Direct evidence for translational regulation by leader RNA and Tat protein of human immunodeficiency virus type 1.

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Abstract
Translational effects of the RNA leader and Tat protein of human immunodeficiency virus type 1 (HIV-1) were investigated in rabbit reticulocyte lysate. Hybrid RNA species with natural or mutated HIV-1 leader fused to human interferon-.gamma. mRNA were produced in vitro from recombinant plasmids. HIV-1 leader RNA was found to inhibit translation through two mechanisms. A 3-fold trans-inhibition of translation was demonstrated by mixing hybrid HIV-1 leader RNA with indicator interferon mRNA. By comparison, HIV-1 leader caused a 50-fold cis-inhibition in lysate in which two trans-inhibitory factors, double-stranded RNA-dependent protein kinase and (2''-5'')oligoadenylate synthetase, were suppressed. In contrast, purified HIV-1 Tat protein production in Escherichia coli enhanced by 4-fold translation from HIV-1 leader-interferon mRNA but not from interferon mRNA lacking HIV sequences or from total poly (A)+ RNA. Translation of mRNA containing either a single base substitution in the loop of the "trans-acting responsive" sequence (TAR) or an alternative stem-loop in TAR was nevertheless stimulated by Tat. The enhancement of translation by Tat was largely due to relief or cis-inhibition, since the effect was found even in lysate in which double-stranded RNA-dependent protein kinase was inhibted with 2-aminopurine. These results suggest that translation is an important level of control in the replication cycle of HIV-1.