Translation Is Not Required To Generate Virion Precursor RNA in Human Immunodeficiency Virus Type 1-Infected T Cells

Abstract

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.

Novel Staufen1 ribonucleoproteins prevent formation of stress granules but favour encapsidation of HIV-1 genomic RNA

“…The interaction between host proteins and vRNA might also provide a molecular switch by suppressing vRNA translation to favour encapsidation (Cimarelli and Luban, 1999). There is still some controversy about whether this type of mechanism exists, because HIV-1 vRNA that is translated might also be encapsidated (Butsch and Boris-Lawrie, 2000;Kaye and Lever, 1999;Poon et al, 2002). Many questions remain concerning how HIV-1 co-opts host protein function and machineries to traffic and encapsidate its RNA genome.…”

Retrovirus Translation Initiation: Issues and Hypotheses Derived from Study of HIV-1

“…Lack of translation is not a prerequisite to qualify HIV-1 unspliced RNA for packaging into progeny virions [16]. Instead, unspliced HIV-1 RNA functions interchangeably as an mRNA template for translation and as genomic RNA that is packaged [16]. A similar conclusion was reached for Rous sarcoma virus (RSV) in a study that evaluated the relationship between translation-dependent nonsense mediated decay and RNA packaging [48].…”

“…In distinction from poliovirus, this activity correlated with a parallel reduction in global RNA level. A collection of gradient analyses of HIV-1 infected T cells determined that HIV-1 infection correlates with small, but reproducible differences in ribosome profiles [16]. These results eliminated the possibility that HIV-1 induces the rapid and dramatic global shutdown of host cell translation that is characteristic of poliovirus infection.…”

“…In the cytoplasm, the unspliced transcript plays a dual role as mRNA template for translation and as genomic RNA that is packaged into assembling virions [17]. Experiments with metabolic inhibitors to address the relationship between HIV-1 RNA packaging and translation have determined that unspliced HIV-1 RNA does not segregate into separate pools for translation and packaging, as has been identified for murine leukemia virus [16,43,54]. Lack of translation is not a prerequisite to qualify HIV-1 unspliced RNA for packaging into progeny virions [16].…”

“…Experiments with metabolic inhibitors to address the relationship between HIV-1 RNA packaging and translation have determined that unspliced HIV-1 RNA does not segregate into separate pools for translation and packaging, as has been identified for murine leukemia virus [16,43,54]. Lack of translation is not a prerequisite to qualify HIV-1 unspliced RNA for packaging into progeny virions [16]. Instead, unspliced HIV-1 RNA functions interchangeably as an mRNA template for translation and as genomic RNA that is packaged [16].…”