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Journal of Virology, December 2000, p. 11531-11537, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Melinda Butsch1,2 and Kathleen Boris-Lawrie1,2,3,4,5,*

Center for Retrovirus Research,1 Departments of Veterinary Biosciences4 and Molecular Virology, Immunology and Medical Genetics,3 The Ohio State Biochemistry Program,2 and Comprehensive Cancer Center,5 The Ohio State University, Columbus, Ohio 43210-1093

Received 18 July 2000/Accepted 25 September 2000

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 [3H]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.

* Corresponding author. Mailing address: Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210-1093. Phone: (614) 292-1392. Fax: (614) 292-6473. E-mail: boris-lawrie.1{at}osu.edu.

Journal of Virology, December 2000, p. 11531-11537, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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