Cellular ribonucleoprotein complexes are generated during transcription, and undergo successive rounds of remodeling (reviewed in ), with their changing protein components dictating their changing fates (reviewed in ). 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 Albendazole largely in complexes 30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first Albendazole 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 made up of 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 as sites of Gag colocalization with ABCE1 and DDX6; Albendazole moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two actions that lead to association of assembling Gag with unspliced HIV-1 RNA. Impartial of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1 1). If a viral-RNA-binding domain name is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2 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 made up of unspliced HIV-1 RNA. Author summary During HIV-1 immature capsid assembly, packaging of the viral genome is initiated when the HIV-1 capsid protein, Gag, first Albendazole associates with unspliced HIV-1 RNA. Although the complex in which this association initially occurs is critical for formation of infectious virus, the identity, composition, and the mechanism by which this complex forms remain unknown. To address this question, we utilized a previously described temporal pathway of intermediates in HIV-1 immature capsid assembly. The late intermediates in this pathway are derived from host RNA granules, which are diverse complexes utilized for cellular RNA storage and degradation. Here we sought to identify the intracellular capsid assembly intermediate in which HIV-1 Gag initially associates with unspliced HIV-1 RNA. We failed to detect an association between the first assembly intermediate, which contains soluble Gag, and unspliced HIV-1 RNA. Instead, the association between Gag and unspliced HIV-1 RNA was observed only in complexes corresponding to the RNA-granule-derived assembly intermediates. We also showed that Gag uses two determinants to form RNA-granule-derived intermediates that contain unspliced HIV-1 RNA. Together, 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. Introduction For released HIV-1 particles to be infectious, they must contain two copies of unspliced (full-length) 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 usually completed. Packaging of the viral genome is initiated when Gag first associates with unspliced viral RNA during assembly, and requires the nucleocapsid domain name (NC) of Gag as well as specific encapsidation signals in unspliced HIV-1 RNA (reviewed in ). Immature capsids subsequently undergo budding, resulting in release of immature virus particles that contain the encapsidated genome and undergo maturation (reviewed in ). In the absence of unspliced HIV-1 RNA, Gag proteins assemble and release properly but the resulting virus-like particles are non-infectious . In addition to being packaged, unspliced HIV-1 RNA is used for translation of Gag and GagPol (reviewed in ). It is generally agreed that translation and packaging are unlikely to occur concurrently, given that translation requires Albendazole melting of secondary structures that are Mouse monoclonal to V5 Tag utilized during packaging; therefore translation and packaging are likely to be mutually exclusive (reviewed in [4,5]). However, the determinants that govern whether an unspliced HIV-1 RNA is usually utilized for translation or for packaging remain unclear. Mechanisms that have been proposed to explain how an unspliced HIV-1 RNA is usually directed towards packaging instead of translation include alternate RNA conformations that mask the translation start site and expose elements that favor packaging (reviewed in [5,6]); alternate 5′ mRNA cap.