Furin cleavage of the HIV-1 Tat protein

Extracellular human immunodeficiency virus-1 (HIV-1) Tat protein and Tat-derived peptides are biologically active but mechanisms of Tat processing are not known. Within the highly conserved basic region of HIV-1 Tat protein (amino acids, a.a. 48-56), we identified two putative furin cleavage sites and showed that Tat protein was cleaved in vitro at the second site, RQRR\ (a.a. 53-56\). This in vitro cleavage was blocked by a monoclonal antibody that binds near the cleavage site or by the furin inhibitor alpha-1 PDX. Monocytoid cells rich in furin also degraded Tat and this process was slowed by the furin inhibitor or the specific monoclonal antibody. Furin processing did not affect the rates for Tat uptake and nuclear accumulation in HeLa or Jurkat cells, but the transactivation activity was greatly reduced. Furin processing is a likely mechanism for inactivating extracellular HIV-1 Tat protein.     

Molecular basis for the relative substrate specificity of human immunodeficiency virus type 1 and feline immunodeficiency virus proteases

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3' region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF/VVNGLVK-NH(2) (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2' position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1', FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2' subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1' subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF/VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVF Psi(CH(2)NH)VVNGL-NH(2.) This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.     

JDV Tat反式激活LTR与HIV-1 Tat采用类似的细胞因子

为分析JDV Tat在反式激活JDV及HIV-1 LTR过程中是否采用与H1V-1 Tat类似的细胞因子,本文构建了包含完整激活域的jTat70和hTat47,同时构建了cyclin T1和CDK9真核表达及反义转译质粒。过量表达hTat47和jTat70对hTat反式激活HIV-1 LTR,jTat反式激活JDV和HIV-1 LTR均有明显的抑制作用推测jTat和hTat的反式激活作用可能涉及类似的细胞因子。通过cyclin T1和CDK9的反义转译质粒对jTat反式激活的抑制作用证实这两种细胞因子参与了jTat对JDV和HIV-1 LTR的反式激活。     

Mutational analysis of HIV-1 Tat minimal domain peptides: identification of trans-dominant mutants that suppress HIV-LTR-driven gene expression

The HIV-1 Tat protein is a potent trans-activator essential for virus replication. We reported previously that HIV-1 Tat peptides containing residues 37-48 (mainly region II), a possible activating region, and residues 49-57 (region III), a nuclear targeting and putative nucleic acid binding region, possess minimal but distinct trans-activator activity. The presence of residues 58-72 (region IV) greatly enhances trans-activation. We postulate that Tat mutant peptides with an inactive region II and a functional region III can behave as dominant negative mutants. We synthesized minimal domain peptides containing single amino substitutions for amino acid residues within region II that are conserved among different HIV isolates. We identify four amino acid residues whose substitution within Tat minimal domain peptides leads to defects in transactivation. Some of these mutants are trans-dominant in several peptide backbones, since they strongly inhibit trans-activation by wild-type Tat protein added to cells or expressed from microinjected plasmid. Significantly, trans-activation of integrated HIV-LTRCAT is blocked by some trans-dominant mutant peptides. These results suggest an attractive approach for the development of an AIDS therapy.