Neerja Kaushik

Anti-TAR Polyamide Nucleotide Analog Conjugated with a Membrane-Permeating Peptide Inhibits Human Immunodeficiency Virus Type 1 Production

ABSTRACT The emergence of drug-resistant variants has posed a significant setback against effective antiviral treatment for human immunodeficiency virus (HIV) infections. The choice of a nonmutable region of the viral genome such as the conserved transactivation response element (TAR element) in the 5′ long terminal repeat (LTR) may potentially be an effective target for drug development. We have earlier demonstrated that a polyamide nucleotide analog (PNA) targeted to the TAR hairpin element, when transfected into cells, can effectively inhibit Tat-mediated transactivation of HIV type 1 (HIV-1) LTR (T. Mayhood et al., Biochemistry 39:11532-11539, 2000). Here we show that this anti-TAR PNA (PNATAR), upon conjugation with a membrane-permeating peptide vector (transportan) retained its affinity for TAR in vitro similar to the unconjugated analog. The conjugate was efficiently internalized into the cells when added to the culture medium. Examination of the functional efficacy of the PNATAR-transportan conjugate in cell culture using luciferase reporter gene constructs resulted in a significant inhibition of Tat-mediated transactivation of HIV-1 LTR. Furthermore, PNATAR-transportan conjugate substantially inhibited HIV-1 production in chronically HIV-1-infected H9 cells. The mechanism of this inhibition appeared to be regulated at the level of transcription. These results demonstrate the efficacy of PNATAR-transportan as a potential anti-HIV agent.

Inhibition of HIV-1 replication by anti-trans-activation responsive polyamide nucleotide analog

Efficient replication and gene expression of human immunodeficiency virus-1 (HIV-1) involves specific interaction of the viral protein Tat, with its trans-activation responsive element (TAR) which forms a highly stable stem-loop structure. We have earlier shown that a 15-mer polyamide nucleotide analog (PNA) targeted to the loop and bulge region of TAR blocks Tat-mediated transactivation of the HIV-1 LTR both in vitro and in cell culture (Mayhood et al., Biochemistry 39 (2000) 11532). In this communication, we have designed four anti-TAR PNAs of different length such that they either complement the entire loop and bulge region (PNA(TAR-16) and PNA(TAR-15)) or are short of few sequences in the loop (PNA(TAR-13)) or in both the loop and bulge (PNA(TAR-12)), and examined their functional efficacy in vitro as well as in HIV-1 infected cell cultures. All four anti-TAR PNAs showed strong affinity for TAR RNA, while their ability to block in vitro reverse transcription was influenced by their length. In marked contrast to PNA(TAR-12) and PNA(TAR-13), the two longer PNA(TARs) were able to efficiently sequester the targeted site on TAR RNA, thereby substantially inhibiting Tat-mediated transactivation of the HIV-1 LTR. Further, a substantial inhibition of virus production was noted with all the four anti-TAR PNA, with PNA(TAR-16) exhibiting a dramatic reduction of HIV-1 production by nearly 99%. These results point to PNA(TAR-16) as a potential anti-HIV agent.

The J-helix of Escherichia coli DNA Polymerase I (Klenow Fragment) Regulates Polymerase and 3′– 5′-Exonuclease Functions

To assess the functional importance of the J-helix region of Escherichia coli DNA polymerase I, we performed site-directed mutagenesis of the following five residues: Asn-675, Gln-677, Asn-678, Ile-679, and Pro-680. Of these, the Q677A mutant is polymerase-defective with no change in its exonuclease activity. In contrast, the N678A mutant has unchanged polymerase activity but shows increased mismatch-directed exonuclease activity. Interestingly, mutation of Pro-680 has a Q677A-like effect on polymerase activity and an N678A-like effect on the exonuclease activity. Mutation of Pro-680 to Gly or Gln results in a 10–30-fold reduction in k cat on homo- and heteropolymeric template-primers, with no significant change in relative DNA binding affinity or K m (dNTP). The mutants P680G and P680Q also showed a nearly complete loss in the processive mode of DNA synthesis. Since the side chain of proline is generally non-reactive, mutation of Pro-680 may be expected to alter the physical form of the J-helix itself. The biochemical properties of P680G/P680Q together with the structural observation that J-helix assumes helical or coiled secondary structure in the polymerase or exonuclease mode-bound DNA complexes suggest that the structural alteration in the J-helix region may be responsible for the controlled shuttling of DNA between the polymerase and the exonuclease sites.

Analysis of the Role of Glutamine 190 in the Catalytic Mechanism of Murine Leukemia Virus Reverse Transcriptase

To determine the catalytic role of Gln190, a member of the highly conserved LPQG motif in Moloney murine leukemia virus reverse transcriptase, we carried out site-directed mutagenesis of this residue to generate Q190N and Q190A. Both mutant proteins exhibited a significant loss in their polymerase and pyrophosphorolysis activities with a more pronounced effect noted with the Gln → Asn substitution. The catalytic efficiencies of the mutants exhibited a 40–70-fold reduction with poly(rC) and poly(dC) templates in the presence of Mg2+ and a 10–20-fold reduction with poly(rA) template in the presence of Mn2+. Interestingly, the K m for NTP exhibited only a moderate 3–10-fold increase irrespective of the template-primer and the metal ion. Photoaffinity labeling of both the mutant and the WT enzymes exhibited an identical affinity for RNA·DNA and DNA·DNA template-primers. However, unlike the WT enzyme, the mutant enzymes exhibited a significantly reduced ability to catalyze the nucleotidyltransferase reaction on the covalently immobilized template-primer. An examination of the rate constants for the first and the second nucleotide for the mutant enzymes indicated dissimilar rates, indicating that Gln190 may be involved in a rate-limiting, conformational change step both before and after the phosphodiester bond formation. Furthermore, the processivity of DNA synthesis by the mutant enzymes was decreased severely, which may result from the lower catalytic efficiency as well as translocation defect.

Insertion of a peptide from MuLV RT into the connection subdomain of HIV-1 RT results in a functionally active chimeric enzyme in monomeric conformation.

The natural form of the human immunodeficiency virus type one reverse transcriptase (HIV‐1 RT) found in virion particles is a heterodimer composed of the p66 and p51 subunits. The catalytic activity resides in the larger subunit in the heterodimeric (p66/p51) enzyme while in the monomeric form it is inactive. In contrast, Murine leukemia virus RT (MuLV RT) is functionally active in the monomeric form. In the primary amino acid sequence alignment of MuLV RT and HIV‐1 RT, we have identified three specific regions in MuLV RT, that were missing in HIV‐1 RT. In a separate study, we have shown that a chimeric RT construct comprising of the polymerase domain of HIV‐1 RT and RNase-H domain of MuLV RT is functionally active as monomer [20]. In this communication, we demonstrate that insertion of a peptide (corresponding to amino acid residues 480–506) from the connection subdomain of MuLV RT into the connection subdomain of HIV‐1 RT (between residues 429 and 430) results in a functionally active monomeric chimeric RT. Furthermore, this chimeric enzyme does not dimerize with exogenously added p51 subunit of HIV‐1 RT. Functional analysis of the chimeric RT revealed template specific variations in its catalytic activity. The chimeric enzyme catalyzes DNA synthesis on both heteropolymeric DNA and homopolymeric RNA (poly rA) template but curiously lacks reverse transcriptase ability on heteropolymeric RNA template. Similar to MuLV RT, the polymerase activity of the chimeric enzyme is not affected by acetonitrile, a reagent which dissociates dimeric HIV‐1 RT into inactive monomers. These results together with a proposed 3‐D molecular model of the chimeric enzyme suggests that the insertion of the missing region may induce a change in the spatial position of RNase H domain such that it is functionally active in monomeric conformation.