification of synthesized –ssDNA were constructed by using proviral plasmid DNA (Z6gptRTwt) diluted serially in NERT stop solution.
Direct identification of HIV genome in cell-free virus particles by the NERT reaction.
We used the NERT reaction product from virus obtained from 1 : 1 co-transfection (Z6gptRTwt : Z6gptRTY181C) to identify the nature of the HIV genome incorporated directly into these virus particles. Intact viruses obtained from 1 : 1 co-transfection were exposed to increasing concentrations of NVP before the NERT reactions, as described above. Products of the NERT reaction were used to amplify the 5' LTR U5 region from the –ssDNA template. Samples of each stopped reaction mixture were amplified using specific oligonucleotides flanking the BamHI site present on the Z6gptRTY181C genome: D1 (5'-GGTCTCTCTGGTTAGACCA-3') and P2-FAM (5'-CTGCTAGAGATTTTTCCACACTGAC-3'). The NERT PCR product was resolved in 1 % agarose gel and quantified. Aliquots of 100 ng of each PCR product were digested with 10 U BamHI. The digested fragments were purified and resolved in a 310 ABI automated sequencer (Applied Biosystems). All samples were run with the Prism GeneScan 500 Tamra size standard (Applied Biosystems) to identify the precise length and peak area of BamHI-digested fragments. The peak areas of digested fragments (181 nt of Z6gptRTwt genome and 38 nt of Z6gptRTY181C genome) were analysed using GENESCAN software (Applied Biosystems). The BamHI digestion fragment patterns of each virus (Z6gptRTwt or Z6gptRTY181C) were used as controls for total digestion. The peak area ratios for Z6gptRTY181C : Z6gptRTwt from NERT reactions were calculated in the presence and absence of NVP.
Statistical analyses.
Differences between Z6gptRTwt and Z6gptRTY181C genome frequencies in Z6gpt+ cell clones submitted or not to NVP selection were compared by using 
2 tests. A P value of 0.05 or less was considered statistically significant. Both linear and non-linear regressions between the plots in the graphics were calculated by using SigmaPlot software, version 8.0. In the non-linear regression, we used the equation of Hill, with four parameters of the sigmoidal function.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Therefore, pseudotyped viruses carrying NVP-resistant RT could be selected specifically by NVP treatment during infection of COS-7 cells and single infected-cell colonies isolated by selection with MPA (Fig. 2).
First, to determine the ability of the Z6gptRTwt and Z6gptRTY181C viruses to express the gpt marker, cells were infected individually with these viruses and colonies of infected cells were selected as described above. Both infections gave rise to the same number of surviving colonies, indicating that both viruses were able to transduce the gpt selective marker (Table 1). From these results, we could conclude that both wild-type Z6gptRTwt and mutant Z6gptRTY181C had a similar replication capacity. Cells were further infected with Z6gptRTwt or Z6gptRTY181C virus in the presence of increasing concentrations of NVP (0–50 µM) and analysed for their survival capacity under the same selective conditions. Z6gptRTwt- and Z6gptRTY181C-infected cells displayed survival curves consistent with the expected patterns for NVP sensitivity and resistance, as described previously (Iglesias-Ussel et al., 2002). The EC50 values for Z6gptRTwt and Z6gptRTY181C were 0.05 and 12.5 µM, respectively (Fig. 3). For infections carried out with viruses obtained from 1 : 1 (Z6gptRTwt : Z6gptRTY181C) co-transfection, the survival curve demonstrated that concentrations of NVP ranging from 0.1 to 0.5 µM would be sufficient to select only viruses carrying a sufficient number of Y181C mutant RTs to ensure its replication capacity (Fig. 3).
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Experiments were conducted by infecting COS-7 cells with virus stocks produced from ratios of 1 : 1 and 5 : 1 (Z6gptRTwt : Z6gptRTY181C) co-transfections in the presence or absence of NVP. Z6gpt+ MPA-resistant cell clones were isolated and expanded for proviral DNA analyses of the RT and 3' LTR regions. For all analyses, at least 100 individual samples were obtained. To test for co-transfection efficiency and incorporation of both Z6gptRTwt and Z6gptRTY181C vRNA into the same virus particle, a mutation was introduced in the 5' LTR U5 region of Z6gptRTY181C to include a new BamHI restriction site (Fig. 1). Synthesis of both minus- and plus-strand DNA involves template switching in which the strong-stop DNA is transferred from the original template to a complementary sequence at the 3' end of either the same or a second homologous copy of the RNA template (Gilboa et al., 1979). Therefore, recombination occurs as a result of RT switching templates between co-packaged RNA molecules during DNA synthesis (Levy et al., 2004). By using BamHI restriction of proviral DNA, it was possible to characterize, in proviral DNA from transduced cells, a high proportion of recombinant genomes between RT and the 3' LTR regions from infections carried out with or without NVP selection, confirming the presence of both vRNAs in single virus particles. In fact, up to 42 % of integrated genomes originated from recombination in transfection ratios of 1 : 1, regardless of NVP selection. These results demonstrated that both RNAs are typically used during HIV-1 reverse transcription and confirmed equal proportions of the two proviral clones (Z6gptRTwt and Z6gptRTY181C) in the same virus particle. In fact, as recombination is very frequent during HIV-1 retrotranscription and, assuming that each virus particle undergoes one recombination event during this process (Zhuang et al., 2002), we could assume that approximately 40 % of the infectious viruses obtained from 1 : 1 co-transfections were composed of one copy each of the Z6gptRTwt and Z6gptRTY181C RNA genomes.
As the rt gene carried the selective marker used to target Gag–Pol, we focused further genetic analyses on this region. The frequencies of the RTwt and RTY181C alleles found in Z6gpt+ cell clones without NVP selection were 0.6 and 0.4, respectively, suggesting that, under no selective pressure, there is an equal distribution of integrated genomes. Next, the genetic composition of proviral DNA obtained from colonies selected with 0.2 µM NVP (a concentration higher than the EC50; see Fig. 3) was analysed. Upon selection, we observed that RTY181C alleles were integrated preferentially into host-cell genomes. In fact, under NVP selection, RTwt and RTY181C allele frequencies were 0.32 and 0.68, respectively, when virus stocks were obtained from ratios of 1 : 1 co-transfection experiments (Fig. 4a). Therefore, we found a consistent twofold increase in frequency of the resistant over the wild-type allele in the presence of NVP treatment. This difference was statistically significant and suggested that Gag–Pol incorporation into Gag/Gag–Pol complexes was specific with its cognate vRNA, as predicted by the in cis model. For experiments carried out with virus stocks obtained from co-transfection ratios of 5 : 1 (Fig. 4b), the increased frequency found for the RTY181C allele in the presence of NVP remained significant (0.45 for the resistant and 0.55 for the wild-type counterpart, compared with 0.18 for the resistant and 0.82 for the wild-type counterpart under no selection). Indeed, even departing from an excess of the wild-type virus present in the infectious stock, the increase in the resistant allele frequency under NVP treatment was statistically significant when compared with the previous situation in the absence of NVP treatment (Fig. 4b), suggesting again that Gag–Pol molecules can interfere in the process of vRNA incorporation.
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; Hooker et al., 2001). As the amphipathic domains of the viral transmembrane protein gp41 render the viral outer coat permeable to a range of small molecules, including dNTPs, intravirion DNA synthesis will occur in the absence of permeabilizing agents (Zhang et al., 1996). The first product of HIV-1 reverse transcription is a 181 nt fragment known as –ssDNA.
A quantitative NERT real-time PCR assay that directly measured the effects of RT inhibitors on early reverse transcription in intact virions was created and the minimum NVP concentration required for selection of viruses harbouring RTY181C-resistant molecules was determined (Fig. 5a). For NERT experiments, we used the env+ versions of each proviral clone (Z6gptRTY181C and Z6gptRTwt). Intact virions were exposed for 2 h to increasing concentrations of the drug prior to NERT reactions. Synthesis of –ssDNA in wild-type viruses was inhibited with 10–50 µM NVP, whereas mutant Z6gptRTY181C virus was resistant to NVP at concentrations up to 256 µM (Fig. 5b). The inhibition profile of NVP in NERT reactions of 1 : 1 co-transfection experiments was similar to that found for integrated proviruses (Fig. 5b). The NVP concentration sufficient to inhibit 50 % of the wild-type –ssDNA synthesis (2 µM) was in agreement with previous studies that evaluated the effects of RT inhibitors during NERT (Hooker et al., 2001). However, it should be noted that residual wild-type RT activity (around 20 %) could still be detected, even with concentrations of NVP as high as 256 mM.
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). Fragments were separated after restriction digestion through sequencing electrophoresis and GENESCAN software was used for analysis of the length and area of each fragment (RFLP). The ratio of digested and non-digested fragments obtained from NERT reactions from 1 : 1 virus with or without NVP selection was representative of the ratio of Z6gptRTwt and Z6gptRTY181C virion-incorporated genomes. Likewise, for integrated HIV genomes, the prevalence of the Z6gptRTY181C genome in the NERT reactions in the presence of NVP demonstrated an intimate association between mutant RT and its cognate vRNA, increasing the efficiency of mutant vRNA reverse transcription both in homodimers of mutants and in heterodimers. Our results demonstrated that, in the absence of NVP, the frequency of Z6gptRTY181C vRNA was slightly lower than its wild-type counterpart (Z6gptRTY181C/Z6gptRTwt=0.8) (Fig. 5c). However, following NVP selection, the frequency of Z6gptRTY181C increased over the frequency of the Z6gptRTwt genome proportionally with the increase in drug concentration, and a twofold higher frequency of the resistant vRNA was observed for the highest NVP concentration used (Fig. 5c). These results confirmed that the incorporation of vRNA and Gag–Pol molecules into virus particles is specific and suggest that Gag–Pol itself could help in coordinating genome incorporation during viral assembly. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In our system, heterozygous virions were obtained from cells co-transfected with two distinct proviral DNAs in equal proportions. Despite the excess of virus produced in this cell system, this is in close proximity to in vivo observations, as splenocytes from HIV-1-infected individuals demonstrate a high number of integrated proviruses per infected cell (Dang et al., 2004). Furthermore, double infections occur frequently in infected cultures of primary and established CD4+ cells, providing the basis for the generation of heterozygous virions (Jung et al., 2002).
Genomic RNA incorporation is a highly specific event during viral assembly and cis-acting sequences within this genomic RNA are essential for its preferential selection over the many other mRNA species present in the cell cytoplasm during assembly. Gag is responsible for interacting with such sequences and specifically selects full-length, non-spliced viral mRNA for incorporation. For HIV-2, a model for genomic RNA incorporation by in cis packaging has been proposed in which unspliced RNA is sequestered by its cognate newly synthesized Gag (Kaye & Lever, 1999). This is necessary and essential for vRNA selection, because the packaging signal of HIV-2 is upstream of the major splice donor and thus exists on both spliced and unspliced RNA species. Controversy exists, however, regarding the initiation mechanism for HIV-1 genomic RNA incorporation. In fact, several studies have demonstrated that HIV-1 Gag incorporates unspliced genomic RNA efficiently both in trans and in cis. However, studies demonstrating that the HIV-1 unspliced mRNA pool can function interchangeably as genomic RNA and as template for Gag and Gag–Pol translation were performed in conditions in which no competition between two distinct RNA species existed (Butsch & Boris-Lawrie, 2000; Griffin et al., 2001; McBride et al., 1997). However, when different unspliced RNA species are present at the same time during viral assembly, it has been demonstrated that the nascent Gag proteins specifically incorporate their cognate RNA (Liang et al., 2002; Poon et al., 2002).
Swanson et al. (2004) demonstrated that the Gag–Pol mRNA export pathways determine the sites of protein translation in the cytoplasm of infected cells influencing the efficiency of virion assembly. These findings agree with the facts that Gag and vRNA interaction occurs at a perinuclear site very early during viral assembly, that it facilitates Gag multimerization and that it helps the assembly complex to migrate to sites of viral budding (Poole et al., 2005, and references therein). Thus, although there is an interchangeable use of HIV-1 unspliced mRNA for both encapsulation and translation, upon specific selective pressure, a preference for cis packaging can be observed. This corroborates well with our results, in which, following NVP selection, mutant Z6gptRTY181C genomic RNA was packaged at a twofold-higher level than the wild-type Z6gptRTwt, given equal expression of both RNAs in co-transfected cells. Our results suggest that, as for Gag, newly synthesized Gag–Pol proteins could establish interactions with Gag multimers very early during translation and, at the same time, with their cognate mRNA that served as template, and this entire complex would be directed efficiently for budding. We observed a significant shift in RT allelic frequencies following NVP selection either in 1 : 1 or 5 : 1 (Z6gptRTwt : Z6gptRTY181C) co-transfection ratios, for both gpt+ clones (Fig. 4a and b) and newly synthesized –ssDNA in NERT reactions (Fig. 5c). Both integrated proviruses and cell-free virions favoured the specific in cis model for vRNA incorporation.
The increase in the proportion of RTY181C compared with RTwt vRNA following NVP selection observed by NERT was slightly lower than that observed for genome integration in transduced cells. This could be due to a significant level of incorporation of single-spliced subgenomic RNA into new virions, as demonstrated previously (Liang et al., 2004). Moreover, the residual activity of wild-type RT, even in the presence of high concentrations of NVP, could also account for the lower frequency of the mutant –ssDNA, as the wild-type counterpart would still be synthesized by its wild-type RT.
Gag molecules are the major components driving genomic RNA incorporation through interaction between the nucleocapsid domain in Gag and the 
site within unspliced RNA. Although such interaction is essential for RNA recruitment, it is not sufficient, and additional viral and/or cellular factors seem to play a role in this process (Aldovini & Young, 1990). In fact, interactions between unspliced genomic RNA and the RT region in the unprocessed Gag–Pol have already been demonstrated and seem to be important for holding Pol components in virions during viral maturation (Cen et al., 2004). Taken together, these results may imply that Gag–Pol molecules also play an important role during RNA packaging, suggesting that, as for Gag, Gag–Pol proteins to a lesser extent can select genomic RNA for incorporation. Importantly, in our system, the observed twofold increase in the levels of resistant Z6gptRTY181C genomic RNA incorporation compared with the susceptible Z6gptRTwt could be explained by the lesser contribution of Gag–Pol to this process. This could be explained by the 20 : 1 ratio of Gag to Gag–Pol proteins present in virions (Jacks et al., 1988). Even considering that Gag–Pol will seldom select its own template as the viral genome, Gag–Pol polyproteins harbouring resistant mutations in the RT gene will selectively pass the resistant genome on to the next infected cell in a selective environment, such as in the presence of anti-RT drugs, demonstrating the importance of this event.
In vitro assembly analyses have shown that viral RNA might function as a scaffold for the multimerization of viral precursor proteins (Khorchid et al., 2002). Furthermore, packaging of the mature dimeric RNA genome is dependent on pol products (Shehu-Xhilaga et al., 2001, 2002). These findings are in agreement with a model in which Gag–Pol also plays an important role for the incorporation of vRNA, suggesting that vRNA interaction with Gag–Pol occurs prior to RNA dimerization.
Together, these results support a cis packaging model in which nascent Gag–Pol proteins are preferentially co-encapsulated with Gag multimers and their cognate RNA into HIV particles, especially in a scenario in which two distinct unspliced genomic RNAs co-exist. This model links translation, packaging and the assembly process, placing Gag–Pol and vRNA components in close proximity where they can interact. This model could also have implications for selecting virions carrying resistant proteins and genomes inside the same particle, thus increasing the likelihood of resistant virus expansion if exposed to anti-retroviral drug selection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Basyuk, E., Galli, T., Mougel, M., Blanchard, J.-M., Sitbon, M. & Bertrand, E. (2003). Retroviral genomic RNAs are transported to the plasma membrane by endosomal vesicles. Dev Cell 5, 161–174.[CrossRef][Medline]
Berkowitz, R. D. & Goff, S. P. (1994). Analysis of binding elements in the human immunodeficiency virus type 1 genomic RNA and nucleocapsid protein. Virology 202, 233–246.[CrossRef][Medline]
Berkowitz, R. D., Ohagen, Å., Höglund, S. & Goff, S. P. (1995). Retroviral nucleocapsid domains mediate the specific recognition of genomic viral RNAs by chimeric Gag polyproteins during RNA packaging in vivo. J Virol 69, 6445–6456.[Abstract]
Berkowitz, R., Fisher, J. & Goff, S. P. (1996). RNA packaging. Curr Top Microbiol Immunol 214, 177–218.[Medline]
Butsch, M. & Boris-Lawrie, K. (2000). Translation is not required to generate virion precursor RNA in human immunodeficiency virus type 1-infected T cells. J Virol 74, 11531–11537.
Caride, E., Brindeiro, R., Hertogs, K. & 10 other authors (2000). Drug-resistant reverse transcriptase genotyping and phenotyping of B and non-B subtypes (F and A) of human immunodeficiency virus type I found in Brazilian patients failing HAART. Virology 275, 107–115.[CrossRef][Medline]
Cen, S., Niu, M. & Kleiman, L. (2004). The connection domain in reverse transcriptase facilitates the in vivo annealing of tRNALys3 to HIV-1 genomic RNA. Retrovirology 1, 33.[CrossRef][Medline]
Clever, J. L., Miranda, D., Jr & Parslow, T. G. (2002). RNA structure and packaging signals in the 5' leader region of the human immunodeficiency virus type 1 genome. J Virol 76, 12381–12387.
Dang, Q., Chen, J., Unutmaz, D., Coffin, J. M., Pathak, V. K., Powell, D., KewalRamani, V. N., Maldarelli, F. & Hu, W.-S. (2004). Nonrandom HIV-1 infection and double infection via direct and cell-mediated pathways. Proc Natl Acad Sci U S A 101, 632–637.
Dorfman, T., Luban, J., Goff, S. P., Haseltine, W. A. & Göttlinger, H. G. (1993). Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein. J Virol 67, 6159–6169.
Gilboa, E., Mitra, S. W., Goff, S. & Baltimore, D. (1979). A detailed model of reverse transcription and tests of crucial aspects. Cell 18, 93–100.[CrossRef][Medline]
Gorelick, R. J., Chabot, D. J., Rein, A., Henderson, L. E. & Arthur, L. O. (1993). The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent. J Virol 67, 4027–4036.
Griffin, S. D. C., Allen, J. F. & Lever, A. M. L. (2001). The major human immunodeficiency virus type 2 (HIV-2) packaging signal is present on all HIV-2 RNA species: cotranslational RNA encapsidation and limitation of Gag protein confer specificity. J Virol 75, 12058–12069.
Halwani, R., Khorchid, A., Cen, S. & Kleiman, L. (2003). Rapid localization of Gag/GagPol complexes to detergent-resistant membrane during the assembly of human immunodeficiency virus type 1. J Virol 77, 3973–3984.
Hill, M. K., Hooker, C. W., Harrich, D., Crowe, S. M. & Mak, J. (2001). Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1. J Virol 75, 6835–6840.
Hooker, C. W. & Harrich, D. (2003). The first strand transfer reaction of HIV-1 reverse transcription is more efficient in infected cells than in cell-free natural endogenous reverse transcription reactions. J Clin Virol 26, 229–238.[CrossRef][Medline]
Hooker, C. W., Lott, W. B. & Harrich, D. (2001). Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription. J Virol 75, 3095–3104.
Iglesias-Ussel, M. D., Casado, C., Yuste, E., Olivares, I. & López-Galíndez, C. (2002). In vitro analysis of human immunodeficiency virus type 1 resistance to nevirapine and fitness determination of resistant variants. J Gen Virol 83, 93–101.
Jacks, T., Power, M. D., Masiarz, F. R., Luciw, P. A., Barr, P. J. & Varmus, H. E. (1988). Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature 331, 280–283.[CrossRef][Medline]
Jung, A., Maier, R., Vartanian, J.-P., Bocharov, G., Jung, V., Fischer, U., Meese, E., Wain-Hobson, S. & Meyerhans, A. (2002). Multiply infected spleen cells in HIV patients. Nature 418, 144.[CrossRef][Medline]
Kaye, J. F. & Lever, A. M. L. (1999). Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation. J Virol 73, 3023–3031.
Khorchid, A., Halwani, R., Wainberg, M. A. & Kleiman, L. (2002). Role of RNA in facilitating Gag/Gag-Pol interaction. J Virol 76, 4131–4137.
Levy, D. N., Aldrovandi, G. M., Kutsch, O. & Shaw, G. M. (2004). Dynamics of HIV-1 recombination in its natural target cells. Proc Natl Acad Sci U S A 101, 4204–4209.
Liang, C., Hu, J., Russell, R. S. & Wainberg, M. A. (2002). Translation of Pr55gag augments packaging of human immunodeficiency virus type 1 RNA in a cis-acting manner. AIDS Res Hum Retroviruses 18, 1117–1126.[CrossRef][Medline]
Liang, C., Hu, J., Russel, R. S., Kameoka, M. & Wainberg, M. A. (2004). Spliced human immunodeficiency virus type 1 RNA is reverse transcribed into cDNA within infected cells. AIDS Res Hum Retroviruses 20, 203–211.[CrossRef][Medline]
McBride, M. S., Schwartz, M. D. & Panganiban, A. T. (1997). Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation. J Virol 71, 4544–4554.[Abstract]
Mulligan, R. C. & Berg, P. (1981). Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A 78, 2072–2076.
Page, K. A., Landau, N. R. & Littman, D. R. (1990). Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity. J Virol 64, 5270–5276.
Poole, E., Strappe, P., Mok, H.-P., Hicks, R. & Lever, A. M. L. (2005). HIV-1 Gag–RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET. Traffic 6, 741–755.[CrossRef][Medline]
Poon, D. T. K., Wu, J. & Aldovini, A. (1996). Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity. J Virol 70, 6607–6616.
Poon, D. T. K., Chertova, E. N. & Ott, D. E. (2002). Human immunodeficiency virus type 1 preferentially encapsidates genomic RNAs that encode Pr55Gag: functional linkage between translation and RNA packaging. Virology 293, 368–378.[CrossRef][Medline]
Provitera, P., Goff, A., Harenberg, A., Bouamr, F., Carter, C. & Scarlata, S. (2001). Role of the major homology region in assembly of HIV-1 Gag. Biochemistry 40, 5565–5572.[CrossRef][Medline]
Richman, D. D., Havlir, D., Corbeil, J. & 10 other authors (1994). Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy. J Virol 68, 1660–1666.
Shehu-Xhilaga, M., Crowe, S. M. & Mak, J. (2001). Maintenance of the Gag/Gag-Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity. J Virol 75, 1834–1841.
Shehu-Xhilaga, M., Hill, M., Marshall, J. A., Kappes, J., Crowe, S. M. & Mak, J. (2002). The conformation of the mature dimeric human immunodeficiency virus type 1 RNA genome requires packaging of Pol protein. J Virol 76, 4331–4340.
Swanson, C. M., Puffer, B. A., Ahmad, K. M., Doms, R. W. & Malim, M. H. (2004). Retroviral mRNA nuclear export elements regulate protein function and virion assembly. EMBO J 23, 2632–2640.[CrossRef][Medline]
Tanuri, A., Jesus da Costa, L., Brindeiro, R., Ramos, C. A., Pau, C.-P. & Rayfield, M. A. (2000). Construction of a selectable nef-defective live attenuated human immunodeficiency virus expressing Escherichia coli gpt gene. Virology 268, 79–86.[CrossRef][Medline]
Zhang, Y. & Barklis, E. (1995). Nucleocapsid protein effects on the specificity of retrovirus RNA encapsidation. J Virol 69, 5716–5722.[Abstract]
Zhang, H., Dornadula, G., Alur, P., Laughlin, M. A. & Pomerantz, R. J. (1996). Amphipathic domains in the C terminus of the transmembrane protein (gp41) permeabilize HIV-1 virions: a molecular mechanism underlying natural endogenous reverse transcription. Proc Natl Acad Sci U S A 93, 12519–12524.
Zhuang, J., Jetzt, A. E., Sun, G., Yu, H., Klarmann, G., Ron, Y., Preston, B. D. & Dougherty, J. P. (2002). Human immunodeficiency virus type 1 recombination: rate, fidelity, and putative hot spots. J Virol 76, 11273–11282.
Received 17 March 2006;
accepted 25 April 2006.
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