Translation is not required to generate virion precursor RNA in human immunodeficiency virus type 1-infected T cells

The retroviral primary transcription product is a multifunctional RNA that is utilized as pre-mRNA, mRNA, and genomic RNA. The relationship between hu man immunodeficiency virus type 1 (HIV-1) unspliced transcripts used as mRN A for viral protein synthesis and as virion precursor RNA (vpRNA) for encap sidation remains an important question. We developed a biochemical assay to evaluate the hypothesis that prior utilization as mRNA template for protei n synthesis is necessary to generate vpRNA. HIV-1-infected T cells were tre ated with translation inhibitors under conditions that maintain virus produ ction. 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 st eady-state Gag are competent for assembly, and the extracellular accumulati on of Gag is proportional to the intracellular abundance of Gag. As early a s 2 h after transcription, newly synthesized RNA is detectable in cell-free virions and encapsidation is sustained upon inhibition of host cell transl ation. Results of both [H-3]uridine incorporation assays and HIV-1-specific RNase protection assays (RPAs) indicate that translation inhibition reduce s the absolute amounts of both cytoplasmic and virion-associated RNA. Evalu ation of encapsidation efficiency by RPA revealed that the cytoplasmic avai lability 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 m RNA and as vpRNA.