Laurence S. Tiley

HIV-1 structural gene expression requires binding of the rev trans-activator to its RNA target sequence

Expression of human immunodeficiency virus type 1 structural proteins requires both the viral Rev trans-activator and its cis-acting RNA target sequence, the Rev response element (RRE). The RRE has been mapped to a conserved region of the HIV-1 env gene and is predicted to form a complex, highly stable RNA stem-loop structure. Site-directed mutagenesis was used to define a small subdomain of the RRE, termed stem-loop II, that is essential for biological activity. Gel retardation assays demonstrated that the Rev trans-activator is a sequence-specific RNA binding protein. The RRE stem-loop II subdomain was found to be both necessary and sufficient for the binding of Rev by the RRE. We propose that the HIV-1 Rev trans-activator belongs to a new class of sequence-specific RNA binding proteins characterized by the presence of an arginine-rich binding motif.

Does the human immunodeficiency virus tat trans-activator contain a discrete activation domain?

Human immunodeficiency virus type 1 (HIV-1) encodes a transcriptional trans-activator, termed Tat, that is absolutely required for viral replication in vitro. By analogy to other known transcription factors, it has been suggested that the HIV-1 Tat protein may contain discrete protein domains that determine sequence specificity and transcriptional activation potential. Here, we report the use of site-directed mutagenesis to examine the functional significance of two candidate activation domains within Tat. A 12 amino acid sequence adjacent to the N-terminus of the Tat protein, which includes a proposed acidic amphipathic alpha-helix activation motif, was found to contribute to, but be dispensable for, Tat function in vivo. In contrast, the integrity of a second potential Tat activation motif, centered on a lysine residue at position 41, was found to be essential for Tat function. However, Tat proteins mutated in this area displayed a fully recessive negative phenotype. Therefore, neither of these two regions of the Tat protein appear to be discrete activation domains. We conclude that previous attempts to categorize Tat as a modular transcription factor have not succeeded and suggest that the functional organization of this complex trans-activator remains to be defined.