The integrated provirus is used as a template for the cellular RNA polymerase II to direct the synthesis of retroviral RNA. Following viral entry, 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. Retroviruses are enveloped, positive-sense, single-stranded RNA viruses that package two copies of their genomes into virions. The first retrovirus discovered, the avian alpharetrovirus Rous sarcoma virus (RSV), has proven to be among the most valuable, launching challenges to existing dogmas that led to the discovery of reverse transcription and cellular oncogenes (reviewed in Parent, 2012). Many retroviruses that infect animals have served as important model systems for unraveling the mechanisms of retroviral replication, pathogenesis, and host defense. Most well-known is the human immunodeficiency virus type 1 (HIV-1), the etiological agent of acquired immunodeficiency syndrome (AIDS). Retroviruses are significant human and animal pathogens, causing cancer and immunodeficiency syndromes in a wide variety of species. 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. 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. We previously reported that RSV Gag nuclear trafficking is required for efficient gRNA packaging. Overexpression of SC35 increased the number of Gag.L219A nucleoplasmic foci, suggesting that SC35 may facilitate the formation of Gag foci. 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. 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. 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. The mechanism by which Gag selectively incorporates unspliced gRNA into virus particles is poorly understood. 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 integrated provirus is used as a template for the transcription of viral RNA. 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. 2Department of Microbiology and Immunology, Penn State College of Medicine, Hershey, PA, USA.1Division of Infectious Diseases and Epidemiology, Department of Medicine, Penn State College of Medicine, Hershey, PA, USA.