Abstract
Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC) assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV) post-transcriptional regulatory element (PRE) mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA) with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.
Highlights
Retrovirus assembly and budding is a highly concerted process mediated by largely undefined spatially- and temporally-regulated interactions between viral proteins and cellular factors
We proposed that the observed distinct Gag assembly patterns could be the result of differential intracellular Gag trafficking as a consequence of the different pathways used for the export of HIV-1 Gag mRNA from the nucleus
Since the bimolecular fluorescence complementation (BiFC) assays used in our previous study only revealed assembled Gag multimers inside cells, in the current study we first visualized the distribution of the total population of Revdependent or posttranscriptional regulatory element (PRE)-dependent HIV-1 Gag-GFP in 293T cells over time using live cell imaging
Summary
Retrovirus assembly and budding is a highly concerted process mediated by largely undefined spatially- and temporally-regulated interactions between viral proteins and cellular factors. During the viral assembly process, thousands of copies of viral structural polyproteins multimerize to form virus particles via an energydependent, multi-step process. The Gag polyprotein consists of matrix (MA), capsid (CA), nucleocapsid (NC), late domain, and spacer proteins and is cleaved into the distinct structural proteins upon virus maturation [1,2]. These Gag domains orchestrate the major steps in virus assembly and budding (reviews [1,2]). Consistent with results published by Malim and colleagues [12,13], our recent work suggests that HIV-1 Gag assembly is regulated at a step as early as nuclear export of its encoding mRNA [14]
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