INTRODUCTION

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The gag and pol genes of human immunodeficiency virus type 1 (HIV-1) are initially translated as polyproteins, which are later cleaved by the viral protease to produce mature virions. Gag is expressed as a 55-kDa precursor (Pr55^Gag), and Pol is expressed in the form of a 160-kDa Gag-Pol fusion protein (Pr160^Gag-Pol). These precursor polyproteins are encoded by the same mRNA but are not synthesized at the same rate. The pol gene lacks its own initiation codon and is in the 1 reading frame with respect to gag. The Gag-Pol fusion protein is synthesized as a result of an infrequent ribosomal frameshifting event, wherein the ribosomes translating gag slip back one nucleotide into the pol reading frame (20). Ribosomal frameshifting generates 20 times as much Gag as Gag-Pol, supporting the greater requirement for structural proteins over enzymatic proteins during viral assembly. Maintenance of the 20:1 ratio of Gag to Gag-Pol is critical for viral infectivity and RNA dimerization (29).

Several lines of indirect evidence suggest that the Gag and Gag-Pol precursor proteins and the viral genomic RNA form an assembly complex in the cytoplasm that is transported as a unit to the cell membrane for viral assembly and budding. Gag and Gag-Pol are translated from the unspliced viral genomic RNA on free polyribosomes in the cell cytoplasm, creating a high local concentration of nascent Gag and Gag-Pol molecules alongside the viral RNA. The finding that the assembly of HIV-1 and other retroviruses is facilitated by nucleic acids (4, 6, 7, 10, 26), combined with the nucleic acid binding properties of the nucleocapsid (NC) region of Gag (for a review, see reference 13), indicates an important role for interactions between Gag and viral genomic RNA in the assembly process. Although retroviral assembly can occur independently of genomic RNA packaging (as shown by HIV-1 packaging signal [Psi] deletion mutants that generate empty particles [for a review, see reference 1]), in these cases assembly may have been promoted by the host cell's nucleic acids (10).

It is generally believed that Gag-Pol is incorporated into the virion via interactions with Gag (19, 31, 33), and there is evidence to suggest that the multimerization of Gag and Gag-Pol occurs prior to their association with the cell membrane. For HIV-1 assembly to occur, Gag must undergo myristoylation (14), a step that is critical for the transport of Gag to the plasma membrane. However, both nonmyristoylated Gag and nonmyristoylated Gag-Pol can be packaged into progeny virions when coexpressed with myristoylated Gag (9, 28, 30). Velocity sedimentation analysis of Gag-only particles has shown that both myristoylated and nonmyristoylated Gag proteins can assemble into multimeric assembly intermediates, but myristoylation is required to complete virus-like particle formation (24, 27). Furthermore, Lee et al. (22, 23) have reported the formation of Gag and Gag-Pol precursor complexes in the cytoplasm of HIV-1-infected CD4⁺ T cells.

Experiments using Moloney murine leukemia virus have shown that the Gag portion of Gag-Pol is not required for the packaging of Pol proteins. However, a lower level of reverse transcriptase (RT) activity was observed in the resulting virions than in wild-type virions (3), suggesting a deficient packaging of Pol. In HIV-1, the provision of Gag in trans to Gag-Pol results in the formation of virus-like particles containing both Gag and Gag-Pol (28, 30). Taken together, these investigations show that the packaging of Gag-Pol does not require Gag and Gag-Pol to be synthesized from the same mRNA. However, these studies do not establish if the multimerization of Gag and Gag-Pol preferentially occurs during protein translation as a means of facilitating the transport and subsequent packaging of Gag-Pol into progeny virions. In this study, we have assessed whether the incorporation of Gag-Pol that is supplied in trans is as efficient as the incorporation of Gag-Pol that is synthesized from the same mRNA as Gag. Using a conditional cotransfection system designed to distinguish between packaged Gag-Pol proteins expressed either from the same construct that expresses Gag or from a separate construct, we observed that Gag and Gag-Pol multimerization during translation from a single mRNA molecule is not critical for virion packaging of the Gag-Pol polyprotein. Furthermore, we found that an interaction between Gag and Gag-Pol proteins generated from the same mRNA is not necessary to generate functional virions.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Construction of DNA plasmids. The HIV-1 DNA constructs used in this study were derived from either the full-length wild-type HIV-1 plasmid HXB2-BH10 (32) or the luciferase reporter plasmid pNL4-3.Luc.R- E- (12). pNL4-3.Luc.R- E- was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institutes of Health, from N. Landau. For all experiments we used two fundamental HIV constructs, one that expresses both Gag and Gag-Pol but with a functional deletion in rev (G:GP), and one that expresses only Gag-Pol due to a frameshift mutation that allows continuous expression of Gag-Pol and bypasses the Gag termination codon (GP). G:GP was created by deleting the majority of the second half of exon 2 of rev in HXB2-BH10 as previously described (29). GP was constructed in HXB2-BH10 as previously described (29) to alter the heptanucleotide sequence that is responsible for 1 ribosomal frameshifting, by direct base changes and the addition of an extra nucleotide (5'-TTTTTTA-3'5'-CTTCCTCA-3').

Cotransfection and virus production. The G:GP and GP plasmids were cotransfected at a ratio of 20 µg of G:GP to 1 µg of GP in order to generate equal levels of Gag-Pol proteins derived from all constructs. The Luc.G:GP and Luc.GP plasmids were also cotransfected at a ratio of 20 to 1, and 5 µg of pVSV-G (a kind gift from J. C. Burns, University of California, San Diego) was included to provide a viral envelope that would enable infection (5). The calcium phosphate coprecipitation method was used for the transient transfection of 293T cells. Supernatants were collected at 36 h posttransfection and centrifuged for 30 min at 3,000 rpm (Beckman model GS-6) to remove cellular debris. Virions were purified and concentrated by ultracentrifugation (Beckman model L-90, SW41 rotor) at 35,000 rpm for 1 h at 4°C through a 20% sucrose cushion. For protein isolation, viral pellets were resuspended in 50 µl of 2× Tris-buffered saline lysis buffer containing 1% Nonidet P-40, 20 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, and 1 µM leupeptin. For use in the reverse transcription assay and the natural endogenous reverse transcription (NERT) assay, viral pellets were resuspended in Tris-EDTA. Virus production from transfections was quantified by measuring p24 antigen levels in the pelleted virion stocks and the cell culture supernatants (HIV-1 p24 assay; Abbott Laboratories).

Western blot analysis. Protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% gel) and transferred to a nitrocellulose membrane (Hybond C; Amersham Pharmacia Biotech). Western blot analysis was carried out as previously described (29), using an -RT antibody (NEN) to detect p66 RT, an -His antibody (Qiagen) to detect the His-tagged Gag-Pol polyproteins, and pooled sera from HIV-infected patients to detect total HIV-1 viral proteins. An ECL (enhanced chemiluminescence) detection kit (Amersham Pharmacia Biotech) was used to visualize the proteins for assessment by laser densitometry.

Cell-free RT assay. The cell-free RT assay was adapted from a method described by Boyer et al. (2). For each sample, 0.2 µl of a 20-µg/ml stock of M13-47 sequencing primer (New England Biolabs) was annealed to 0.25 µg of single-stranded M13mp18 DNA (New England Biolabs) by heating to 95°C for 5 min and then slowly cooling to room temperature. In a final volume of 60 µl, each sample contained 10 µl of Nonidet P-40, pelleted virus (5 ng of p24), 25 mM Tris-Cl (pH 8.0), 75 mM KCl, 8.0 mM MgCl₂, 2 mM dithiothreitol, 100 µg of bovine serum albumin per ml, 10 mM CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 10 µM each dATP, dGTP, and dCTP, 5 µM dTTP, 0.5 µCi of [-³³P]dTTP (NEN), and 3TC-triphosphate (Moravek Biochemicals), which was routinely used at a concentration of 2.5, 5, or 15 µM. The samples were incubated for 1 h at 37°C, and then 8 µl of each reaction was spotted onto DE81 filter paper, which was washed six times in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate and then analyzed on a microbeta counter (Wallac).

NERT assay. The NERT assay was carried out as previously described (17). Briefly, sucrose-purified virus (typically 100 pg of p24) was supplemented with MgCl₂ and DNase I and incubated at 37°C for 90 min in the presence and absence of deoxynucleoside triphosphates. Reverse transcription was terminated by the addition of EDTA and proteinase K followed by boiling for 10 min. Stopped reactions were assayed for negative-strand strong-stop DNA by PCR. Standard curves were generated by amplifying serial dilutions of an HIV-1 proviral plasmid, and all PCRs were performed in the linear range of the assay.

Single-round infection analyses with the pNL4-3-Luc.R- E- constructs. Cleared viral supernatants containing 10 ng of p24 were adjusted to 200 µl with culture medium and incubated with 10⁶ MT-2 cells for 2 h. The virus was then removed by washing the infected cells once with phosphate-buffered saline without calcium chloride and magnesium chloride and resuspending them in 1 ml of culture medium. The luciferase activity in the cells was determined 48 h postinfection using a luciferase assay kit (Promega). In experiments conducted in the presence of 3TC (obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institutes of Health), the MT-2 cells were incubated with 50 nM 3TC for 4 h prior to infection, 2 h during infection, and 48 h postinfection. The mean 50% inhibitory concentration of 3TC, which was shown to inhibit several different strains of HIV-1 in primary cells, ranges between 2.5 nM and 0.67 µM (11). Trypan blue staining was used to monitor cytotoxic effects of 3TC, and no differences were observed between control and 3TC-treated cultures.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

G:GP and GP were cotransfected to determine whether the interaction between Gag and Gag-Pol is initiated during protein translation. If Gag and Gag-Pol interact during their synthesis from a single mRNA molecule to facilitate Gag-Pol packaging, the progeny virions would be expected to contain primarily Gag-Pol from the plasmid synthesizing both Gag and Gag-Pol. Conversely, the progeny virions will contain similar levels of Gag-Pol proteins from both plasmids if the Gag/Gag-Pol interaction can occur efficiently when Gag-Pol is supplied in trans.

Development of a conditional cotransfection system. The conditional cotransfection system allows viral particle formation only when both cotransfected plasmids are simultaneously expressed in the same cell. This system takes advantage of the fact that both Gag and Rev are required for viral particle formation. As Rev is required for the transport of genomic RNA from the nucleus to the cytoplasm (for a review, see reference 18), transfection of a Rev-deficient HIV-1 plasmid (G:GP) alone results in viral mRNA being trapped in the nucleus. Similarly, viral particles will not be formed when GP is transfected alone, as Gag is required for virion production. Accordingly, when either G:GP or GP was transfected into 293T cells, no pelletable HIV-1 proteins were detected (data not shown). As the natural ratio of Gag to Gag-Pol synthesis is 20:1, the two plasmids were cotransfected at a ratio of 20 G:GP to 1 GP (based on DNA concentration) so that equivalent amounts of Gag-Pol proteins would be derived from all constructs. This enables a direct comparison of packaged Gag-Pol synthesized from either G:GP or GP. The HIV-1 constructs used in these analyses are depicted in Fig. 1A.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   (A) The HIV-based constructs used for all cotransfections. (B) Schematic representation of the Gag and Gag-Pol polyproteins showing the major domains and the sites of labeling with either the His epitope or the K65R mutation. LTR, long terminal repeat; MA, matrix; CA, capsid; NC, nucleocapsid; PR, protease.

Gag and Gag-Pol do not interact during translation from the same mRNA to facilitate Gag-Pol packaging. Epitope tagging of the Gag-Pol proteins was used to ascertain the amounts of packaged Gag-Pol derived from either the G:GP or the GP plasmid. G:GP_His/GP and G:GP/GP_His viral particles obtained by cotransfection of 293T cells were concentrated by ultracentrifugation and resuspended in protein lysis buffer. A protein dilution series of each virus was resolved by SDS-PAGE for Western blotting, and the resultant blots were hybridized successively with -RT, -His, and pooled sera from HIV-1-infected patients. RT levels provided a comparative measure of the total amount of HIV-1 Gag-Pol polyprotein in each virus stock, and the concentration of -His reactive protein reflects the amount of Gag-Pol packaged from a particular plasmid (Fig. 2). For the representative experiment depicted in Fig. 2, the His signals (standardized to equivalent levels of RT) were 993, 421, and 309 (densitometry units) for the three dilutions of G:GP/GP_His and 906, 478, and 270 for the three dilutions of G:GP_His/GP, indicating similar efficiencies of Gag-Pol packaging from the two plasmids. Hybridization with the pooled patient sera indicated that there were no obvious differences in total HIV-1 protein expression levels or patterns between the two viruses (data not shown).

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Western blot analysis of virion lysates produced from cotransfections in 293T cells to produce two virion types, G:GPHis/GP and G:GP/GPHis. Virion lysates were analyzed by SDS-PAGE with twofold dilutions of each lysate alongside the mock and the wild-type HXB2-BH10 control. RT (p66) is indicative of the overall level of Gag-Pol protein. His-tagged IN reflects the level of one particular Gag-Pol population in each virus type.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Cell-free RT assay performed in the presence and absence of 3TC-triphosphate on four virion types: the drug-sensitive G:GP/GP and drug-resistant G:GPK65R/GPK65R controls and the two test cases, G:GP/GPK65R and G:GPK65R/GP. Virions were produced by co-transfection in 293T cells and were normalized on the basis of p24 levels. Inhibition of RT activity is presented relative to a no-drug control for three 3TC-triphosphate concentrations. The data shown are the averages of assays performed with virions produced from three separate transfections.

Gag and Gag-Pol interaction during translation from the same mRNA is not required for endogenous reverse transcription or viral infectivity. To determine whether or not an interaction between Gag and Gag-Pol at the time of translation is important for correct protein folding and the proper molecular arrangement of proteins within the virion, functional competence of the K65R labeled viruses was assessed by NERT and viral infectivity assays. The NERT assay measures the endogenous RT activity within intact virions, which is thought to reflect the viruses' ability to successfully initiate infection (35, 36). The NERT assays (Fig. 4A) show that the drug-sensitive (G:GP/GP) and the drug-resistant (G:GP_K65R/GP_K65R) controls are affected by 3TC-triphosphate as anticipated, with G:GP_K65R/GP_K65R being two- to threefold more resistant to 3TC-triphosphate than G:GP/GP (Fig. 4A). However, the two test cases, G:GP/GP_K65R and G:GP_K65R/GP, displayed similar levels of NERT activity; both were approximately 1.3-fold more resistant than the drug-sensitive control and 0.5-fold less resistant than the drug-resistant control (Fig. 4A). Viral infectivity was assessed from the infection of MT-2 cells in the presence and absence of 3TC via the luciferase reporter gene. The controls were clearly separable on the basis of drug response, with the Luc.G:GP/Luc.GP virions being 2- to 2.5-fold more susceptible to 3TC than the Luc.G:GP_K65R/Luc.GP_K65R virions. However, the two test samples, Luc.G:GP/Luc.GP_K65R Luc.G:GP_K65R/Luc.GP, consistently generated similar levels of luciferase activity, with both approximately 1.3-fold more resistant than the drug-sensitive control and 0.5-fold less resistant than the drug-resistant control (Fig. 4B).

View larger version (19K): [in this window] [in a new window]   FIG. 4.   (A) Inhibition of NERT activity in the presence of 10 µM 3TC-triphosphate, relative to no-drug controls, in the four virus types, G:GP/GP, G:GPK65R/GPK65R, G:GP/GPK65R, and G:GPK65R/GP, that were produced by cotransfection in 293T cells and normalized on the basis of p24 levels. The results obtained in duplicate assays performed with two independent virus stocks. Standard deviation from the mean is indicated. (B) Inhibition of luciferase activity by 3TC when the four virion types, Luc.G:GP/Luc.GP, Luc.G:GPK65R/Luc.GPK65R, Luc.G:GP/Luc.GPK65R, and Luc.G:GPK65R/Luc.GP, were produced by cotransfection in 293T cells, normalized on the basis of p24 levels, and used to infect MT-2 cells in the presence and absence of 50 nM 3TC. The results are presented relative to a no-drug control.