title: Direct evidence for translational regulation by leader RNA and Tat protein of human immunodeficiency virus type 1.
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 produced 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 of cis-inhibition, since the effect was found even in lysate in which double-stranded RNA-dependent protein kinase was inhibited with 2-aminopurine. These results suggest that translation is an important level of control in the replication cycle of HIV-1.
authors.affiliation: Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799.
authors.forename: D N
authors.initials: DN
authors.lastname: SenGupta
authors.forename: B
authors.initials: B
authors.lastname: Berkhout
authors.forename: A
authors.initials: A
authors.lastname: Gatignol
authors.forename: A M
authors.initials: AM
authors.lastname: Zhou
authors.forename: R H
authors.initials: RH
authors.lastname: Silverman
mesh_terms: D000818:Animals; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000096:biosynthesis / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012156:Reticulocytes / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D015696:Gene Products, tat; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D007371:Interferon-gamma