Incorporation of Pol into Human Immunodeficiency Virus Type 1 Gag Virus-Like Particles Occurs Independently of the Upstream Gag Domain in Gag-Pol

By using particle-associated reverse transcriptase (RT) activityas an assay for Pol incorporation into human immunodeficiencyvirus type 1 (HIV-1) Gag virus-like particles (VLPs), it hasbeen found that truncated, protease-negative, Gag-Pol missingcis Gag sequences is still incorporated into Gag VLPs, albeitat significantly reduced levels (10 to 20% of the level of wild-typeGag-Pol). In this work, we have directly measured the incorporationof truncated Gag-Pol species into Gag VLPs and have found thattruncated Gag-Pol that is missing all sequences upstream ofRT is still incorporated into Gag VLPs at levels approximating70% of that achieved by wild-type Gag-Pol. Neither proteasenor integrase regions in Pol are required for its incorporation,implying an interaction between Gag and RT sequences in thePol protein. While the incorporation of Gag-Pol into Gag VLPsis reduced 12-fold by the replacement of the nucleocapsid withinGag with a leucine zipper motif, this mutation does not affectPol incorporation. However, the deletion of p6 in Gag reducesPol incorporation into Gag VLPs four- to fivefold. Pol showsthe same ability as Gag-Pol to selectively package tRNA^Lys intoGag VLPs, and primer tRNA₃^Lys is found annealed to the viralgenomic RNA. These data suggest that after the initial separationof Gag from Pol during cleavage of Gag-Pol by viral protease,the Pol species still retains the capacity to bind to both Gagand tRNA₃^Lys, which may be required for Pol and tRNA₃^Lys tobe retained in the assembling virion until budding is completed.

INTRODUCTION

Top

Introduction

During human immunodeficiency virus type 1 (HIV-1) assembly,the major tRNA^Lys isoacceptors in mammalian cells, tRNA₃^Lysand tRNA_1,2^Lys (44), are selectively packaged into the virion(28), where tRNA₃^Lys serves as the primer for the reverse transcriptase(RT)-catalyzed synthesis of minus-strand cDNA (37). The annealingof tRNA₃^Lys to the primer binding site of the viral RNA genomeis proportional to the amount of tRNA₃^Lys packaged in the viralpopulation (14). tRNA^Lys packaging into HIV-1 depends upon thepresence of the precursor protein Gag-Pol (36) and may dependdirectly upon sequences in the RT thumb domain. Thus, C-terminaldeletions of Gag-Pol which include sequences coding for integraseand the RNase H and connection domain of RT do not affect tRNA^Lyspackaging, but larger deletions that also include the thumbdomain inhibit packaging (31). While deletion of the RT thumbdomain could induce conformational changes affecting tRNA^Lysbinding elsewhere in the molecule, cross-linking studies betweenpurified HIV-1 RT and tRNA₃^Lys indicate a direct interactionbetween the thumb domain and tRNA₃^Lys (12).

Extracellular virus-like particles (VLPs) can be produced byHIV-1 Gag alone (17, 21, 29, 50), and putative regions of interactionsbetween Gag molecules have been identified within the C-terminalhalf 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 obtainthe interactions required for assembly, the Gag molecules mustfirst be concentrated at a cellular site. Membrane has beenproposed to play a role in the concentration and alignment ofGag molecules, and in HIV-1-transfected COS cells, almost allsteady-state Gag is membrane bound (22, 24, 52). In many retroviruses,the Gag precursor protein attaches to the lipid membrane viaa myristic acid moiety added posttranslationally to the aminoterminus of the matrix (4, 18, 23, 45, 48) and through ionicinteractions between the phospholipids in the membrane and asequence of basic amino acid residues within the matrix (46,47, 53, 56). However, a minimal, 16-kDa Gag construct lackingmatrix has been shown to be capable of forming VLPs in vivoand is composed of an N-terminal myristylation signal, the C-terminalhalf of CA, p2, a leucine zipper domain of yeast GCN4 replacingNC, and a PPPPY motif replacing p6 (1).

The incorporation of Gag-Pol into extracellular particles requiresthe participation of Gag, since the expression of Gag-Pol alonedoes not readily generate such particles (22). Gag appears tobe responsible for bringing Gag-Pol to the membrane, since unmyristylatedGag or Gag-Pol molecules can also be rescued into assembly complexesby myristylated Gag (39, 41, 50). The direct interaction ofGag with Gag-Pol has been demonstrated by immunoprecipitationof this complex from lysates of HIV-1-transfected cells withanti-integrase (22). While RNA appears to be important in facilitatingGag-to-Gag interactions (2, 7, 19), RNA is not required forthe direct interaction of Gag-Pol with multimeric Gag (30).

The amino acid sequences within Gag and Gag-Pol that facilitatetheir interaction have been less studied than those involvedin Gag/Gag multimerization. It is believed that Gag sequenceswithin Gag-Pol play an important role in the interaction ofGag-Pol with Gag. In particular, deletions of the major homologyregion and the adjacent C-terminal half of the capsid regionin Gag-Pol were shown to inhibit Gag-Pol incorporation intoGag particles (25, 51). More recent work has indicated thatthe Gag sequences in Gag-Pol may not be essential for Pol incorporationinto Gag VLPs in either murine leukemia virus or in HIV-1 (5,8). In HIV-1 (8), the incorporation of both protease (PR)-positiveand PR-negative Gag-Pol constructs into Gag VLPs was examined.By using PR-positive constructs, Pol incorporation was determinedby measuring the amount of processed viral proteins in the particle.However, in cells overexpressing viral proteins, there is prematureprocessing of viral proteins in the cytoplasm, and their presencein VLPs is therefore not necessarily a direct measure of incorporatedGag-Pol. By using PR-negative virions, the presence of RT activityin 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 plusPR, from Gag-Pol, the truncated Pol species were still incorporated.Incorporation was, however, only 10 to 20% of that of wild-typeGag-Pol. On the other hand, when murine leukemia virus Gag andPol were coexpressed from separate plasmids in cells, RT activityassociated with Gag VLPs was about 25% lower when Pol was incorporatedthan when Gag-Pol was incorporated (5). The incorporation ofPol into retroviral Gag particles has a natural precedent. Inhuman foamy virus, Pol is made from a separate spliced mRNAthan that used for producing Gag (54), i.e., no Gag-Pol speciesis found. Pol is incorporated into human foamy virus, and whiletRNA_1,2^Lys appears to be the primer for reverse transcriptionin this virion (33), no studies have been done yet upon whetherit is selectively packaged into the virus.

The interaction of Gag with Gag-Pol is clearly an importantpart of the retroviral life cycle since Gag-Pol carries essentialenzymes involved in viral replication, as well as the tRNA₃^Lysrequired for priming reverse transcription. The nature of thisinteraction is therefore of interest, both from a theoreticaland a therapeutic viewpoint. However, because of difficultiesin purifying Gag-Pol, the interaction between Gag and Gag-Polis most easily examined in vivo by studying the effects of mutationsin Gag-Pol upon its interaction with Gag and its packaging intoGag VLPs. In the report herein, we have directly measured theincorporation of truncated forms of Gag-Pol and observe thateven after removal of Gag and PR from Gag-Pol, the truncatedspecies are incorporated at levels equal to 70% of that of wild-typeand, furthermore, still retained the biological function ofselectively packaging tRNA^Lys.

Specific incorporation of Pol into HIV-1 is independent of Gag within Gag-Pol.

Top

Specific incorporation of pol...

In this report, HIV-1 proteins were produced in transfectedHEK-293T cells in the absence of an active viral PR. The productionof Gag alone is sufficient to produce extracellular VLPs. Lysatesof cells transfected with various plasmids (see Table 1) orVLPs produced by these cells were analyzed by Western blots(Fig. 1A and B) probed with anti-RT (upper panels), anti-ß-actin(Fig. 1A, lower panel), or anti-CA (Fig. 1B, lower panel). Lanes1 and 2 show cell or VLP proteins produced from cells transfected,respectively, with BH10.P-, a plasmid which codes for all wild-typeHIV-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 cellscotransfected with BH10.FS- and either hGag-Pol ${Delta}$ FS ${Delta}$ PR-, whichcodes for Gag-Pol (Fig. 1A and B, lanes 3) or hPol, which codesfor Pol (Fig. 1A and B, lanes 4). In Gag VLPs, Pol is packagedapproximately 70% as efficiently as Gag-Pol. Cells transfectedwith a single plasmid coding for either Gag-Pol (Fig. 1A and B,lanes 5) or Pol (lanes 6) alone did not produce either Gagor extracellular VLPs, although they are effectively producedin the cell as shown by the Western blots of cell lysate.

View this table:
[in this window]
[in a new window]
TABLE 1. Plasmids used in this study

View larger version (34K):
[in this window]
[in a new window]
FIG.1. Incorporation of Pol and Gag-Pol into Gag VLPs. 293T cells were transfected or cotransfected with different plasmids coding for wild-type Gag and wild-type or mutant Gag-Pol proteins, using Lipofectamine. The plasmids used are listed along the top of each panel. BH10.P- is a simian virus 40-based vector that contains full-length wild-type HIV-1 proviral DNA containing an inactive viral PR (D25G) and was a gift from E. Cohen, University of Montreal. BH10.FS- contains mutations at the frameshift site, i.e., nucleic acid sequence 2082-TTTTTT-2087 replaces 2082-CTTCCT-2087, which prevents frameshifting during the translation of the Gag protein and generates particles that contain only Gag, not Gag-Pol (35). hGag-Pol ${Delta}$ FS ${Delta}$ PR was constructed by deleting five thymidines in the frameshift site and codes for Gag-Pol. The humanized proteins have identical amino acid sequences to their viral counterparts, but the mRNAs coding for them have had their codons optimized for mammalian cell codon usage, which results in more efficient translation and protein production and also makes nuclear export of these mRNAs Rev-independent through modification of the multiple inhibitory sequences (26, 43). Both hPol and hGag-Pol ${Delta}$ FS ${Delta}$ PR contain an inactive PR due to an R42G mutation in the active site. hPol-V5, hPol. ${Delta}$ PR-V5, and hPol. ${Delta}$ IN-V5 code for Pol, Pol missing PR, and Pol missing integrase, respectively; and all proteins contain a C-terminal V5 tag. Plasmid pSV.Myr1.3 h was a gift from R. Craven and J. Wills (University of Pennsylvania, Philadelphia) and was constructed as previously described (9). We have renamed it RSV.Gag.P-. It encodes a truncated Myr1 protein that carries RSV Gag sequences and only the first seven amino acids of RSV PR, followed by one foreign amino acid, and produces RSV extracellular Gag VLPs containing Gag but no Gag-Pol. HEK-293T cells were cultured as previously described (20), transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. At 48 h posttransfection, Gag VLPs were purified from the media as previously described (14). Cells or Gag VLPs were lysed in radioimmunoprecipitation assay buffer (10 mM Tris [pH 7.4], 100 mM NaCl, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1% NP-40, 2 mg of aprotinin/ml, 2 mg of leupeptin/ml, 1 mg of pepstatin A/ml, 100 mg of phenylmethylsulfonyl fluoride/ml). Proteins in the cell or viral lysates were resolved by SDS-PAGE (10% acrylamide), followed by blotting onto nitrocellulose membranes (Amersham Pharmacia). Detection of protein by Western blotting utilized monoclonal antibodies that are specifically reactive with HIV-1 CA (Zepto Metrocs Inc.), RT (National Institutes of Health, AIDS Research and Reference Reagent Program) and ß-actin (Sigma). Rabbit antiserum to RSV was a kind gift from R. Craven (University of Pennsylvania). Detection of proteins was performed by enhanced chemiluminescence (NEN Life Sciences Products) by using as secondary antibodies anti-mouse (for CA, RT, and ß-actin) and anti-rabbit (for RSV), both obtained from Amersham Life Sciences. (A and B) Lysates of cells transfected with various plasmids or VLPs produced by these cells were analyzed by Western blots probed with anti-RT (upper panels), anti-ß-actin (A, lower panel), or anti-CA (B, lower panel). (C) Western blots of Gag VLPs, probed with anti-RT (upper panel) or anti-CA (lower panel). (D) Western blots of lysates of RSV Gag VLPs probed with either RSV anti-CA (lower panel) or HIV anti-RT (upper panel). (E and F) Lysates of cells transfected with various plasmids or Gag VLPs produced by these cells were analyzed by Western blots probed with anti-V5 and either anti ß-actin (E) or anti-CA (F). Bands in Western blots were quantitated with the UN-SCAN-IT gel automated digitizing system.

The lower efficiency of incorporation of Pol into Gag VLPs isfound even when viral Gag is replaced with Gag synthesized fromhumanized Gag plasmid (hGag). Cells were transfected with thehGag plasmid coding for Gag alone or cotransfected with thehGag plasmid and a humanized plasmid coding for either Gag-Pol(Fig. 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 stillincorporated into Gag VLPs, albeit with only about 70% efficiencycompared to levels for Gag-Pol.

To test for the specificity of interaction between Gag and Pol,similar transfection experiments were performed in which wereplaced the plasmids coding for HIV-1 Gag with one coding forGag from Rous sarcoma virus (RSV). This plasmid (RSV.Gag.P-)codes for RSV Gag but not Gag-Pol (9). Figure 1D shows Westernblots of lysates of RSV Gag VLPs probed with either anti-CA(for RSV) or anti-RT (for HIV-1). While the lysates of the VLPsshow a strong signal for RSV capsid, little or no Gag-Pol orPol is incorporated into the particles, indicating that thisincorporation is specific for HIV-1 Gag and not due to randomincorporation.

The sequences in Pol required for incorporation into Gag VLPswere 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. ${Delta}$ PR-V5), or Pol protein lackingintegrase (hPol. ${Delta}$ IN-V5). Lysates of cells transfected with variousplasmids (Fig. 1E) or Gag VLPs produced by these cells (Fig.1F) were analyzed by Western blots probed with anti-V5 and eitheranti-ß-actin (Fig. 1E) or anti-CA (Fig. 1F). The Pol/Gagratios indicate that neither PR nor integrase sequences arerequired for incorporation of Pol sequences into Gag VLPs. Sincewe have previously reported that the incorporation of both Gag-Poland tRNA^Lys into HIV-1 does not require the C-terminal Gag-Polsequences coding for integrase or the RNase H and connectionsubdomains of RT (31), it seems likely that the Pol sequencesthat bind to Gag lie somewhere within the fingers, palm, orthumb subdomain of RT.

The role of NC and p6 in Gag interactions with Gag-Pol and Pol.

Top

The role of nc...

The ability of Gag-Pol or Pol to be incorporated into wild-typeor mutant forms of Gag VLPs was monitored. HEK-293T cells werecotransfected with either hGag-Pol ${Delta}$ FS ${Delta}$ P- (coding for Gag-Pol,but not Gag) or hPol and with a second plasmid coding for wild-typeGag (BH10.FS-) or mutant Gag (ZWt or ZWt-p6). The Gag expressedfrom each plasmid is shown in the results in Fig. 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 NCsequence has been replaced with a yeast leucine zipper domainto allow for protein-to-protein interactions; ZWt codes fora mutant Gag similar to ZWt-p6, except for the further deletionof the p6 region. These two mutant Gag species remain efficientin forming VLPs (1), even with the deletion of p6, which canplay an important role in viral release. However, several studieshave shown that a role for p6 in viral budding can be overriddenin high-efficiency transfection systems and in virions containingan inactive PR (for a review, see reference 13). Western blotsof lysates of viruses produced from cells cotransfected withthese different plasmids were probed with either anti-RT oranti-CA, and the results are shown in the upper and lower partsof Fig. 2B, respectively. These results indicate that thereis a 12-fold reduction in the incorporation of Gag-Pol intoVLPs composed of mutant Gag in which NC has been replaced bya yeast leucine zipper domain, while the incorporation of Polinto these Gag VLPs is not significantly altered. This resultindicates that NC plays a more prominent role in the incorporationof Gag-Pol into Gag VLPs than in the incorporation of Pol. Onthe other hand, Pol incorporation into Gag VLPs is reduced four-to fivefold in the absence of p6 in Gag (Fig. 2B).

View larger version (56K):
[in this window]
[in a new window]
FIG. 2. Sequences in Gag binding Gag-Pol or Pol. 293T cells were cotransfected with a plasmid coding for wild-type or mutant Gag and with a plasmid coding for either hGag-Pol (hGag-Pol ${Delta}$ FS ${Delta}$ PR) or hPol (hPol). ZWt and ZWt-p6 were a gift of Heinrich Göttlinger (Dana- Farber Cancer Institute) and were constructed as previously described (1). The plasmids used are listed along the top of each panel. At 48 h posttransfection, purified Gag VLPs were lysed in radioimmunoprecipitation assay buffer, and Gag and Gag-Pol proteins were detected with Western blots probed with antibodies to HIV-1 RT or CA. (A) Cartoon of wild-type and mutant Gag expressed. (B) Lysates of viruses analyzed by Western blots probed with anti-RT (upper panel) or anti-CA (lower panel). Bands were quantitated with the UN-SCAN-IT gel automated digitizing system.

Pol can facilitate the selective packaging of tRNA^Lys into Gag VLPs and its annealing onto the viral RNA genome.

Top

Pol can facilitate the...

The incorporation of tRNA^Lys isoacceptors into HIV-1 requiresGag-Pol (36). C-terminal deletions which include sequences codingfor integrase and the RNase H and connection domains of RT donot inhibit tRNA^Lys packaging, but further deletions into thethumb domain of RT inhibit tRNA^Lys selective packaging (31).We have investigated whether Pol sequences alone can facilitatethe selective incorporation of tRNA^Lys isoacceptors into thevirus. Cells were transfected with BH10.P- alone or BH10.FS-alone or were cotransfected with BH10.FS- and either hGag-Pol ${Delta}$ FS ${Delta}$ PRor hPol. Western blot analysis similar to that shown in Fig.1 was used to determine the Gag-Pol or Pol/Gag ratios in theVLPs produced from these transfected cells, and these ratiosare listed in Table 2. As previously demonstrated by the resultsshown in Fig. 1, Pol incorporation into Gag VLPs is somewhatlower (70 to 80%) than Gag-Pol incorporation.

View this table:
[in this window]
[in a new window]
TABLE 2. The effect of Gag-Pol or Pol incorporation upon selective packaging and annealing of tRNA₃^Lys in HIV-1^a

Total RNA was then isolated from virions, and analyzed by dotblot hybridization with probes specific for tRNA₃^Lys or viralgenomic RNA, as previously described (14). These results areshown in Fig. 3A, which also includes standard curves of differentconcentrations of genomic RNA or tRNA₃^Lys to show that the dotblot signals of samples fall within the linear range of thecurves. The ratios of tRNA₃^Lys/genomic RNA were then determined,and these ratios, normalized to BH10P-, are also listed in Table2. It can be seen that the lower amount of tRNA₃^Lys packagedreflects the lower amount of Pol incorporated into VLPs, indicatingthat Pol sequences can efficiently replace Gag-Pol in facilitatingtRNA₃^Lys packaging into VLPs.

View larger version (37K):
[in this window]
[in a new window]
FIG. 3. Incorporation of tRNA₃^Lys into Gag VLPs and its annealing to viral RNA. HEK-293T cells were transfected or cotransfected with different plasmids coding for wild-type Gag and wild-type or mutant Gag-Pol proteins with Lipofectamine. The plasmids used are listed along the top of each panel. At 48 h posttransfection, Gag VLPs were purified, and total viral RNA was extracted using guanidiumisothiocynate, as previously described (28). (A) Incorporation of tRNA₃^Lys into virions. Dot blots of viral RNA were hybridized with DNA probes complementary to either viral genomic RNA (upper panel) or tRNA₃^Lys (lower panel), as previously described (14). Hybridization signals were analyzed by phosphorimaging, and the ratio of tRNA₃^Lys/genomic RNA was determined for each sample (see Table 1). The standard curves shown in the left part of each blot contain known amounts of in vitro transcribed HIV-1 genomic RNA (5' end fragment) or in vitro transcribed tRNA₃^Lys and were hybridized with the DNA probes complementary to either tRNA₃^Lys or genomic RNA to show the linearity of the signal. (B) 2D PAGE analysis of low-molecular-weight viral RNA. Total viral RNA was extracted from virions, 3'-end-labeled with [³²P]pCp and electrophoresed in 11% polyacrylamide in the first dimension and 20% polyacrylamide in the second dimension, as previously described (28). Only low-molecular-weight RNA moves into the gel and is detected by autoradiography. Spot 3, tRNA₃^Lys; spots 1 and 2, tRNA_1,2^Lys. The text below each panel lists plasmids used to express each viral type analyzed. (C) tRNA₃^Lys annealing to viral RNA. Total viral RNA was used as the source of primer tRNA₃^Lys/genomic RNA template in an in vitro reverse transcription reaction, carried out in the presence of [ ${alpha}$ -³²P]dGTP, dCTP, dTTP, and ddATP, as previously described (27). This process results in a six-base extension product since the first six bases incorporated are CTGCTA. Products were resolved by one-dimensional PAGE, with different samples containing equal amounts of genomic RNA. The standard curve shown in the left part of the blot in panel C contains a dilution series of total BH10 viral RNA, which is used as the source of primer or template to show the linearity of the signal.

The ability of Pol sequences to selectively package tRNA^Lysisoacceptors into Gag VLPs was further analyzed by using two-dimensionalpolyacrylamide gel electrophoresis (2D-PAGE), as previouslydescribed (28). Total viral RNA was 3'-end-labeled with [³²P]pCpand analyzed by 2D-PAGE. Only low-molecular-weight RNA can enterthe gel, and Fig. 3B shows the 2D-PAGE patterns of this RNA.Panel I shows the pattern of RNA in virions produced from cellstransfected with the BH10.P- plasmid. The labeled spots havepreviously been identified (28); spot 3 is tRNA₃^Lys, the primertRNA for RT, and spots 1 and 2 represent tRNA_1,2^Lys. A similarpattern of low-molecular-weight RNA is seen in VLPs producedfrom cells cotransfected with BH10.FS-, and either hGag-Pol ${Delta}$ FS ${Delta}$ PR(Fig. 3B, panel II), or hPol (panel III). In virions producedfrom cells transfected only with BH10.FS-, no selective packagingof tRNA^Lys isoacceptors is seen (panel IV).

The ability of Pol to selectively package primer tRNA₃^Lys isalso reflected in the ability of the total viral RNA to supportreverse transcription. We have shown that the annealing of tRNA₃^Lysto viral genomic RNA is proportional to the viral concentrationof tRNA₃^Lys (14). To measure the amount of tRNA₃^Lys annealedto the primer binding site in vivo, total viral RNA is usedas the source of primer or template in an in vitro reverse transcriptionreaction with exogenous HIV-1 RT, dCTP, dTTP, ${alpha}$ -³²P-dGTP, andddATP. This assay measures the amount of extendable tRNA₃^Lysplaced on the viral genome (it is not known if all annealedtRNA₃^Lys is extendable). Since the sequence of the first sixdNTPs incorporated is CTGCTA, annealed primer tRNA₃^Lys willbe extended by only six bases in the presence of ddATP, andthe extended tRNA₃^Lys can be resolved and detected by one-dimensionalPAGE. The electrophoretic results are shown in Fig. 3C and listedin Table 2. The first four lanes in Fig. 3C use increasing amountsof genomic RNA in the viral RNA to show that the signal undergoesa linear increase with increasing RNA. The amount of viral genomicRNA labeled 1.00 in lane 4 is the amount of genomic RNA usedin the remaining five lanes. The ratios of extension of primertRNA₃^Lys/genomic RNA, relative to that found for BH10P-, arelisted in Table 2. tRNA₃^Lys placement in Gag VLPs containingPol is significantly reduced compared to VLPs containing Gag-Pol.In part this result is probably due to the reduced incorporationof tRNA₃^Lys, since we have shown that the amount of tRNA₃^Lysannealed to viral genomic RNA is proportional to the amountof tRNA₃^Lys packaged (14). However, the reduction in tRNA₃^Lysannealing is greater than expected, which may reflect factorsother than the concentration of tRNA₃^Lys in the virion, includingthe presence of Pol rather than Gag-Pol in the virion.

In this report, we have shown that Pol can be efficiently incorporatedinto Gag VLPs independently of upstream Gag sequences in Gag-Pol.Although this process has been studied in a cellular systemin which larger amounts of viral proteins are being synthesizedcompared to normally infected T cells, it is unlikely that Polincorporation is the result of random packaging. Thus, the resultsof the experiment shown in Fig. 1D demonstrate that Rous sarcomaGag VLPs do not package HIV Pol species. Also, the results ofthe experiments shown in Fig. 2 demonstrate a specific requirementfor particular Gag sequences. Replacement of NC in Gag witha leucine zipper motif (plasmid Zwtp6) prevents Gag-Pol, butnot Pol, incorporation into Gag VLPs, while an additional deletionof p6 in Gag (plasmid Zwt) reduces Pol incorporation into GagVLPs four- to fivefold.

It is possible, however, that with lower viral protein concentrationsin the cell, the interaction of Gag-Pol with Gag p6 plays aminor role in the incorporation of Gag-Pol into virions, withhomologous interactions between Gag and Gag (Gag-Pol) beingsufficient for this process. The interaction between Gag-Poland Gag p6 may be more important at a later stage of assembly.Thus, previous reports have presented data that suggest thatthe HIV-1 Gag p6 domain functions to retain the Pol domain proteinswithin the assembling virus after the initial activation ofthe viral PR (11, 55). Current evidence suggests that priorto viral budding, the viral PR-induced cleavage of Gag-Pol atthe cell membrane is initiated in trans within a Gag-Pol dimerto produce an N-terminal Gag fragment terminating in p2 anda C-terminal fragment containing NC-transframe-PR-RT-integrase(42). Thus, this first PR cleavage will separate that part ofGag sequences within Gag-Pol which contain major sites of bindingfor Gag, i.e., the major homology region and C-terminal portionof capsid. The binding of Pol sequences to p6 sequences in Gagcould therefore be important for retaining Pol sequences andtRNA^Lys until viral budding is complete. This interpretationis further supported by the observation reported above thatPol can facilitate the selective packaging of tRNA^Lys isoacceptorswith an efficiency reflecting the incorporation of Pol intothe Gag particles (approximately 70% of that found using intactGag-Pol) (Table 2).

ACKNOWLEDGMENTS

This work was supported by grants from the Canadian Institutesfor Health Research and the Canadian Foundation for AIDS Research.

We thank Heinrich Göttlinger and Rebecca Craven for thegift of plasmids used in this work.

FOOTNOTES

* Corresponding author. Mailing address: Lady Davis Institute for Medical Research-Jewish General Hospital, 3755 Côte Ste-Catherine Rd., Montreal, Quebec, Canada H3T 1E2. Phone: (514) 340-8260. Fax: (514) 340-7502. E-mail: lawrence.kleiman{at}mcgill.ca.

REFERENCES

Top