Potent Inhibition of HIV-1 Replication by a Tat Mutant

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.

A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+T Cells

“…However, unlike previously reported mutants (Green et al, 1989;Pearson et al, 1990;Modesti et al, 1991;Ulich et al, 1996), Nullbasic inhibited reverse transcription and also effectively suppressed the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity (Meredith et al, 2009). HeLa CD4 + cells constitutively expressing Nullbasic were strongly protected from a spreading infection by HIV-1 (Meredith et al, 2009). This study investigated whether Nullbasic could be used to effectively protect human T cells from HIV-1 infection.…”

“…pLOX-CW-Nullbasic-EGFP, pLOX-CW-EGFP, pCMVDR8.91, and pCMV-VSV-G plasmids used for production of lentiviral VLPs (virus-like particles) have been described elsewhere (Zufferey et al, 1997;Salmon et al, 2000). The pCDN3.1-Nullbasic-FLAG plasmid was previously described (Meredith et al, 2009). The plasmid pGCsamEN has been described (Treisman et al, 1995).…”

“…The plasmid pGCsamEN has been described (Treisman et al, 1995). The pGCsamEN-Nullbasic-EGFP construct was made by PCR of Nullbasic-EGFP from pcDNA3.1-Nullbasic-EGFP (Meredith et al, 2009), using primers P1 and P2, which introduce unique NotI and SnaBI restriction sites compatible with the multiple cloning site of pGCsamEN (Supplementary Table S1). Similarly, pGCsamEN-EGFP was made by PCR of an EGFP cassette from pCDN3.1-EGFP, using primers P2 and P3, and pGCsamEN-ZsGreen1 was made by PCR of ZsGreen1 from pLVX-IRES-ZsGreen1 (Clontech, Mountain View, CA), using primers P4 and P5.…”

“…Expression of EGFP and Nullbasic-EGFP was confirmed by Western blot using lysates from cells prepared and electrophoresed as previously described (Meredith et al, 2009). Lysates were equalized for total protein concentration.…”

“…We described a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that inhibited multiple steps of the HIV-1 replication cycle (Meredith et al, 2009), a unique feature compared with other antiviral proteins. Nullbasic was created by replacing the entire arginine-rich basic domain of wild-type Tat with glycine/alanine residues.…”