A Bipartite Late-Budding Domain in Human Immunodeficiency Virus Type 1

Abstract

Human immunodeficiency virus type 1 (HIV-1) encodes a PTAP motif within the p6 domain of Gag that recruits Tsg101 and associated factors to facilitate virion budding. In this study, we use trans-complementation assays to demonstrate that the PTAP motif acts synergistically with additional p6 sequences to mediate the formation of infectious extracellular HIV-1 virions. These studies suggest that Tsg101 recruitment is necessary but not sufficient to account for late-budding activity exhibited by HIV-1 p6.

All retroviruses and some otherwise unrelated rhabdo- and filoviruses encode a so-called late-budding domain or “L-domain” whose disruption results in a specific defect in particle release (4, 9-13, 27, 28). Viral L-domains fall into three classes, based on the presence of characteristic PT/SAP, PPXY, or YPDL sequence motifs. In several cases, viral L-domains are functionally interchangeable, can exert their activity when positioned at different locations within a retroviral Gag protein, and can function in trans (4, 17, 20, 29). These observations strongly suggest that L-domains act by recruiting host cell factors to facilitate egress, rather than directly influencing virion morphogenesis. In fact, a variety of findings indicate that the human immunodeficiency virus type 1 (HIV-1) and Ebola virus PTAP-type L-domains act by recruiting Tsg101 (5, 8, 17, 18, 26), a component of “endosomal sorting complex required for transport-I” (ESCRT-I) that normally functions in the budding of vesicles into the endosomal lumen (14). Specifically, there is an almost perfect correlation between the abilities of HIV-1 p6 mutants to bind Tsg101 and to mediate virion production (8, 17, 26). In addition, depletion of Tsg101 with small interfering RNA results in an HIV-1 budding defect that recapitulates the phenotype of a PTAP-defective virus (8). Moreover, expression of HIV-1 Gag or Ebola virus VP40 results in PTAP-dependent recruitment of Tsg101 and VPS28, another ESCRT-I component, to sites of particle assembly at the plasma membrane (17, 18).

We have previously described constructs and assays that demonstrated that artificial tethering of Tsg101 to sites of HIV-1 particle assembly, by fusion to Gag, reverses the HIV-1 budding defect that results from mutational inactivation of the PTAP L-domain (17, 18). This type of assay is represented schematically in Fig. 1A and employs a Gag precursor protein lacking the p6 domain (Gagδp6) that encodes a synthetic linker at its C terminus. Candidate L-domains or Tsg101 is fused to this truncated Gag protein and is expressed with an L-domain-defective proviral plasmid. Because HIV-1 Gag efficiently multimerizes at the plasma membrane (16, 25), expression of a Gagδp6 protein that is fused to functional L-domain or is directly fused to Tsg101 can functionally complement a PTAP-defective proviral construct, resulting in infectious virion production (17, 18). A result from this type of assay is shown in Fig. 1B. For this experiment, 5 × 10⁴ HOS cells seeded in 24-well plates were cotransfected with 300 ng of a PTAP-defective HIV-1 proviral construct (pNL[LTAL]) (17) and 200 ng of a pGagδp6-Tsg101 expression plasmid by using Lipofectamine Plus, according to the manufacturer's instructions. Infectious virus production was measured by inoculating P4-R5 cells, with clarified supernatant from the transfected HOS cells. β-Galactosidase expression resulting from activation of the integrated HIV-1 long terminal repeat-lacZ indicator gene in P4-R5 cells was measured 48 h later, as previously described (3, 17). As can be seen in Fig. 1B, coexpression of Gagδp6-Tsg101 resulted in an enhancement of NL(LTAL) infectious virion production by 10- to 15-fold. Conversely, in an almost identical experiment also shown in Fig. 1B, but in which the HIV-1 provirus was rendered defective by the introduction of a translational termination codon in place of the N-terminal p6 codon (NLδp6), the Gagδp6-Tsg101 fusion protein was, surprisingly, unable to restore infectious virion production.

FIG. 1.

Tsg101 recruitment is not sufficient to account for the L-domain activity exhibited by HIV-1 p6. (A) The complementation assay. A schematic representation of the complementing Gagδp6 protein is shown. It contains MA, CA, and NC domains and the N-terminal 4 residues of p6 (LQSR) to permit cleavage by the HIV-1 protease. The synthetic linker sequence (LEFGGRLE) also encodes EcoRI, NotI, and XhoI restriction sites (underlined) for reinsertion of full-length or truncated versions of p6, the PTAP sequence, or Tsg101. For the assay itself, Gag-Pol and mutant Gag proteins are expressed by the defective proviral construct and are depicted in black; the complementing fusion protein, in this case Gagδp6-Tsg101, is shown in white. Tsg101 is recruited to sites of virion production by virtue of Gag multimerization. (B) Infectious virus production by defective provirus (NL[LTAL], black bars; or NLδp6, white bars) complemented by Gagδp6-Tsg101. The chart shows β-galactosidase activity in HeLa P4/R5 cells following inoculation with supernatant harvested from HOS cells transfected with defective provirus constructs and the indicated complementing expression plasmid. RLU, relative light units. (C) Same information as that given for panel B applies, except that only the pNLδp6 proviral plasmid was used, complemented with either pGagδp6-p6 or pGagδp6-Tsg101, as indicated. (D) Western blot analysis, with an anti-CA monoclonal antibody, of cell lysates and virion pellets following transfection of HOS cells with the pNLδp6 provirus plus either pGagδp6 (lane 1), pGagδp6-p6 (lane 2), or pGagδp6-Tsg101 (lane 3).

As is shown in Fig. 1C, the inability of Gagδp6-Tsg101 to complement NLδp6 was not due to any cis-acting defect in the NLδp6 provirus. Indeed, this defective virus was efficiently complemented in trans by coexpression of a Gagδp6-p6 fusion protein. Moreover, as shown in Fig. 1D, the overall particle release and cell-associated Gag processing defect exhibited by NLδp6, which are characteristic of L-domain defective HIV-1 constructs, were reversed by coexpression of Gagδp6-p6 but not by coexpression of Gagδp6-Tsg101. In contrast, we have previously shown that similar defects induced by point mutations in the PTAP motif could be reversed by Gagδp6-Tsg101 coexpression (17, 18). Thus, these results indicate that recruitment of Tsg101 is sufficient to account for the activity exhibited by the HIV-1 PTAP motif but does not account for the entire effect on virion morphogenesis that is exerted by HIV-1 p6.

To test whether p6 sequences outside the PTAP motif could exhibit L-domain activity, either alone or in conjunction with PTAP, we again used a complementation approach. This strategy, shown schematically in Fig. 2A, was elaborated to include two complementing Gagδp6-fusion proteins, coexpressed with the defective pNLδp6 proviral construct. First, we compared the ability of a 10-residue HIV-1 p6-derived peptide containing the PTAP motif (P₅EPTAPPEES₁₄) versus the entire p6 domain to function in trans in the context of a Gagδp6-fusion protein to restore infectious virion production by NLδp6. For these experiments, 5 × 10⁴ 293T cells seeded in 24-well plates were cotransfected with 300 ng of NLδp6 along with 100 ng of pGagδp6 and 100 ng of either pGagδp6-p6, pGagδp6-PTAP, or pGagδp6-p6 (15-51) expression plasmids by using Lipofectamine Plus. Analysis of infectious virion production revealed that the intact p6 domain functioned substantially more efficiently than did the PTAP-containing peptide sequence. Indeed, as shown in Fig. 2B, expression of Gagδp6-p6 increased infectious virion production by NLδp6 approximately 270-fold. In contrast, Gagδp6-PTAP was less than 10% as active as Gagδp6-p6 and enhanced infectious virion formation by only 24-fold.

FIG. 2.

The PTAP motif and additional p6 sequences can act synergistically and in trans to mediate infectious HIV-1 virion production. (A) Schematic representation of the complementation assay. Gag-Pol and Gagδp6 proteins expressed by the defective proviral construct (NLδp6) are depicted in black; the complementing fusion proteins, in this case Gagδp6-PTAP and Gagδp6-p6 (15-51), are shown in white. (B) Infectious virion production measured as for Fig. 1 by using HeLa P4/R5 cells. Forty-eight hours of transfection of 293T cells with 300 ng of NLδp6 was carried out, and the indicated quantities (in nanograms) of pGagδp6, pGagδp6-p6, pGagδp6-PTAP and/or pGagδp6-p6 (15-51) are indicated. RLU, relative light units. (C and D) The same information as that given for panel A applies, except that HOS (C) or TE671 (D) was transfected.

Previous observations have suggested that HIV-1 replication and particle release are partly defective when sequences C terminal to the PTAP motif are deleted from p6 (6, 9). Moreover, a PTAP motif cannot support particle budding in the context of an HIV-1-based artificial mini-Gag construct (24). These data and the findings depicted in Fig. 2B suggested that sequences outside the PTAP motif in HIV-1 p6 are required for efficient HIV-1 virion release. In principle, the additional (non-PTAP-encoding) p6 sequences could modulate PTAP function by influencing the structure or presentation of the PTAP motif. However, it has been previously shown that short PTAP-containing peptides and the entire p6 domain bind to Tsg101 with essentially identical affinity (8, 21). Alternatively, p6 sequences outside the PTAP motif might function independently, perhaps by recruiting additional cellular cofactors to facilitate HIV-1 budding. Nonetheless, Gagδp6-p6 (15-51), which contains all p6 sequences C terminal to the PTAP-containing peptide described above, was inactive and did not detectably increase virion formation when coexpressed with NLδp6 (Fig. 2B). Notably, however, when used in combination, Gagδp6-PTAP and Gagδp6-p6 (15-51) complemented NLδp6 efficiently and supported infectious virion formation almost as efficiently as the intact Gagδp6-p6 protein (Fig. 2B). These data indicate that the PTAP motif and p6 residues 15 to 51 can complement each other in trans to mediate the formation of infectious HIV-1 particles. This strongly suggests that p6 residues outside the PTAP motif do not accentuate HIV-1 budding by modulating PTAP structure but instead contribute an additional activity that synergizes with PTAP to induce HIV-1 virion morphogenesis. To confirm that the activity ascribed to p6 residues 15 to 51 was not simply a cell line-specific phenomenon, we performed experiments essentially identical to those depicted in Fig. 2A but used additional HIV-1-permissive cell lines. As is shown in Fig. 2B and C, a similar trans-complementing phenotype between PTAP and p6 (15-51) was observed in both HOS and TE671 cells. Similar results were also obtained with HeLa cells (data not shown).

We further examined Gag processing and particle production phenotypes in 293T cells by using the same complementation assay in conjunction with Western blot analysis. As shown in Fig. 3, cells expressing NLδp6 and Gagδp6 released undetectable levels of viral particles and analysis of cell-associated Gag revealed a profound processing defect with little mature p24 being present. A substantial reversion of this defect was observed when Gagδp6-p6 was coexpressed. Although significant levels of partly processed p25 (CA-p2) were observed in these cell lysates, this is likely due to a suboptimal level of p6 and Pol expressed in these cells: because this elaboration of the trans-complementation assay requires cotransfection with three expression plasmids, only approximately one-fifth of the total Gag expressed encodes p6 and about three-fifths of the normal level of Pol is expressed. Nonetheless, this analysis clearly demonstrates the ability of Gagδp6-p6 to enhance NLδp6Gag processing and particle release. Gagδp6-PTAP had marginal effects on Gag processing but did support particle release. However, while the total level of Gagδp6-PTAP-induced particle release was only slightly reduced (<2-fold) compared to that induced by Gagδp6-p6, approximately one-third of the particle-associated capsid protein was in an immature, p25CA-p2 form. This latter finding probably explains why Gagδp6-PTAP-complemented virions appear significantly less infectious than do Gagδp6-p6-complemented counterparts (Fig. 2A). The Gagδp6-p6 (15-51) fusion protein appeared inactive when present as the sole complementing construct; however, its expression clearly enhanced the ability of Gagδp6-PTAP to mediate fully processed virion formation. Indeed, the combination of Gagδp6-PTAP and Gagδp6-p6 (15-51) was as active as the intact Gagδp6-p6 protein (Fig. 3). Thus, these results are similar to those obtained by use of the infectivity assay of virion production (Fig. 2A) and confirm that the PTAP motif and p6 residues 15 to 51 act synergistically and in trans to mediate the formation of mature HIV-1 virions. A further Western analysis of cell lysates and virion pellets derived from similarly transfected HOS cells revealed results essentially identical to those obtained with 293T cells (data not shown).

FIG. 3.

Western blot analysis of the effect of PTAP and p6 (15-51) on Gag processing and particle release. 293T cells were transfected with 300 ng of pNLδp6 and the indicated complementing pGagδp6-fusion protein expression plasmids, as was done for Fig. 2. Forty-eight hours after transfection, cell lysates and pelleted extracellular virions (harvested by ultracentrifugation through a 20% sucrose cushion) were analyzed by Western blotting with an anti-p24CA monoclonal antibody. The number(s) below each lane indicates the levels of mature p24 CA protein and the incompletely processed p25 CA-p2 intermediate in virion pellets, quantitated by analysis of the blots with NIH Image. ND, not detectable.

We next used electron microscopy to visualize HIV-1 budding by NLδp6 virions complemented in trans by coexpression of either Gagδp6-PTAP or Gagδp6-p6. These experiments, shown in Fig. 4, revealed that pNLδp6-transfected cells synthesized immature virions that largely remained tethered to each other and to cells (Fig. 4A). This phenotype was expected but was somewhat more dramatic than has been previously reported for PTAP point mutants. Conversely, NLδp6 complemented with Gagδp6-p6 did not exhibit this dramatic L-domain-defective phenotype. Budding structures as well as immature, and occasionally mature, extracellular virions were observed (Fig. 4B). Complementation with Gagδp6-PTAP resulted in an apparently intermediate phenotype: while the late-budding defect appeared somewhat less severe than in the absence of a complementing Gagδp6-fusion protein, immature cell-associated virions were observed and mature extracellular virions were only very rarely observed (Fig. 4C). These results suggest that the L-domain activity exhibited by the PTAP motif is accentuated by the presence of additional p6 sequences.

FIG. 4.

Electron microscopy analysis of p6- and PTAP-mediated virion budding. 293T cells were transfected with pNLδp6 and pGagδp6 (A), pNLδp6 and pGagδp6-p6 (B), or pNLδp6 and pGagδp6-PTAP (C), in each case in a 3:2 molar ratio. Cells were harvested and processed for microscopy 48 h after transfection. Each panel shows an overview of part of a transfected cell (left) as well as a higher magnification of budding structures and virions (right).

Taken together, these findings suggest that HIV-1 encodes a bipartite L-domain whose function is only partly due to the PTAP-Tsg101 interaction. While previous studies have documented that the late-budding defect is more profound when all of p6 is deleted, compared to when only PTAP is abolished (6, 9), this study explicitly indicates that p6 encodes an additional activity that can act synergistically with the PTAP/Tsg101 interaction to mediate HIV-1 particle formation. While p6 residues 15 to 51 did not exhibit detectable L-domain activity when present alone, the likeliest explanation for these results is that p6 (15-51) contributes to the overall L-domain activity exhibited by HIV-1 p6. Clearly, these sequences were required for full expression of the PTAP-induced phenotype. At present we cannot exclude a postbudding effect of p6 (15-51) on virion maturation, but the electron microscopy analysis (Fig. 4) suggested that the PTAP motif is only partly effective in inducing HIV-1 particle release, and previous findings indicate that PTAP is insufficient and that an intact p6 is essential for particle release in an artificial mini-Gag context (24). Moreover, maturation defects in extracellular particles (Fig. 3) are likely to be consequent to partially defective L-domain function. Indeed, in some contexts L-domain defects can result in the formation of extracellular virions that remained tethered to each other and, consequently, immature (6). In addition, late-budding defects induced by p6 deletion or PTAP mutation result in reduced incorporation of pol gene products into virions and deleterious effects on maturation (7, 9). Because neither PTAP nor p6 (15-51) appears sufficient for full L-domain activity, these findings strongly suggest that HIV-1 encodes a bipartite L-domain. Importantly, however, we show that these two p6 domains are not required to be present in cis to mediate virion formation. Thus, it seems likely that the two components of the HIV-1 L-domain act independently of each other, and it is therefore unlikely that p6 (15-51) acts by influencing the structure of the Tsg101 binding site. Moreover, because Gagδp6-Tsg101 does not complement NLδp6, it is also unlikely that p6 (15-51) acts by facilitating Tsg101 recruitment. This effectively excludes the possibility that p6 ubiqutination and consequent enhancement of Tsg101 binding (8) are the underlying mechanism by which p6 (12-51) enhances HIV-1 particle formation. Indeed, it has been previously shown that lysine residues in p6 are completely dispensable for HIV-1 budding and replication (19). While it is possible that ubiquitination at alternative HIV-1 Gag sites might facilitate Tsg101 recruitment (8, 23), the presence of this proven L-domain cofactor at sites of particle assembly appears insufficient to account for the virion morphogenesis activity exhibited by HIV-1 p6. One possibility, currently under investigation, is that p6 (12-51) acts by recruiting distinct cellular cofactors, perhaps additional ESCRT components or other class E VPS factors that are known to mediate the budding of vesicles within cells (1, 2, 15, 22).