Human immunodeficiency virus type 1 Gag polyprotein modulates its own translation

Abstract

The full-length viral RNA of human immunodeficiency virus type 1 (HIV-1) functions both as the mRNA for the viral structural proteins Gag and Gag/Pol and as the genomic RNA packaged within viral particles. The packaging signal which Gag recognizes to initiate genome encapsidation is in the 5' untranslated region (UTR) of the HIV-1 RNA, which is also the location of translation initiation complex formation. Hence, it is likely that there is competition between the translation and packaging processes. We studied the ability of Gag to regulate translation of its own mRNA. Gag had a bimodal effect on translation from the HIV-1 5' UTR, stimulating translation at low concentrations and inhibiting translation at high concentrations in vitro and in vivo. The inhibition was dependent upon the ability of Gag to bind the packaging signal through its nucleocapsid domain. The stimulatory activity was shown to depend on the matrix domain of Gag. These results suggest that Gag controls the equilibrium between translation and packaging, ensuring production of enough molecules of Gag to make viral particles before encapsidating its genome.

FIG. 1.

A. Schematic of pJHIV-1 mRNA, including HIV-1 5′ UTR secondary structures. B. Schematic of GST-Gag polyprotein with GST and the domains of Gag indicated: MA, capsid (CA), p2, NC, p1, and p6. C. Translation of pJHIV-1 RNA in the presence of 0, 0.05, 0.075, 0.1, 0.2, 0.4, and 0.8 μM GST-Gag (top panels); 0, 0.05, 0.1, 0.2, 0.4, and 0.8 μM GST-GagΔp6 (middle panels); and 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 μM GST (lower panels). Autoradiographs are shown on the left; Western blots probed with anti-HIV-1 p17 antibody are shown on the right. D. Graphical representation of translation from pJHIV-1 in the presence of different levels of Gag protein. Error bars represent standard errors of the means; n > 3. E. ³²P-labeled pJHIV-1 RNA (lane 1, input) was extracted from translation reaction mixtures that contained 0, 0.1, 0.2, 0.4 and μM Gag (lanes 2 to 5).

FIG. 2.

A. Schematics of Gag, GagΔZn1 + 2, and GagC28/49S proteins, with deletions and mutations shown. B. Gel mobility shift assays showing the HIV-1 5′ UTR probe alone (lanes 1, 5, and 9) or with 0.05, 0.1, or 0.2 μM GST-Gag (lanes 2 to 4), GST-GagΔZn1 + 2 (lanes 6 to 8), or GST-GagC28/49S (lanes 10 to 12). The positions of monomeric and dimeric HIV-1 5′ UTRs are indicated, and the positions of RNA-protein complexes are shown by arrowheads. C. Translation of pJHIV-1 RNA in the presence of 0, 0.1, 0.2, 0.and 4 μM GST-Gag, GST-GagΔZn1 + 2, or GST-GagC28/49S. Autoradiographs, in which the position of NS′ is indicated, are shown in the upper panels; Western blots probed with anti-HIV-1 p17 are shown in the lower panels. D. Graphical representation of translation results. Diamonds, GST-Gag; squares, GST-GagΔZn1 + 2; triangles, GST-GagC28/49S. Error bars represent standard errors of the means; n > 3. Asterisks indicate a significant difference from wild-type GST-Gag (P < 0.005).

FIG. 3.

A. Schematic of the secondary structure of the HIV-1 5′ UTR, with the TAR and packaging signal (Ψ) indicated. B. GST-Gag (0, 0.1, 0.2, or 0.4 μM) was added to translation reaction mixtures programmed with pJHIV-1ΔTAR, pJHIV-1ΔΨ, pJ(CAA)₁₉, and cyclin and pJHIV-1 RNAs. The RNA is shown above each panel. The positions of NS′ and cyclin products are shown to the right of the autoradiographs. C. Graphical representation of translation results. Diamonds, HIV-1-NS′; open squares, ΔTAR-NS′; triangles, ΔΨ-NS′, gray squares, (CAA)₁₉; circles, cyclin. Error bars represent standard errors of the means; n > 2. Asterisks indicate a significant difference from HIV-1-NS′ (P < 0.005).

FIG. 4.

A. Schematics of the matrix-deleted Gag protein (GagΔMA) and MA. B. Gel mobility shift assays showing HIV-1 5′ UTR probe alone (lanes 1 and 5) or with 0.05, 0.1, and 0.2 μM GagΔMA (lanes 2 to 4) or MA (lanes 6 to 8). The positions of monomeric and dimeric HIV-1 5′ UTR are indicated, and the positions of RNA-protein complexes are shown by arrowheads. C. Translation of pJHIV-1 RNA in the presence of 0, 0.1, and 0.4 μM GagΔMA or MA. The position of the NS′ product is shown to the right of the autoradiographs. D. Graphical representation of the translation results. Diamonds, GST-Gag; squares, GST-GagΔMA; triangles, GST-MA. Error bars represent standard errors of the means; n > 3. Asterisks indicate a significant difference from wild-type GST-Gag (P < 0.01).

FIG. 5.

Translation of pHIVluc RNA (black bars) and pJluc RNA (gray bars) when cotransfected into COS-1 cells with increasing amounts of pJGag RNA (4 to 250 ng). Error bars represent standard errors of the means; n > 3. Asterisks indicate a significant difference from results with 0 ng Gag RNA (P < 0.005).

FIG. 6.

Model of HIV-1 translation modulated by Gag. Initially, in the absence of Gag, there is low-level translation initiation and Gag starts to be produced. Gag stimulates translation initiation, resulting in greater Gag production. Increasing Gag levels result in oligomerization of Gag and subsequent coating of the HIV-1 5′ UTR. Translation is inhibited, and viral RNA is sequestered for dimerization and encapsidation. Ψ represents the packaging signal (high-affinity Gag binding site).