Missense mutations in an infectious human immunodeficiency viral genome: functional mapping of tat and identification of the rev splice acceptor.

Abstract

Single nucleotide alterations were introduced into an infectious clone of human imnmunodeficiency virus type 1 to create a series of missense mutants in the tat coding region. Although mutations in a proline-rich region and a basic lysine-arginine-rich region resulted in wild-type phenotypes, five of six mutations in a cysteine-rich domain completely abolished tat activity and virus replication. One cysteine mutant retained tat activity but was negative for virus expression. Surprisingly, this mutant could not be complemented by tat, and virus expression was restored only by cotransfection with a plasmid expressing the rev gene. Another mutant with an alteration toward the C-terminal region showed significantly reduced tat activity and required complementation by a combination of tat and rev for virus replication. Further analysis revealed that a previously unrecognized splice acceptor site within this region, apparently used to generate the rev mRNA, had been altered. We provide evidence suggesting that tat and rev proteins are encoded by distinct mRNA species.Human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS) contains three major structural genes (gag, pol, and env) in common with all retroviruses (1). In addition, HIV contains several accessory genes-namely, vif (virion infectivity factor) (2), vpr (viral protein R) (3), tat (trans-activator) (4), rev (regulator of expression of virion proteins) (5), and nef (negative factor) (6); these designations were proposed recently to standardize the nomenclature for known human retrovirus accessory genes (7). The tat-and rev-encoded proteins have both been shown to be required for virus replication. Tat activates transcription from the viral long terminal repeat (LTR), resulting in increased levels of viral RNA and proteins (8). The rev gene product allows accumulation of steady-state levels of gag-pol and env mRNAs (5) and, in addition, has been shown to negatively affect transcription (8). Also rev has been proposed to function as an anti-repressor at the translational level (9). The trans-regulatory genes, tat and rev, are expressed from largely overlapping reading frames. To identify domains of these proteins that are important for function, we have generated a series of HIV-1 proviruses containing altered coding sequences in the tat and rev genes. Site-directed mutagenesis was targeted to the three major structural motifs of the functional first coding exon of the tat gene-the N-terminal proline-rich region, the middle cysteine-rich region, and the C-terminal basic amino acid stretch. Alterations in the last of these regions affected the first rev coding exon as well.The mutants were evaluated for phenotypic changes by transfection into COS-1 cells and assaying (i) their ability to trans-activate the homologous LTR by chloramphenicol acetyltransferase (CAT) assay, (ii) viral mRNA synthesis by Northern (RNA) blot, (iii) viral protein synthesis by radioimmunoprecipitation (RIP) assay, (iv) virus production ...

Expression of alternatively spliced human T-lymphotropic virus type I pX mRNA in infected cell lines and in primary uncultured cells from patients with adult T-cell leukemia/lymphoma and healthy carriers.

“…3). The complex alternative splicing of HTLV-I regulatory gene messages is similar to the situation with human immunodeficiency virus type 1 (HIV-1) (28)(29)(30)(31)(32)(33). In fact, alternative splicing is a common feature of retroviruses (34).…”

Presence of Exon Splicing Silencers within Human Immunodeficiency Virus Type 1 tat Exon 2 and tat-rev Exon 3: Evidence for Inhibition Mediated by Cellular Factors

“…The spliced products resulting from the use of 3Ј splice sites 4c, 4a, 4b, and 5 in substrate HS1-6026 are detected only with longer exposures. The two rev 3Ј splice sites, 4a and 4b, appear as one band because of the close proximity of their 3Ј splice sites (20,42). Splice site 4c is used infrequently in vivo (37) and is detected upon longer autoradiographic exposures.…”

Jembrana Disease Virus Tat Can Regulate Human Immunodeficiency Virus (HIV) Long Terminal Repeat-Directed Gene Expression and Can Substitute for HIV Tat in Viral Replication

“…Two tat mutant proviral clones, pMtat(Ϫ) and pMtat30, were obtained from the AIDS Research and References Program National Institute of Allergy and Infectious Diseases [NIAID], National Institutes of Health, Bethesda, Md. ): pMtat(Ϫ) contains a termination codon (TGA) in place of the ATG initiation codon and is unable to synthesize Tat (49); pMtat30, which carries a Cys3Gly substitution within the cysteine-rich region, produces a missense Tat (50).…”