Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging

Abstract

The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and transacting elements within the Gag polyprotein. The packaging signal , at the 5 end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.The encapsidation of the RNA genome is essential for retrovirus replication. Because the viral genomic RNA (gRNA) constitutes only a small fraction of the total cellular mRNA, a specific Gag-RNA interaction is thought to be required for viral genome packaging (2). The determinants of virus-specific gRNA incorporation include the cis-acting element at the 5Јend of the viral gRNA, known as the packaging signal (), and the nucleocapsid (NC) domain of the Gag polyprotein (3, 14, 62). In Rous sarcoma virus (RSV), the NC domain contains basic residues that are required for the recognition of and binding to , as well as two Cys-His motifs that maintain the overall conformation of NC and are essential for RNA packaging (30, 31).Packaging of gRNA into progeny virions requires that the unspliced viral mRNA be exported from the nucleus. However, cellular proofreading mechanisms ensure that unspliced or intron-containing mRNAs are retained in the nucleus until splicing occurs. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) overcome this export block of unspliced genomes by encoding the Rev protein, which interacts with a cis-acting sequence in the viral RNA (the Revresponsive element [RRE]) to facilitate cytoplasmic accumulation of intron-containing viral mRNA (16,35). The export of the Rev-viral RNA complex is mediated through the interaction of a leucine-rich nuclear export signal (NES) in Rev with the CRM1 nuclear export factor (17,18,37,41). Simple retroviruses do no...

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

“…Parent and colleagues have shown in a series of studies that Rous sarcoma virus (RSV) Gag shuttles (6,17,20,(45)(46)(47), and experiments with RSV Gag mutants that do not undergo nuclear cycling provided evidence that this is needed for efficient gRNA encapsidation (17,45). RSV Gag nuclear import is mediated by NLSs in MA and NC (45).…”

“…(K to Q) Subcellular localization of FIV MA-CFP, CA-CFP, and NCp2-CFP. Transfected HeLa cells were treated or not with LMB for 3 h. The nuclear/total MFI (Q) was determined as described above for panel J for 158 cells (6,17,25,23,15,15,21,16,11, and 9 cells for columns from left to right, respectively). Images are representative of at least three independent experiments.…”

“…The protein is translated on free polysomes and targeted to the plasma membrane, where virus particles are assembled (16). A central question is whether Gag and the viral genomic RNA (gRNA) associate at the plasma membrane or earlier during assembly and, if so, whether this occurs in the cytosol; in association with organelles, e.g., cotranslationally; or even in the nucleus, as was described previously for an alpharetrovirus (17). Human immunodeficiency virus type 1 (HIV-1) gRNAs become anchored at the plasma membrane before particle assembly is detectable, but it is not clear whether the Gag-gRNA complexes of this and other lentiviruses first form in the cytoplasm and then transit to the plasma membrane or the gRNA traffics there independently (26,27).…”

T-body formation precedes virus-like particle maturation inS. cerevisiae

“…19,20 This suggests that Gag-mediated T-body formation may play a central role in the initial assembly of VLPs. First, Ty1 features required for VLP-and T-body-formation are similar.…”

Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA

“…RSV Gag is synthesized on cytosolic ribosomes, transiently traffics through the nucleus, and then travels to the plasma membrane, where complexes of Gag bound to the viral RNA (vRNA) genome form virus particles that emerge from the cell via budding (6,7). Nuclear trafficking of RSV Gag is necessary for efficient incorporation of vRNA into nascent particles, suggesting that Gag might bind its genome in the nucleus (8). This model represents a unique paradigm for how retroviruses package their genomes.…”