Abstract
Herein we describe a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that potently inhibits multiple steps of the HIV-1 replication cycle. Nullbasic was created by replacing the entire arginine-rich basic domain of wild type Tat with glycine/alanine residues. Like similarly mutated one-exon Tat mutants, Nullbasic exhibited transdominant negative effects on Tat-dependent transactivation. However, unlike previously reported mutants, we discovered that Nullbasic also strongly suppressed the expression of unspliced and singly-spliced viral mRNA, an activity likely caused by redistribution and thus functional inhibition of HIV-1 Rev. Furthermore, HIV-1 virion particles produced by cells expressing Nullbasic had severely reduced infectivity, a defect attributable to a reduced ability of the virions to undergo reverse transcription. Combination of these inhibitory effects on transactivation, Rev-dependent mRNA transport and reverse transcription meant that permissive cells constitutively expressing Nullbasic were highly resistant to a spreading infection by HIV-1. Nullbasic and its activities thus provide potential insights into the development of potent antiviral therapeutics that target multiple stages of HIV-1 infection.
Highlights
Tat is an essential HIV-1 regulatory protein whose best described role is to promote high levels of viral gene expression via interactions with the HIV-1 transactivation response element (TAR) RNA [1,2]
The first exon is organized into two major domains: the activation domain, which interacts with numerous cellular proteins including cyclin T1, and the basic domain, which is primarily comprised of arginine and lysine residues
To see if Nullbasic localizes to the cytoplasm, HeLa cells expressing Nullbasic or the wild type Tat-FLAG fusion protein were visualized by indirect immunofluorescence microscopy using anti-FLAG antibody
Summary
Tat is an essential HIV-1 regulatory protein whose best described role is to promote high levels of viral gene expression via interactions with the HIV-1 transactivation response element (TAR) RNA [1,2]. The basic domain (amino acids 49–57) is required for many of Tat’s activities including nuclear localization [3,4] and TAR binding [5]. A transdominant negative mutant is typically an altered form of a protein that can inhibit the normal function of its wild type counterpart. Engineered Tat proteins with altered basic domains possess transdominant negative phenotypes against wild type Tat. most studies of Tat transdominance have used one-exon tat mutants encoding truncated proteins of 72 amino acids or less. Tat truncated at residue 53 can suppress transactivation initiated by wild type Tat [9]. Mutations in the activation domain of the Tat mutant, which normally suppress the transactivation function of wild type Tat, can suppress transdominance [10]
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