The human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site

Abstract

Several retroviruses have recently been shown to promote translation of their gag gene products by internal ribosome entry. In this report, we show that mRNAs containing the human immunodeficiency virus type 1 (HIV-1) gag open reading frame (ORF) exhibit internal ribosome entry site (IRES) activity that can promote translational initiation of Pr55(gag). Remarkably, this IRES activity is driven by sequences within the gag ORF itself and is not dependent on the native gag 5'-untranslated region (UTR). This cap-independent mechanism for Pr55(gag) translation may help explain the high levels of translation of this protein in the face of major RNA structural barriers to scanning ribosomes found in the gag 5' UTR. The gag IRES activity described here also drives translation of a novel 40-kDa Gag isoform through translational initiation at an internal AUG codon found near the amino terminus of the Pr55(gag) capsid domain. Our findings suggest that this low-abundance Gag isoform may be important for wild-type replication of HIV-1 in cultured cells. The activities of the HIV-1 gag IRES may be an important feature of the HIV-1 life cycle and could serve as a novel target for antiretroviral therapeutic strategies.

FIG. 1

RIPA analysis of Pr55^gag from plasmid-transfected and poliovirus-infected 293T cells. Cells were transfected with pEMCV-CAT (A) or pCAT-GAG (B) for 40 h and then infected with freshly amplified poliovirus stock for the times shown. Cells were then detached, radiolabeled with [³⁵S]Cys-Met for 45 min, lysed, and subjected to immunoprecipitation with anti-HIV–anti-CAT antibody mixture. Unprecipitated bulk lysate of cells transfected with pCAT-GAG is shown in panel C. Panel D presents a summary of PhosphorImager analysis of five independent transfection-infection experiments. The y axis represents the fold inhibition of band intensity with respect to the band seen at 1 h post-poliovirus infection for the protein indicated. In two of the five experiments summarized in panel D, the CAT and Pr55^gag bands were quantitated in cells cotransfected with pCAT and pENV-ΔUTR-GAG instead of pCAT-GAG. The intensity of signal of lysate bands was determined by examining a 28- to 33-kDa window found between prominent poliovirus VP3 and VP1 bands.

FIG. 2

Northern blotting of bicistronic vector pCAT-GAG. 293T cells were transfected with the plasmid expression vector indicated above each lane and subjected to Northern blotting. Kilobase RNA size markers are shown on the left. The predicted sizes the full-length transcript of each vector are given on the right.

FIG. 3

Analysis of Gag expression using bicistronic Env and Gag vectors. RIPA analysis of COS-7 cells transfected with the indicated plasmid expression vector was done using either anti-HIV-1 antibody stock (A) or anti-CAT antibody stock (B). The numbers below each lane in panel A represent the p24 ELISA signal from lysates of a separatedly transfected population of COS-7 cells.

FIG. 3

Analysis of Gag expression using bicistronic Env and Gag vectors. RIPA analysis of COS-7 cells transfected with the indicated plasmid expression vector was done using either anti-HIV-1 antibody stock (A) or anti-CAT antibody stock (B). The numbers below each lane in panel A represent the p24 ELISA signal from lysates of a separatedly transfected population of COS-7 cells.

FIG. 4

Manipulation of p40 expression. Each lane represents comparable numbers of Cos-7 cells transfected with the indicated plasmid vector and subjected to RIPA with anti-HIV antibody stock. The numbers below each lane represent p24 ELISA signal from lysates of a separatedly transfected population of Cos-7 cells.

FIG. 5

p40^sac expression in HIV-1-infected PBMCs. PHA-activated, CD8⁺-depleted PBMC were mock infected (lane 1) or were infected with wild-type viruses vLAI or vATG (lanes 2, 4, and 5) or with p40^sac knockout virus vATC for 5 days. Cells were treated with (+) or without (−) indinavir, an HIV-1 protease inhibitor, and then radiolabeled and subjected to RIPA with polyclonal anti-p24 serum.

FIG. 6

Rescue of codon 142 mutant virus vATC by expression of p40^sac and p145^sac-pol in trans. Infectious supernatants from 293T cells transfected with codon 142 mutant provirus pVATC or isogenic wild-type control provirus pVATG were normalized for p24 content and added to Jurkat cells, the p40^sac and p145^sac-pol stable Jurkat cell line Jurkat+sac, or the control Jurkat cell line JurkatΔsac. Cells were washed at 24 h postinfection, and the supernatants were sampled at the times shown and subjected to p24 ELISA.