A Murine Retrovirus Co-Opts YB-1, a Translational Regulator and Stress Granule-Associated Protein, To Facilitate Virus Assembly

Abstract

The Gag protein of the murine retrovirus mouse mammary tumor virus (MMTV) orchestrates the assembly of immature virus particles in the cytoplasm which are subsequently transported to the plasma membrane for release from the cell. The morphogenetic pathway of MMTV assembly is similar to that of Saccharomyces cerevisiae retrotransposons Ty1 and Ty3, which assemble virus-like particles (VLPs) in intracytoplasmic ribonucleoprotein (RNP) complexes. Assembly of Ty1 and Ty3 VLPs depends upon cellular mRNA processing factors, prompting us to examine whether MMTV utilizes a similar set of host proteins to facilitate viral capsid assembly. Our data revealed that MMTV Gag colocalized with YB-1, a translational regulator found in stress granules and P bodies, in intracytoplasmic foci. The association of MMTV Gag and YB-1 in cytoplasmic granules was not disrupted by cycloheximide treatment, suggesting that these sites were not typical stress granules. However, the association of MMTV Gag and YB-1 was RNA dependent, and an MMTV RNA reporter construct colocalized with Gag and YB-1 in cytoplasmic RNP complexes. Knockdown of YB-1 resulted in a significant decrease in MMTV particle production, indicating that YB-1 plays a role in MMTV capsid formation. Analysis by live-cell imaging with fluorescence recovery after photobleaching (FRAP) revealed that the population of Gag proteins localized within YB-1 complexes was relatively immobile, suggesting that Gag forms stable complexes in association with YB-1. Together, our data imply that the formation of intracytoplasmic Gag-RNA complexes is facilitated by YB-1, which promotes MMTV virus assembly. IMPORTANCECellular mRNA processing factors regulate the posttranscriptional fates of mRNAs, affecting localization and utilization of mRNAs under normal conditions and in response to stress. RNA viruses such as retroviruses interact with cellular mRNA processing factors that accumulate in ribonucleoprotein complexes known as P bodies and stress granules. This report shows for the first time that mouse mammary tumor virus (MMTV), a mammalian retrovirus that assembles intracytoplasmic virus particles, commandeers the cellular factor YB-1, a key regulator of translation involved in the cellular stress response. YB-1 is essential for the efficient production of MMTV particles, a process directed by the viral Gag protein. We found that Gag and YB-1 localize together in cytoplasmic granules. Functional studies of Gag/YB-1 granules suggest that they may be sites where virus particles assemble. These studies provide significant insights into the interplay between mRNA processing factors and retroviruses.

Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction

“…Interestingly, another cold shock domain containing protein, Lin28, binds pre-miRNAs of the Let7 family via hairpin-loop structures (Nam, Chen, Gregory, Chou, & Sliz, 2011). YBX1 also binds hairpin-loops in a murine retrovirus, leading to stabilization of the viral RNA genome and increased particle production (Bann, Beyer, & Parent, 2014). YBX1 binding of viral RNA also increases production of other retroviruses, including HIV (Bann et al, 2014;W.…”

“…YBX1 also binds hairpin-loops in a murine retrovirus, leading to stabilization of the viral RNA genome and increased particle production (Bann, Beyer, & Parent, 2014). YBX1 binding of viral RNA also increases production of other retroviruses, including HIV (Bann et al, 2014;W. Li, Wang, & Gao, 2012;Mu, Li, Wang, & Gao, 2013).…”

HIV-1 RNA genomes initiate host protein packaging in the cytosol independently of Gag capsid proteins

“…In this context, we speculate that all successful retroviruses and endogenous elements must adapt to subvert, suppress, or avoid PB- or SG-associated activities. Indeed, HIV-1 42,43 , other retroviruses 44,45 , and endogenous retroelements including LINEs 46 and yeast Ty elements 47 are thought to exploit non-membranous RNP complexes related to PBs/SGs in order to compartmentalize activities including mRNA translation and genome packaging. Regarding suppression, intriguing recent work from Mouland and colleagues has shown HIV-1 to actively inhibit SG formation, proposed to reduce the potential for stress signaling triggered by Gag NC interacting with host RNA species 33,34 .…”

Pericentriolar Targeting of the Mouse Mammary Tumor Virus GAG Protein

“…A recent report proposes that Gag co-localizes with the ribosomal protein L9 in a subset of MMTV-infected cells suggesting that nucleolar localization maybe required for virion assembly [ 25 ]. In the cytoplasm, MMTV Gag co-localizes with viral RNA and YB-1, a translational regulator associated with P bodies and stress granules [ 26 ]. Notably, YB-1 plays an important role in centriolar and centrosome maturation [ 27 ] and its knockdown results in diminished MMTV particle production [ 26 ].…”