A longstanding unknown in viral RNA biology is the relationship between translation and packaging of genomic RNA. For retroviruses, an extensive body of work has characterized nuclear export of the unspliced genome-length transcript (5,8,15), but the cytoplasmic trafficking of the RNA has remained relatively undefined. An elegant experimental approach that was initiated over 25 years ago has been updated and extended to human retroviruses during the last year. A consensus on the relationship between translation and packaging of retroviral RNA has been reached. An unexpected finding was that retroviruses have adapted two divergent approaches to manage the cytoplasmic fate of genomic RNA. This minireview introduces the interdependent relationship between translation and packaging of retroviral RNA, postulates models of retroviral RNA trafficking in the cytoplasm, summarizes experimental results that address the models, and discusses the recent consensus.Unspliced genome-length retroviral RNA is utilized for both translation and packaging. Retroviruses are a unique family of RNA viruses that utilize virally encoded reverse transcriptase (RT) to replicate genomic RNA through a proviral DNA intermediate (40). The provirus is permanently integrated into the host cell chromosome and, like a cellular gene, is expressed by the host cell transcription, RNA processing, and translation machinery. The primary transcription product interacts with the cellular RNA processing machinery and, similar to a typical cellular pre-mRNA, is spliced, exported to the cytoplasm, and translated by the host protein synthesis machinery. A proportion of the pre-mRNA subverts typical RNA processing and interacts with viral and/or cellular nucleocytoplasmic shuttle proteins that activate nuclear export, despite the lack of intron removal (8). In the cytoplasm, the unspliced genome-length

The retroviral primary transcription product is a multifunctional RNA that is utilized as pre-mRNA, mRNA, and genomic RNA. The relationship between human immunodeficiency virus type 1 (HIV-1) unspliced transcripts used as mRNA for viral protein synthesis and as virion precursor RNA (vpRNA) for encapsidation remains an important question. We developed a biochemical assay to evaluate the hypothesis that prior utilization as mRNA template for protein synthesis is necessary to generate vpRNA. HIV-1-infected T cells were treated with translation inhibitors under conditions that maintain virus production. Immunoprecipitation of newly synthesized HIV-1 Gag protein revealed that de novo translation is not necessary to sustain assembly, release, or processing of Gag structural protein. Both newly synthesized protein and steady-state Gag are competent for assembly, and the extracellular accumulation of Gag is proportional to the intracellular abundance of Gag. As early as 2 h after transcription, newly synthesized RNA is detectable in cell-free virions and encapsidation is sustained upon inhibition of host cell translation. Results of both [ 3 H]uridine incorporation assays and HIV-1-specific RNase protection assays (RPAs) indicate that translation inhibition reduces the absolute amounts of both cytoplasmic and virion-associated RNA. Evaluation of encapsidation efficiency by RPA revealed that the cytoplasmic availability of vpRNA is increased, indicating that HIV-1 unspliced mRNA can be rerouted to function as vpRNA. Our data contrast with results from the HIV-2 and murine leukemia virus systems and indicate that HIV-1 unspliced RNA constitutes a single functional pool that can function interchangeably as mRNA and as vpRNA.

Previous work has shown that spleen necrosis virus (SNV) long terminal repeats (LTRs) are associated with Rex/Rex-responsive element-independent expression of bovine leukemia virus RNA and supports the hypothesis that SNV RNA contains a cis-acting element that interacts with cellular Rex-like proteins. To test this hypothesis, the human immunodeficiency virus type 1 (HIV) Rev/RRE-dependent gag gene was used as a reporter to analyze various SNV sequences. Gag enzyme-linked immunosorbent assay and Western blot analyses reveal that HIV Gag production is enhanced at least 20,000-fold by the 5′ SNV LTR in COS, D17, and 293 cells. Furthermore, SNV RU5 in the sense but not the antisense orientation is sufficient to confer Rev/RRE-independent expression onto a cytomegalovirus-gag plasmid. In contrast, the SNV 3′ LTR and 3′ untranslated sequence between env and the LTR did not support Rev-independent gag expression. Quantitative RNase protection assays indicate that the SNV 5′ RNA terminus enhances cytoplasmic accumulation and polysome association of HIV unspliced and spliced transcripts. However, comparison of the absolute amounts of polysomal RNA indicates that polysome association is not sufficient to account for the significant increase in Gag production by the SNV sequences. Our analysis reveals that the SNV 5′ RNA terminus contains a unique cis-acting posttranscriptional control element that interacts with hypothetical cellular Rev-like proteins to facilitate HIV RNA transport and efficient translation.