The current model of HIV-1 morphogenesis and release can be divided into multiple sequential and well-orchestrated steps. The MA domain mediates Gag interactions with the plasma membrane, the site of particle assembly. This step is followed by Gag-Gag multimerization into a spherical shell that requires the CA domain but also involves the NC domain by virtue of its ability to interact with RNA. The viral egress function was believed to be confined to the p6 region and carried out by the PTAP and LYPXnL L domain motifs, the two sequences endowed with the function of recruiting components of the ESCRT pathway that promote virus release. Although initially considered autonomous, L domain motifs are greatly weakened when moved away from their native location (38
) and cease to function when the NC domain of Gag is mutated or deleted (17
). These observations indicated that p6 does not function autonomously and suggested a functional interdependence between the p6 and NC domains in promoting virus release. Here we present evidence supporting a role for NC in HIV-1 release. Following mutations of basic residues in NC, fully assembled virus particles remained tethered to the plasma membrane, demonstrating a clear defect in virus abscission from the cell. Such phenotypes were seen for both 293T and T cells and were similar to budding defects observed for the HIV-1 PTAP−
mutant in 293T cells. As these NC mutant viruses retained all viral morphogenetic properties, including binding to the cell's membrane and RNA and the assembly of spherical particles, we concluded that NC is involved in virus budding, possibly by participating in the recruitment and/or the function of components of the ESCRT pathway, the host cell fission machinery members that promote HIV-1 release.
Previous studies reported that mutations of basic residues in NC result in the inhibition of HIV-1 production. Such a phenotype was attributed to various defects, including Gag's inability to interact with RNA (56
), to multimerize and form particles (17
), or to assemble stable virions (52
). These seemingly conflicting explanations reflected the difficulty in capturing the multiple and interconnected functions of the NC domain's basic residues in virus morphogenesis and release. In fact, mutation of basic residues in NC seems to have inhibited an aspect of HIV-1 morphogenesis other than assembly, since NC mutant Gag proteins were previously shown to retain the ability to multimerize (17
). Consistent with these observations, we found that Gag proteins carrying mutations of basic residues in either the N- or C-terminal regions of NC (RKI and RKII mutants) formed fully assembled spherical HIV-1 particles at the plasma membrane (Fig. and ), where they remained arrested at late stages of particle budding, suggesting a failure to release. NC mutant budding defects were ascertained by a careful examination of dozens of distinct fields and morphogenetic characterizations of 100 to 200 particles. These enumerations showed that NC mutants exhibited tethered budding particles in numbers comparable to those seen with the PTAP−
mutant in 293T cells and to those seen with the PTAP−
double mutant in T cells (Fig. and ), further supporting a budding defect phenotype.
The NC mutant Gag polyproteins (RKI and RKII) formed spherical particles at the membrane and coassembled with WT Gag proteins (Fig. ), further attesting to their ability to assemble virus. In fact, the proper particle assembly of these NC mutants was not surprising and is most likely the result of a combination of factors: (i) mutations introduced into NC were confined to either the N- or the C-terminal region, leaving half of the basic residues in NC available to engage in interactions with cellular RNAs that were reported to be specifically incorporated into HIV-1 particles (59
) (Fig. ) and can substitute for genomic RNA by serving as a scaffold for Gag-Gag multimerization (11
); (ii) NC mutant proteins retained an intact MA domain, in which basic residues can also bind RNA (53
) and indirectly favor Gag-Gag multimerization at the plasma membrane (15
); and (iii) NC mutants bound the plasma membrane with efficiencies comparable to that of the wild-type protein (Fig. ), an HIV-1 morphogenetic property that is known to compensate for defects caused by a disruption in the NC domain, by serving as a scaffold for Gag-Gag assembly (30
). By retaining these properties, NC mutants were able to efficiently multimerize, further excluding an assembly defect as a cause for their inability to release. NC mutants formed stable virus particles that tethered to the plasma membrane, thus exhibiting a clear release defect. Together, our findings present the first evidence that the NC domain of Gag plays a role in HIV-1 budding and release.
Both of the NC mutants RKI and RKII failed to release virus particles from 293T cells despite having an intact PTAP L domain motif. Because the majority of HIV-1 particle release from 293T cells is mediated through the PTAP motif, we concluded that an intact NC domain is critical for HIV-1 release through the PTAP/Tsg101 pathway. Remarkably, NC mutant viruses remained tethered to the plasma membrane with long membranous stalks that are morphologically similar to those seen following the disruption of the PTAP L domain motif (Fig. and ). In addition, the mutation of basic residues in NC or the PTAP L domain motif in p6 led to an intracellular Gag processing defect, as illustrated by the accumulation of the p25/24CA doublet, a hallmark of HIV-1 budding defects. These data indicate that the PTAP L domain motif in p6 requires the basic residues in its adjacent NC domain to facilitate virus release, thus establishing the first functional link between these two adjacent domains of Gag in virus budding.
NC mutants were arrested in late stages of virus budding and remained attached to the plasma membrane of 293T and T cells with long membranous stalks, indicating a failure to abscise their particles away from the cell. To achieve the latter, HIV-1 requires the recruitment of ESCRT-I through the Tsg101-PTAP motif interaction, members of ESCRT-III that promote membrane fission, and the activity of the VPS4 ATPase. Both NC mutant Gag proteins retained binding to Tsg101 (Fig. ), thereby excluding the disruption of Tsg101 recruitment as a cause for the failure of NC mutants to release virus. These findings suggested that mutations in NC impeded the ability of Gag to interact with a stable ESCRT-I complex and/or with the fission machinery factors that act downstream of ESCRT-I (i.e., ESCRT-III and VPS4). Alternatively, a mutation in NC could have prevented Gag from utilizing ESCRT members that were bound to its adjacent p6 domains. In either case, NC mutants remained arrested in late stages of virus budding at the cell surface, indicating improper release most likely due to the lack of ESCRT function. This notion was supported by the critical finding that the NC mutants’ release defects were remedied by providing Gag with an alternative access to the host's ESCRT pathway, through the overexpression of Nedd4.2s (Fig. ), an E3 ubiquitin ligase that links Gag to the cell's abscission complexes independently of both NC and p6 but in an ESCRT-dependent manner (16
). Such a rescue was specific for viruses that completed assembly, since Nedd4.2s failed to rescue an assembly-defective virus (DelNC), indicating that its activity can remedy only postassembly events and strongly suggesting that Nedd4.2s triggered the fission of the NC mutant particles from the cell's membrane. Since Nedd4.2s rescues virus release in a cellular ESCRT-dependent manner (16
) and is considered to act as an alternative link between Gag and the cellular ESCRT pathway to promote membrane fission (16
), we concluded that the NC domain is involved in postassembly events necessary for HIV-1 budding.
Other evidence in this study also supported a role for NC in virus budding and release. In fact, the NC mutants’ release defects were relieved when the mutants coexpressed with release-defective HIV-1 carrying a WT NC but lacking all L domain functions. This result not only indicated that NC mutants retained the ability to coassemble with WT NC-Gag proteins but also indicated that NC can act in trans as an “L domain-like” domain to promote virus budding and release. Interestingly, only a small amount of WT NC was needed to trigger virus release (12.5%) (Fig. ), suggesting that only a subset of NC proteins is involved in these late events of virus budding. One would thus envision that NC domains within Gag proteins localized at the budding neck would be engaged in a dynamic process with its adjacent p6 domain to promote late stages of virus abscission from the cell.
We previously reported that an interaction between the Bro1 domain of the ESCRT-associated Alix protein and NC is involved in the recruitment of the ESCRT-III member CHMP4 that is critical for virus release (20
), implicating NC in HIV-1 release mediated through the LYPXnL/Alix budding pathway. Here we showed evidence of a role for NC in HIV-1 release through the PTAP/Tsg101 pathway. The nature of the involvement of NC in this pathway is not clear. Evidence presented in this report, however, suggested that NC participates in the recruitment and/or utilization of ESCRT proteins and bears a cis
-acting “L domain-like” region that functions in concert with the PTAP L domain in p6. Further studies are warranted to elucidate the nature of the involvement of NC in these processes.