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By using particle-associated reverse transcriptase (RT) activity as an assay for Pol incorporation into human immunodeficiency virus type 1 (HIV-1) Gag virus-like particles (VLPs), it has been found that truncated, protease-negative, Gag-Pol missing cis Gag sequences is still incorporated into Gag VLPs, albeit at significantly reduced levels (10 to 20% of the level of wild-type Gag-Pol). In this work, we have directly measured the incorporation of truncated Gag-Pol species into Gag VLPs and have found that truncated Gag-Pol that is missing all sequences upstream of RT is still incorporated into Gag VLPs at levels approximating 70% of that achieved by wild-type Gag-Pol. Neither protease nor integrase regions in Pol are required for its incorporation, implying an interaction between Gag and RT sequences in the Pol protein. While the incorporation of Gag-Pol into Gag VLPs is reduced 12-fold by the replacement of the nucleocapsid within Gag with a leucine zipper motif, this mutation does not affect Pol incorporation. However, the deletion of p6 in Gag reduces Pol incorporation into Gag VLPs four- to fivefold. Pol shows the same ability as Gag-Pol to selectively package tRNALys into Gag VLPs, and primer tRNA3Lys is found annealed to the viral genomic RNA. These data suggest that after the initial separation of Gag from Pol during cleavage of Gag-Pol by viral protease, the Pol species still retains the capacity to bind to both Gag and tRNA3Lys, which may be required for Pol and tRNA3Lys to be retained in the assembling virion until budding is completed.
During human immunodeficiency virus type 1 (HIV-1) assembly, the major tRNALys isoacceptors in mammalian cells, tRNA3Lys and tRNA1,2Lys (44), are selectively packaged into the virion (28), where tRNA3Lys serves as the primer for the reverse transcriptase (RT)-catalyzed synthesis of minus-strand cDNA (37). The annealing of tRNA3Lys to the primer binding site of the viral RNA genome is proportional to the amount of tRNA3Lys packaged in the viral population (14). tRNALys packaging into HIV-1 depends upon the presence of the precursor protein Gag-Pol (36) and may depend directly upon sequences in the RT thumb domain. Thus, C-terminal deletions of Gag-Pol which include sequences coding for integrase and the RNase H and connection domain of RT do not affect tRNALys packaging, but larger deletions that also include the thumb domain inhibit packaging (31). While deletion of the RT thumb domain could induce conformational changes affecting tRNALys binding elsewhere in the molecule, cross-linking studies between purified HIV-1 RT and tRNA3Lys indicate a direct interaction between the thumb domain and tRNA3Lys (12).
Extracellular virus-like particles (VLPs) can be produced by HIV-1 Gag alone (17, 21, 29, 50), and putative regions of interactions between Gag molecules have been identified within the C-terminal half of Gag and include the C-terminal half of capsid (CA) (3, 15, 34, 38), p2 (1, 32, 40), nucleocapsid (NC) (6, 7, 10, 49), and p6 (16). It is generally assumed that in order to obtain the interactions required for assembly, the Gag molecules must first be concentrated at a cellular site. Membrane has been proposed to play a role in the concentration and alignment of Gag molecules, and in HIV-1-transfected COS cells, almost all steady-state Gag is membrane bound (22, 24, 52). In many retroviruses, the Gag precursor protein attaches to the lipid membrane via a myristic acid moiety added posttranslationally to the amino terminus of the matrix (4, 18, 23, 45, 48) and through ionic interactions between the phospholipids in the membrane and a sequence of basic amino acid residues within the matrix (46, 47, 53, 56). However, a minimal, 16-kDa Gag construct lacking matrix has been shown to be capable of forming VLPs in vivo and is composed of an N-terminal myristylation signal, the C-terminal half of CA, p2, a leucine zipper domain of yeast GCN4 replacing NC, and a PPPPY motif replacing p6 (1).
The incorporation of Gag-Pol into extracellular particles requires the participation of Gag, since the expression of Gag-Pol alone does not readily generate such particles (22). Gag appears to be responsible for bringing Gag-Pol to the membrane, since unmyristylated Gag or Gag-Pol molecules can also be rescued into assembly complexes by myristylated Gag (39, 41, 50). The direct interaction of Gag with Gag-Pol has been demonstrated by immunoprecipitation of this complex from lysates of HIV-1-transfected cells with anti-integrase (22). While RNA appears to be important in facilitating Gag-to-Gag interactions (2, 7, 19), RNA is not required for the direct interaction of Gag-Pol with multimeric Gag (30).
The amino acid sequences within Gag and Gag-Pol that facilitate their interaction have been less studied than those involved in Gag/Gag multimerization. It is believed that Gag sequences within Gag-Pol play an important role in the interaction of Gag-Pol with Gag. In particular, deletions of the major homology region and the adjacent C-terminal half of the capsid region in Gag-Pol were shown to inhibit Gag-Pol incorporation into Gag particles (25, 51). More recent work has indicated that the Gag sequences in Gag-Pol may not be essential for Pol incorporation into Gag VLPs in either murine leukemia virus or in HIV-1 (5, 8). In HIV-1 (8), the incorporation of both protease (PR)-positive and PR-negative Gag-Pol constructs into Gag VLPs was examined. By using PR-positive constructs, Pol incorporation was determined by measuring the amount of processed viral proteins in the particle. However, in cells overexpressing viral proteins, there is premature processing of viral proteins in the cytoplasm, and their presence in VLPs is therefore not necessarily a direct measure of incorporated Gag-Pol. By using PR-negative virions, the presence of RT activity in the viral particle was used to indicate Pol incorporation, even though no processed, mature RT was present in the Gag VLPs. The authors concluded that after removal of Gag, or Gag plus PR, from Gag-Pol, the truncated Pol species were still incorporated. Incorporation was, however, only 10 to 20% of that of wild-type Gag-Pol. On the other hand, when murine leukemia virus Gag and Pol were coexpressed from separate plasmids in cells, RT activity associated with Gag VLPs was about 25% lower when Pol was incorporated than when Gag-Pol was incorporated (5). The incorporation of Pol into retroviral Gag particles has a natural precedent. In human foamy virus, Pol is made from a separate spliced mRNA than that used for producing Gag (54), i.e., no Gag-Pol species is found. Pol is incorporated into human foamy virus, and while tRNA1,2Lys appears to be the primer for reverse transcription in this virion (33), no studies have been done yet upon whether it is selectively packaged into the virus.
The interaction of Gag with Gag-Pol is clearly an important part of the retroviral life cycle since Gag-Pol carries essential enzymes involved in viral replication, as well as the tRNA3Lys required for priming reverse transcription. The nature of this interaction is therefore of interest, both from a theoretical and a therapeutic viewpoint. However, because of difficulties in purifying Gag-Pol, the interaction between Gag and Gag-Pol is most easily examined in vivo by studying the effects of mutations in Gag-Pol upon its interaction with Gag and its packaging into Gag VLPs. In the report herein, we have directly measured the incorporation of truncated forms of Gag-Pol and observe that even after removal of Gag and PR from Gag-Pol, the truncated species are incorporated at levels equal to 70% of that of wild-type and, furthermore, still retained the biological function of selectively packaging tRNALys.
In this report, HIV-1 proteins were produced in transfected HEK-293T cells in the absence of an active viral PR. The production of Gag alone is sufficient to produce extracellular VLPs. Lysates of cells transfected with various plasmids (see Table Table1)1) or VLPs produced by these cells were analyzed by Western blots (Fig. 1A and B) probed with anti-RT (upper panels), anti-β-actin (Fig. (Fig.1A,1A, lower panel), or anti-CA (Fig. (Fig.1B,1B, lower panel). Lanes 1 and 2 show cell or VLP proteins produced from cells transfected, respectively, with BH10.P−, a plasmid which codes for all wild-type HIV-1 proteins except PR, which is inactive, or with the BH10.FS− plasmid, which codes for all HIV-1 proteins except Gag-Pol. Lanes 3 and 4 show cell or VLP proteins produced from cells cotransfected with BH10.FS− and either hGag-PolΔFSΔPR−, which codes for Gag-Pol (Fig. 1A and B, lanes 3) or hPol, which codes for Pol (Fig. 1A and B, lanes 4). In Gag VLPs, Pol is packaged approximately 70% as efficiently as Gag-Pol. Cells transfected with a single plasmid coding for either Gag-Pol (Fig. 1A and B, lanes 5) or Pol (lanes 6) alone did not produce either Gag or extracellular VLPs, although they are effectively produced in the cell as shown by the Western blots of cell lysate.
The lower efficiency of incorporation of Pol into Gag VLPs is found even when viral Gag is replaced with Gag synthesized from humanized Gag plasmid (hGag). Cells were transfected with the hGag plasmid coding for Gag alone or cotransfected with the hGag plasmid and a humanized plasmid coding for either Gag-Pol (Fig. (Fig.1C,1C, lane 2) or Pol (lane 3). The Western blots shown, probed with anti-RT (upper panel) or anti-CA (lower panel), indicate that Pol protein missing the Gag sequences is still incorporated into Gag VLPs, albeit with only about 70% efficiency compared to levels for Gag-Pol.
To test for the specificity of interaction between Gag and Pol, similar transfection experiments were performed in which we replaced the plasmids coding for HIV-1 Gag with one coding for Gag from Rous sarcoma virus (RSV). This plasmid (RSV.Gag.P−) codes for RSV Gag but not Gag-Pol (9). Figure Figure1D1D shows Western blots of lysates of RSV Gag VLPs probed with either anti-CA (for RSV) or anti-RT (for HIV-1). While the lysates of the VLPs show a strong signal for RSV capsid, little or no Gag-Pol or Pol is incorporated into the particles, indicating that this incorporation is specific for HIV-1 Gag and not due to random incorporation.
The sequences in Pol required for incorporation into Gag VLPs were determined by cotransfecting HEK-293T cells with BH10.FS− as the source of Gag, with a plasmid coding for either Pol (hPol-V5), Pol protein lacking PR (hPol.ΔPR-V5), or Pol protein lacking integrase (hPol.ΔIN-V5). Lysates of cells transfected with various plasmids (Fig. (Fig.1E)1E) or Gag VLPs produced by these cells (Fig. (Fig.1F)1F) were analyzed by Western blots probed with anti-V5 and either anti-β-actin (Fig. (Fig.1E)1E) or anti-CA (Fig. (Fig.1F).1F). The Pol/Gag ratios indicate that neither PR nor integrase sequences are required for incorporation of Pol sequences into Gag VLPs. Since we have previously reported that the incorporation of both Gag-Pol and tRNALys into HIV-1 does not require the C-terminal Gag-Pol sequences coding for integrase or the RNase H and connection subdomains of RT (31), it seems likely that the Pol sequences that bind to Gag lie somewhere within the fingers, palm, or thumb subdomain of RT.
The ability of Gag-Pol or Pol to be incorporated into wild-type or mutant forms of Gag VLPs was monitored. HEK-293T cells were cotransfected with either hGag-PolΔFSΔP− (coding for Gag-Pol, but not Gag) or hPol and with a second plasmid coding for wild-type Gag (BH10.FS−) or mutant Gag (ZWt or ZWt-p6). The Gag expressed from each plasmid is shown in the results in Fig. Fig.2.2. BH10.FS− codes for wild-type HIV-1 proteins (with an inactive viral PR), including Gag; ZWt-p6 codes for a mutant Gag in which the NC sequence has been replaced with a yeast leucine zipper domain to allow for protein-to-protein interactions; ZWt codes for a mutant Gag similar to ZWt-p6, except for the further deletion of the p6 region. These two mutant Gag species remain efficient in forming VLPs (1), even with the deletion of p6, which can play an important role in viral release. However, several studies have shown that a role for p6 in viral budding can be overridden in high-efficiency transfection systems and in virions containing an inactive PR (for a review, see reference 13). Western blots of lysates of viruses produced from cells cotransfected with these different plasmids were probed with either anti-RT or anti-CA, and the results are shown in the upper and lower parts of Fig. Fig.2B,2B, respectively. These results indicate that there is a 12-fold reduction in the incorporation of Gag-Pol into VLPs composed of mutant Gag in which NC has been replaced by a yeast leucine zipper domain, while the incorporation of Pol into these Gag VLPs is not significantly altered. This result indicates that NC plays a more prominent role in the incorporation of Gag-Pol into Gag VLPs than in the incorporation of Pol. On the other hand, Pol incorporation into Gag VLPs is reduced four- to fivefold in the absence of p6 in Gag (Fig. (Fig.2B2B).
The incorporation of tRNALys isoacceptors into HIV-1 requires Gag-Pol (36). C-terminal deletions which include sequences coding for integrase and the RNase H and connection domains of RT do not inhibit tRNALys packaging, but further deletions into the thumb domain of RT inhibit tRNALys selective packaging (31). We have investigated whether Pol sequences alone can facilitate the selective incorporation of tRNALys isoacceptors into the virus. Cells were transfected with BH10.P− alone or BH10.FS− alone or were cotransfected with BH10.FS− and either hGag-PolΔFSΔPR or hPol. Western blot analysis similar to that shown in Fig. Fig.11 was used to determine the Gag-Pol or Pol/Gag ratios in the VLPs produced from these transfected cells, and these ratios are listed in Table Table2.2. As previously demonstrated by the results shown in Fig. Fig.1,1, Pol incorporation into Gag VLPs is somewhat lower (70 to 80%) than Gag-Pol incorporation.
Total RNA was then isolated from virions, and analyzed by dot blot hybridization with probes specific for tRNA3Lys or viral genomic RNA, as previously described (14). These results are shown in Fig. Fig.3A,3A, which also includes standard curves of different concentrations of genomic RNA or tRNA3Lys to show that the dot blot signals of samples fall within the linear range of the curves. The ratios of tRNA3Lys/genomic RNA were then determined, and these ratios, normalized to BH10P−, are also listed in Table Table2.2. It can be seen that the lower amount of tRNA3Lys packaged reflects the lower amount of Pol incorporated into VLPs, indicating that Pol sequences can efficiently replace Gag-Pol in facilitating tRNA3Lys packaging into VLPs.
The ability of Pol sequences to selectively package tRNALys isoacceptors into Gag VLPs was further analyzed by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), as previously described (28). Total viral RNA was 3′-end-labeled with [32P]pCp and analyzed by 2D-PAGE. Only low-molecular-weight RNA can enter the gel, and Fig. Fig.3B3B shows the 2D-PAGE patterns of this RNA. Panel I shows the pattern of RNA in virions produced from cells transfected with the BH10.P− plasmid. The labeled spots have previously been identified (28); spot 3 is tRNA3Lys, the primer tRNA for RT, and spots 1 and 2 represent tRNA1,2Lys. A similar pattern of low-molecular-weight RNA is seen in VLPs produced from cells cotransfected with BH10.FS−, and either hGag-PolΔFSΔPR (Fig. (Fig.3B,3B, panel II), or hPol (panel III). In virions produced from cells transfected only with BH10.FS−, no selective packaging of tRNALys isoacceptors is seen (panel IV).
The ability of Pol to selectively package primer tRNA3Lys is also reflected in the ability of the total viral RNA to support reverse transcription. We have shown that the annealing of tRNA3Lys to viral genomic RNA is proportional to the viral concentration of tRNA3Lys (14). To measure the amount of tRNA3Lys annealed to the primer binding site in vivo, total viral RNA is used as the source of primer or template in an in vitro reverse transcription reaction with exogenous HIV-1 RT, dCTP, dTTP, α-32P-dGTP, and ddATP. This assay measures the amount of extendable tRNA3Lys placed on the viral genome (it is not known if all annealed tRNA3Lys is extendable). Since the sequence of the first six dNTPs incorporated is CTGCTA, annealed primer tRNA3Lys will be extended by only six bases in the presence of ddATP, and the extended tRNA3Lys can be resolved and detected by one-dimensional PAGE. The electrophoretic results are shown in Fig. Fig.3C3C and listed in Table Table2.2. The first four lanes in Fig. Fig.3C3C use increasing amounts of genomic RNA in the viral RNA to show that the signal undergoes a linear increase with increasing RNA. The amount of viral genomic RNA labeled 1.00 in lane 4 is the amount of genomic RNA used in the remaining five lanes. The ratios of extension of primer tRNA3Lys/genomic RNA, relative to that found for BH10P−, are listed in Table Table2.2. tRNA3Lys placement in Gag VLPs containing Pol is significantly reduced compared to VLPs containing Gag-Pol. In part this result is probably due to the reduced incorporation of tRNA3Lys, since we have shown that the amount of tRNA3Lys annealed to viral genomic RNA is proportional to the amount of tRNA3Lys packaged (14). However, the reduction in tRNA3Lys annealing is greater than expected, which may reflect factors other than the concentration of tRNA3Lys in the virion, including the presence of Pol rather than Gag-Pol in the virion.
In this report, we have shown that Pol can be efficiently incorporated into Gag VLPs independently of upstream Gag sequences in Gag-Pol. Although this process has been studied in a cellular system in which larger amounts of viral proteins are being synthesized compared to normally infected T cells, it is unlikely that Pol incorporation is the result of random packaging. Thus, the results of the experiment shown in Fig. Fig.1D1D demonstrate that Rous sarcoma Gag VLPs do not package HIV Pol species. Also, the results of the experiments shown in Fig. Fig.22 demonstrate a specific requirement for particular Gag sequences. Replacement of NC in Gag with a leucine zipper motif (plasmid Zwtp6) prevents Gag-Pol, but not Pol, incorporation into Gag VLPs, while an additional deletion of p6 in Gag (plasmid Zwt) reduces Pol incorporation into Gag VLPs four- to fivefold.
It is possible, however, that with lower viral protein concentrations in the cell, the interaction of Gag-Pol with Gag p6 plays a minor role in the incorporation of Gag-Pol into virions, with homologous interactions between Gag and Gag (Gag-Pol) being sufficient for this process. The interaction between Gag-Pol and Gag p6 may be more important at a later stage of assembly. Thus, previous reports have presented data that suggest that the HIV-1 Gag p6 domain functions to retain the Pol domain proteins within the assembling virus after the initial activation of the viral PR (11, 55). Current evidence suggests that prior to viral budding, the viral PR-induced cleavage of Gag-Pol at the cell membrane is initiated in trans within a Gag-Pol dimer to produce an N-terminal Gag fragment terminating in p2 and a C-terminal fragment containing NC-transframe-PR-RT-integrase (42). Thus, this first PR cleavage will separate that part of Gag sequences within Gag-Pol which contain major sites of binding for Gag, i.e., the major homology region and C-terminal portion of capsid. The binding of Pol sequences to p6 sequences in Gag could therefore be important for retaining Pol sequences and tRNALys until viral budding is complete. This interpretation is further supported by the observation reported above that Pol can facilitate the selective packaging of tRNALys isoacceptors with an efficiency reflecting the incorporation of Pol into the Gag particles (approximately 70% of that found using intact Gag-Pol) (Table (Table22).
This work was supported by grants from the Canadian Institutes for Health Research and the Canadian Foundation for AIDS Research.
We thank Heinrich Göttlinger and Rebecca Craven for the gift of plasmids used in this work.