Abstract
During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.
Highlights
For released HIV-1 particles to be infectious, they must contain two copies of unspliced HIV-1 RNA that are packaged during assembly of the immature HIV-1 capsid
These studies support a novel model for HIV-1 genome packaging, in which the first association between HIV-1 Gag and unspliced HIV-1 RNA occurs within a host RNA granule
Confirmation of phenotypes for proviruses expressing WT Gag and Gag mutants To study the association of HIV-1 Gag with unspliced HIV-1 RNA in HIV-1 capsid assembly intermediates, we used a variety of previously published HIV-1 expression systems (Fig 1A, sets I—IV); these produce WT Gag or Gag mutants with known phenotypes for production of viruslike particles (VLPs) that either do or do not contain the genome (Fig 1B)
Summary
For released HIV-1 particles to be infectious, they must contain two copies of unspliced (fulllength) HIV-1 RNA that are packaged during assembly of the immature HIV-1 capsid. Each immature capsid is composed of ~3000 copies of the HIV-1 structural protein Gag, which initially oligomerize in the cytoplasm and subsequently target to the plasma membrane (PM), where Gag multimerization is completed. Packaging of the viral genome is initiated when Gag first associates with unspliced viral RNA during assembly, and requires the nucleocapsid domain (NC) of Gag as well as specific encapsidation signals in unspliced HIV-1 RNA (reviewed in [1]). In the absence of unspliced HIV-1 RNA, Gag proteins assemble and release properly but the resulting virus-like particles are non-infectious [3]. In aggregate, the data argue that non-translating unspliced HIV-1 RNA undergoes packaging, and that one or more regulatory mechanisms play a role in determining whether a particular unspliced HIV-1 RNA is translated or packaged
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