Depending on the producer cell type examined, HIV-1 p6 mutants with defective PTAP L domains exhibit severe defects in either virion-cell or virion-virion detachment (16
). In adherent cell lines and in primary human macrophages, the defect at the level of virion-cell detachment predominates, resulting in inefficient virus release (16
). It was therefore surprising that certain chimeric HIV-1 Gag constructs did not require p6 for the efficient production of VLP in adherent cells (1
). In these Gag constructs, HIV-1 NC was replaced by foreign protein-protein interaction domains to substitute for its assembly function. Remarkably, this led to near wild-type levels of particle production even in cases where the entire NC-p1-p6 region was replaced and all consensus L domain motifs in HIV-1 Gag were therefore eliminated (1
One possible explanation for these observations was that the NC-p1-p6 region of HIV-1 Gag has an inhibitory effect on virus release that is counteracted by the L domain function of p6, and the results of the present study support this model. First, we found that replacing NC-p1-p6 with a heterologous dimerization domain in a Gag construct termed ZWT
caused resistance to dominant-negative CHMP3 or Vps4 under conditions where particle production by WT HIV-1 Gag was nearly abolished. We note that in a previous study, dominant-negative Vps4 had no effect on p6-deficient HIV-1 lacking active PR (19
). However, in our hands p6-deficient HIV-1 exhibited significant defects in particle formation even after PR was inactivated, whereas the ZWT
Gag construct produced nearly wild-type levels of particles in our study (1
). Since dominant-negative versions of the CHMPs or of Vps4 block the function of all known L domains, our observations argue against the use of an alternative L domain by ZWT
Gag that would explain its efficient release despite the absence of p6.
In principle, the apparent L domain independence of the chimeric ZWT Gag construct could have been caused by the presence of a foreign leucine zipper domain rather than by the absence of C-terminal HIV-1 Gag sequences. However, L domain dependence was restored in a Gag construct that harbored both the leucine zipper domain and NC-p1-p6, since efficient particle production by this construct depended on the Tsg101 binding site in p6. Moreover, particle production was substantially inhibited when NC-p1 rather than NC-p1-p6 was added back to ZWT. Together, these results suggested that the presence or absence of NC-p1 determined whether an L domain was needed to promote particle release.
Our data suggest that the p6 domain itself does not confer a requirement for an L domain, since the Tsg101 binding site within p6 was not required for efficient particle production by HIV-1 Gag when NC-p1 was replaced by a leucine zipper. Furthermore, particle production by this p6-containing construct was largely resistant to CHMP3-RFP, indicating that ESCRT-mediated membrane scission was not required. Additionally, the finding that p6 by itself did not confer sensitivity to CHMP3-RFP suggested that dominant-negative ESCRT pathway components do not simply arrest budding by tethering Gag to aberrant ESCRT complexes via the Tsg101 and ALIX binding sites in p6.
Remarkably, replacing only the NC domain by a leucine zipper had effects very different from replacing NC-p1. In the former case, the Tsg101 binding site in p6 became crucial for efficient particle production, and CHMP3-RFP had a profound inhibitory effect on particle release. Thus, the presence of p1-p6 but not of p6 alone at the C terminus of the leucine zipper sequence conferred both L domain dependence and ESCRT pathway dependence that resembled that exhibited by authentic HIV-1 Gag. Although the mechanism by which p1 contributed to this phenotype remains unknown, it does not appear to involve the role of the p1 coding sequence in ribosomal frameshifting, because the slippery sequence at which frameshifting occurs (30
) was not required to confer sensitivity to CHMP3-RFP (data not shown). We cannot exclude the possibility that the presence of p1 positioned the p6 domain in a manner that allowed the entrapment of Gag by aberrant ESCRT polymers induced by CHMP3-RFP. However, we consider it more likely that p1 conferred a bona fide requirement for a functional ESCRT pathway in the chimeric Gag context, since in the presence of p1 the Tsg101 binding site in p6 became critical for particle production.
A recent three-dimensional analysis of immature HIV-1 particles and of HIV-1 budding sites indicated that ESCRT-III assists Gag in the membrane bending required to complete the budding process (9
). Consistent with this model, upon overexpression certain ESCRT-III components have been shown to assemble into circular arrays that bend the plasma membrane into buds that emerge from the cell surface (25
). It is possible that certain Gag-leucine zipper chimeras do not need the assistance of ESCRT-III because the zipper domain improves the ability of Gag to bend membranes on its own. According to this scenario, the fusion of NC-p1 or of p1-p6 to the zipper domain might simply compromise this ability and thus restore ESCRT dependence. However, we also observed that the sensitivity of authentic HIV-1 Gag to inhibition of the ESCRT pathway could be reduced by certain deletions in the NC-p1 region. In the absence of ESCRT pathway inhibition, these deletions reduced the efficiency of Gag release, which argues against the possibility that ESCRT pathway independence was merely a consequence of an improved ability to assemble into a lattice capable of bending membranes.
Particle production by authentic HIV-1 Gag became only moderately less sensitive to inhibition of the ESCRT pathway when specific NC residues crucial for the encapsidation of the viral genomic RNA were mutated. However, it was previously demonstrated that cellular mRNA can substitute for viral genomic RNA during retroviral assembly (45
). In addition to its ability to specifically recognize genomic viral RNA, NC exhibits a nonspecific RNA binding activity that is critical for its function in particle assembly (5
). This nonspecific RNA binding activity depends on clusters of basic residues but not on the integrity of the zinc fingers in NC. Notably, it has been shown that the CC28,49
SS and FW16,37
AA mutations have only a minor effect on the ability of NC to bind RNA in vitro
, despite their drastic effects on the packaging of viral genomic RNA (13
). It is thus likely that the CC28,49
SS and FW16,37
AA mutant particles packaged cellular RNA rather than viral RNA. Therefore, our results do not exclude the possibility that the role of NC in the ESCRT pathway dependence of budding is related to its function in RNA encapsidation. We note that particles produced by the ZWT
Gag construct, which are released in a fully ESCRT-independent manner (this study), contain no detectable cellular RNA and are therefore thought to assemble without incorporating any form of nucleic acid (14
In the authentic Gag context, deleting p1 alone had little effect on the inhibition of particle production by dominant-negative CHMP3. Rather, it was necessary to additionally delete a portion of NC to mitigate the effect of CHMP3-RFP. On the other hand, p1 conferred sensitivity to dominant-negative CHMP3 in a chimeric Gag context lacking NC. Together, these observations suggest that NC and p1 can independently contribute to a requirement for ESCRT pathway engagement for optimal particle production. An understanding of how NC and p1 confer such a requirement may provide further insights into why retroviruses use L domains, given that efficient particle release can in principle be achieved in an ESCRT pathway-independent manner.