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.

FIG. 1

Incorporation of [³⁵S]cysteine/methionine is inhibited by incubation with pac, chx, and aniso. Inhibition of [³⁵S]cysteine/methionine incorporation occurs 0.5 h posttreatment and is sustained over a 4-h period. CEM(A)/HIV-1 T cells were incubated with cysteine/methionine-free RPMI medium for 0.5 h, followed by the addition of [³⁵S]cysteine/methionine with pac, chx, or aniso. Total cell lysates were collected at 0.5, 2, or 4 h posttreatment, and [³⁵S]cysteine/methionine incorporation was quantified by TCA precipitation assay. Average results of at least four experiments are shown; error bars indicate standard deviations.

FIG. 2

Ribosomal profile analysis in response to pac, chx, and aniso. HIV-1 infected or uninfected CEM(A) T cells were treated for 4 h with or without pac, chx, or aniso, and cytoplasmic extracts were placed on 10-ml linear gradients of 15 to 45% sucrose. After ultracentrifugation, the gradients were fractionated and monitored at A₂₅₄ using an ISCO gradient fractionation system.

FIG. 3

Pulse-chase analysis of Gag incorporation into virions. CEM(A)/HIV-1 T cells were incubated with cysteine/methionine-free medium for 0.5 h, followed by a 1-h incubation with of 10 μCi of [³⁵S]cysteine/methionine per ml and 5 × 10⁻⁸ M pac in control plates. Cells were washed and incubated in complete medium with or without pac. Cell lysates and virion lysates were collected at intervals between 1 and 8 h postchase. TCA precipitation assay was performed with 50 ng of cellular protein or virion lysate equivalent to 30 ng of Gag. Representative results of three experiments are shown.

FIG. 4

Pac, chx, and aniso significantly inhibit Gag synthesis. CEM(A)/HIV-1 T cells were incubated with cysteine/methionine-free RPMI medium for 0.5 h, followed by the addition of 10 μCi of [³⁵S]cysteine/methionine per ml with or without pac, chx, or aniso. Cell lysates and cell-free supernatants were collected 4 h posttreatment, and virions were isolated by centrifugation. Fifty-nanogram samples of ³⁵S-labeled cell lysate and virions equivalent to 100 ng Gag were subjected to radioimmunoprecipitation assay with Gag p24 antibody followed by SDS-PAGE and PhosphorImager analysis.

FIG. 5

Incorporation of [³H]uridine into newly synthesized virions. CEM(A)/HIV-1 cells were incubated for 2, 4, and 6 h in medium containing [³H]uridine (30 μCi/ml) with or without actD (0.5 μg/ml). [³H]uridine levels in cytoplasmic RNA and in virion RNA were quantified by TCA precipitation analysis. Representative results of at least three experiments are shown.

FIG. 6

HIV-1 vpRNA remains available for encapsidation during translation inhibition. CEM(A)/HIV-1 cells were incubated for 4 h in medium containing 30 μCi of [³H]uridine per ml with or without pac, chx, or aniso. [³H]uridine levels in cytoplasmic RNA and in virion RNA were quantified by TCA precipitation analysis. Average results of three experiments are shown; error bars indicate standard deviations.

FIG. 7

Encapsidation efficiency is not reduced upon inhibition of de novo translation. (A) Representative RPA of cytoplasmic and virion RNA that was harvested after 4 h of incubation with or without pac, chx, or aniso. Labels indicate the sizes of the protected RNAs and control 100-bp DNA fragment used to control for viral RNA sample processing (virus control), cell treatment, and undigested probes. (B) Summary of four RPAs. Average results are shown; error bars indicate standard deviations. Encapsidation efficiency was determined by dividing virion RNA level by the corresponding cytoplasmic RNA level.