Abstract
Retroviruses are positive-sense, single-stranded RNA viruses that reverse transcribe their RNA genomes into double-stranded DNA for integration into the host cell chromosome. The integrated provirus is used as a template for the transcription of viral RNA. The full-length viral RNA can be used for the translation of the Gag and Gag-Pol structural proteins or as the genomic RNA (gRNA) for encapsidation into new virions by the Gag protein. The mechanism by which Gag selectively incorporates unspliced gRNA into virus particles is poorly understood. Although Gag was previously thought to localize exclusively to the cytoplasm and plasma membrane where particles are released, we found that the Gag protein of Rous sarcoma virus, an alpharetrovirus, undergoes transient nuclear trafficking. When the nuclear export signal of RSV Gag is mutated (Gag.L219A), the protein accumulates in discrete subnuclear foci reminiscent of nuclear bodies such as splicing speckles, paraspeckles, and PML bodies. In this report, we observed that RSV Gag.L219A foci appeared to be tethered in the nucleus, partially co-localizing with the splicing speckle components SC35 and SF2. Overexpression of SC35 increased the number of Gag.L219A nucleoplasmic foci, suggesting that SC35 may facilitate the formation of Gag foci. We previously reported that RSV Gag nuclear trafficking is required for efficient gRNA packaging. Together with the data presented herein, our findings raise the intriguing hypothesis that RSV Gag may co-opt splicing factors to localize near transcription sites. Because splicing occurs co-transcriptionally, we speculate that this mechanism could allow Gag to associate with unspliced viral RNA shortly after its transcription initiation in the nucleus, before the viral RNA can be spliced or exported from the nucleus as an mRNA template.
Highlights
Retroviruses are significant human and animal pathogens, causing cancer and immunodeficiency syndromes in a wide variety of species
Co-expression of Clk1 Enhances Co-localization of Gag.L219A with SC35 and SF2 Phosphorylation is a major mechanism for regulating the localization of SR proteins in the nucleus (Yeakley et al, 1999), we examined whether the degree of phosphorylation of splicing factors SC35 and SF2 influenced their association with Gag.L219A
Rous sarcoma virus (RSV) is unique among retroviral Gag proteins because the protein accumulates in the nucleus when nuclear export is blocked by treatment of cells with leptomycin B (LMB) and a CRM1-mediated nuclear export signal (NES) was identified in the p10 region of Gag (Scheifele et al, 2002, 2005)
Summary
Retroviruses are significant human and animal pathogens, causing cancer and immunodeficiency syndromes in a wide variety of species. Retroviruses are enveloped, positive-sense, single-stranded RNA viruses that package two copies of their genomes into virions. The retroviral genomic RNA (gRNA) undergoes reverse transcription to generate a complementary, double-stranded DNA that integrates into the host cell genome to form the provirus. The genome-length retroviral transcript may be spliced to create subgenomic mRNAs, which are exported from the nucleus to synthesize other viral proteins. The full-length RNA may remain unspliced with two potential outcomes: it may serve as the mRNA template for the translation of the Gag and Gag-Pol structural proteins or it may be bound by the Gag protein for packaging into new virions as the gRNA. The Gag protein selects the gRNA for encapsidation through a high-affinity interaction between the nucleocapsid (NC) domain of Gag and the psi ( ) packaging sequence in the 5′ untranslated region of the viral RNA (Shank and Linial, 1980; Aronoff and Linial, 1991; Aronoff et al, 1993; Berkowitz et al, 1995, 1996; Butsch and Boris-Lawrie, 2002; Lee et al, 2003; Lee and Linial, 2004; Zhou et al, 2005, 2007)
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