Retrovirus Assembly and Maturation

Division Of Basic Sciences - Nci

Investigators

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Abstract

As in previous years, we have used a combination of in vivo approaches with cultured cells and in vitro approaches with recombinant viral protein in our analysis of the molecular mechanisms underlying the assembly of retrovirus particles, including those of HIV-1. We originally reported a role for inositol hexakisphosphate (IP6) in HIV-1 assembly over 20 years ago (Campbell et al., PNAS 2001). We have extended this analysis using differential scanning fluorometry, which provides very quantitative information on particle assembly in vitro under different conditions. We found that the ability of IP6 to induce the Gag protein (the building block of retrovirus particles) to assemble into particles requires the presence of positive charges near the C-terminus of the protein; this requirement is normally fulfilled by the nucleocapsid (NC) domain. Presumably, the presence of these positive charges causes Gag molecules to coalesce around IP6, which has an extraordinarily high density of negative charges. IP6 also catalyzes the assembly of "pre-oligomerized" chimeric Gag molecules in which the NC domain has been replaced by a leucine-zipper dimerizing domain or an isoleucine-zipper trimerizing domain. These domains are not highly charged, but engage in high-affinity homotypic interactions. In still another case, the nucleocapsid domain is replaced by another trimerizing domain, taken from the bacteriophage "foldon" protein. All of our results support the general hypothesis that IP6 promotes assembly by causing Gag oligomers (especially trimers) to coalesce into hexamers, beginning at their C-termini. We also explored the role of IP6 in the replication of Moloney murine leukemia virus (MLV), a member of the gammaretrovirus genus. Unlike lentiviruses such as HIV-1, gammaretroviruses cannot infect non-dividing cells; this is because their DNA and integration machinery cannot penetrate the interphase nucleus, but only gain access to host DNA when the nuclear membrane breaks down during mitosis. We found that like HIV-1, MLV particles contain IP6 and that IP6 stabilizes the core of the mature virion, enabling it to copy the viral RNA into DNA. One notable difference between MLV and HIV-1 is that MLV DNA synthesis and infection is impaired if the target cells are deficient in IP5/6, while this is not true for HIV-1. This is probably because the MLV core partially disassembles in the cytoplasm of the newly infected cell and may depend on IP5/6 in the cytoplasm, while that of HIV-1 remains intact until it is inside the nucleus. We analyzed the effects of HIV-1 Gag protein upon the surface tension of aqueous solutions. Our data showed that Gag behaves as a surfactant, and proposed that this property contributes to the membrane curvature required for virus formation in vivo.