In this study, the ability of HIV-1 Gag to associate with different viral glycoproteins within the same cell was examined. It was expected that a producer cell containing two viral spikes could give rise to virions containing both virions (). Though the producer cell was able to generate viral particles that incorporated each viral spike, these glycoproteins segregated to distinct lipid raft-like microdomains and ultimately to separate pseudotyped lentiviral vectors (–). This finding suggests that a single Gag particle associates with one lipid raft that is homogeneous with respect to trimeric viral envelope. This observation was made for both Ebola and HIV glycoproteins, and we have observed it between HIV-1 Env and other viral glycoproteins as well, including HIV-1 Env and SARS, HIV-1 Env and FeLV, and HIV-1 and MuLV (data not shown). Previous studies have demonstrated that Envs derived from closely related lentiviruses, such as HIV-1 variants or HIV-2 and SIV, can form mixed heterotrimers (Boulay et al., 1988
; Doms et al., 1990
; Yang et al., 2005a
; Yang et al., 2005b
). Such heterotrimers would be expected to co-locate to the same lipid raft, as confirmed here (, left panels) but viral spikes from different glycoproteins did not co-localize to the same lipid rafts (, right panel). These observations suggest that this association is dependent on the coiled-coil region of the viral spike, which may also contribute to the formation of trimeric oligomers within the lipid raft. While each raft is homogeneous with respect to the viral spike, other host membrane glycoproteins are not likely excluded from the raft because many such cellular proteins are incorporated into mature virus particles (Arthur et al., 1992
Models of segregation of viral spikes with lentiviral ribonucleoprotein complexes
The assembly of virus particles requires packaging of viral proteins and RNA. For enveloped viruses like HIV and Ebola, assembly and budding occurs at the plasma membrane of infected cells (Cimarelli and Darlix, 2002
; Maruyama et al., 1999
; Hartlieb and Weissenhorn, 2006
) and possibly through endosomal membranes (Martin-Serrano et al., 2001
; Ono and Freed, 2004
; Dong et al., 2005
; Grigorov et al., 2006
), though virus located at this latter site could arise by endocytosis of recently released virus (Jouvenet et al., 2006
). In HIV-infected T-cells, newly synthesized Env transits from the ER to the Golgi, where nascent N-linked polysaccharide side chains are modified, and the protein traffics to the cell surface. Previous studies have shown that HIV-1, and possibly Ebola virus, interacts with Tsg101, an endosomal protein sorting factor, as well as other members of the ESCRT pathway (reviewed in (Goff et al., 2003
; Martin-Serrano et al., 2001
)) to facilitate budding of the assembled virus from cells. Lipid rafts have been estimated at ~100–200 nm in diameter (Pike, 2003
), while an HIV virion is ~125 nm (Briggs et al., 2006
). These sizes, along with the finding that virions packaged in cells contain only one of the two expressed viral glycoproteins, are consistent with a model in which a virion arises from a single lipid raft domain that associates with an assembled Gag particle.
The replication-defective pseudotyped lentiviral vectors examined here facilitate the analysis of the molecular mechanisms of this process. It was generally known that pseudotyped viral particles could be made with the core of one virus surrounded by heterologous envelope proteins, but the segregation of different spikes to distinct viral particles has not been previously appreciated, and this finding was unexpected. Confocal microscopy revealed distinct subcellular distribution of Env, GP, and Gag, though at the plasma membrane, Env and GP co-localized separately with Gag at the presumed site of budding. Furthermore, Gag expression did not alter the subcellular distribution of envelope proteins by confocal imaging analysis (, Fig. S2
). A similar distribution of Env or GP was observed intracellularly in cells co-transfected with or without HIV-1 Gag. The association of a single viral spike with each particle suggests that the viral ribonucleoprotein complex associates with each lipid raft. This finding could arise if Gag oligomerization occurred prior to its association with an individual raft. Alternatively, the interaction of Gag with its relevant viral glycoproteins on a lipid raft may recruit additional Gag molecules to complete the formation of a single viral particle.
A recent paper by Brass et al. (Brass et al., 2008
) employed small interfering RNA screening to identify more than 250 host proteins implicated in HIV replication. Among them, it is interesting to note that Caveolin-2 (Cav-2) is a lipid raft associated host protein previously shown to inhibit HIV virion production more than 90% in 293T cells (Llano et al., 2002
). This observation raises the possibility that this caveolin-related pathway may be involved in the segregation of viral spikes to distinct particles. Elucidation of such mechanisms will provide further understanding of this process and possible targets for antagonists of HIV replication.
Concurrent viral infections are well documented to occur in nature, and the ability of viral spike proteins to associate with their cognate viral ribonucleoprotein complex is critical to the generation of replication-competent viruses. The inclusion of the appropriate glycoproteins and exclusion of heterologous envelope proteins would, in fact, be crucial for the survival of a virus species. Here, the analysis of two envelopes expressed in a producer cell also served to define the mechanism of viral assembly further. The quantal nature of the spike association with Gag provides fundamental insight into the nature of HIV-1 assembly and suggests that the virus exploits normal cellular substructures and pathways of protein trafficking to allow its disparate components to interact selectively and to complete assembly. Knowledge of this mechanism also facilitates the development of antagonists of HIV-1 that could inhibit productive viral replication and lends insight into strategies for AIDS vaccine design.