Methionine 184 of HIV-1 RT is a constituent of the catalytically crucial and highly conserved YXDD motif in the reverse transcriptase class of enzymes. We investigated the role of this residue by substituting it with Ala and Val by site-directed mutagenesis followed by extensive characterization of the two mutant enzymes. The kinetic parameters governing DNA synthesis directed by RNA and DNA templates indicated that both M184A and M184V mutants are catalytically as efficient as the wild type enzyme. Photoaffinity labeling of both the mutant and the wild type enzyme exhibited an identical affinity for RNA-DNA and DNA-DNA template primers. We further demonstrate that M-->V substitution at 184 position significantly increases the fidelity of DNA synthesis while M-->A substitution results in a highly error-prone enzyme without having compromised its efficiency of DNA synthesis. The M184V mutant exhibited a 25-45-fold increase in mismatch selectivity (ratio of k(cat)/K(m) of correct versus incorrect nucleotides) as compared to the WT enzyme. This pattern of error-prone synthesis is also confirmed by examining the abilities of the enzyme-(template-primer) covalent complexes to incorporate correct versus incorrect nucleotide onto the immobilized template-primer. The nature of error-prone synthesis by the M184A mutant shows an increase in both the mismatch synthesis and extension of the mismatched primer termini. Using a three-dimensional molecular model of the ternary complex of HIV-1 RT, template-primer, and dNTP, we observe that the strategic location of M184 may allow it to interact with the sugar moiety of either the primer nucleotide or the dNTP substrate.

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In order to clarify the role(s) of the individual member of the carboxylate triad in the catalytic mechanism of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, we carried out site-directed mutagenesis of D185, D186, and D110, followed by the extensive characterization of the properties of the individual mutant enzymes. We find that all three residues participate at or prior to the chemical step of bond formation. The incorporation pattern seen with phosphorothioate analogs of dNTP on both RNA-DNA and DNA-DNA template-primers indicated that D186 may be the residue that coordinates with the alpha-phosphate group of dNTP in the transition-state ternary complex. Further support for the role assigned to D186 was obtained by examination of the ability of the individual carboxylate mutants to catalyze the reverse of the polymerase reaction (pyrophosphorolysis). Mutants of D185 exhibited near-normal pyrophosphorolysis activity, while those of D186 were completely devoid of this activity. Thus, D185 appears to participate only in the forward reaction, probably required for the generation of nucleophile by interacting with the 3'-OH of the primer terminus, while D186 seems to be involved in both the forward and the reverse reactions, presumably by participating in the pentavalent intermediate transition state. Lack of any elemental effects during polymerization with mutant enzymes of residue D110, together with their inability to catalyze pyrophosphorolysis, suggest its probable participation in the metal-coordinated binding to the beta-gamma-phosphate of dNTP or PPi in the forward and reverse reactions, respectively. A molecular model of the ternary complex based on these results is also presented.

Chronic infection by hepatitis C virus (HCV) is the leading cause of severe hepatitis that often develops into liver cirrhosis and hepatocellular carcinoma. The molecular mechanisms underlying HCV replication and pathogenesis are poorly understood. Similarly, the role(s) of host factors in the replication of HCV remains largely undefined. Based on our knowledge of other RNA viruses, it is likely that a number of cellular factors may be involved in facilitating HCV replication. It has been demonstrated that elements within the 3-nontranslated region (3-NTR) of the (؉) strand HCV genome are essential for initiation of (؊) strand synthesis. The RNA signals within the highly conserved 3-NTR may be the site for recruiting cellular factors that mediate virus replication/pathogenesis. However, the identities of putative cellular factors interacting with these RNA signals remain unknown. In this report, we demonstrate that an RNA affinity capture system developed in our laboratory More than a decade ago, hepatitis C virus (HCV) 1 was discovered as the major causative agent of parenteral non-A non-B hepatitis (1, 2), and the number of HCV-infected individuals worldwide is currently estimated at 170 million. Although some infected individuals are able to clear the virus without treatment, most infections persist leading in about 50% of all cases to chronic hepatitis, which may develop into chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma. The main therapeutic regimen currently in clinical use is a combination treatment consisting of high doses of interferon-␣ and the nucleoside analog ribavirin, and a large percentage of patients receiving this regimen are not responsive. These stark facts underscore the importance of expediently developing new strategies to combat this viral infection.The hepatitis C virus is a positive strand RNA virus of the flaviviridae family having a genome roughly 9.6 kb in length (3). The RNA genome contains a single ORF that encodes a polyprotein of ϳ3000 amino acids that is processed by a combination of host-and virus-encoded proteases (4) into four structural proteins (core, E1, E2, and p7) and six nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. Replication of HCV is initiated at the 3Ј-NTR of the RNA genome, although little is known about the mechanism of initiation or the factors required for this process. The NS5B protein is the RNA-dependent RNA polymerase, or HCV replicase, responsible for replication of the HCV genome whose structure and enzymatic activities have been well characterized (5, 6). Although it has been established that NS5B can initiate primer-independent RNA synthesis, the mechanistic details of this process remain the source of some controversy (7-9). Hong et al. (10) have proposed that a structural motif within NS5B positions the terminal nucleotides of the genome in such a way that de novo synthesis is initiated from the 3Ј-end of the genome. The initiation of HCV virus replication may very well involve the aid of cellular factors in...

In order to determine the catalytic role of Arg72 of HIV-1 reverse transcriptase (RT), we carried out site-directed mutagenesis at codon 72. Two mutant proteins (R72A and R72K) were purified and characterized. With Arg to Ala substitution the kcat of the polymerase reaction was reduced by nearly 100-fold with poly(rA) template, but only about 5-15-fold with poly(rC) and poly(dC) templates. The Arg to Lys substitution exhibited a qualitatively similar pattern, although the overall reduction in kcat was less severe. Most interestingly, we noted a large difference in the rate constant of the first and second nucleotide incorporation by R72A, suggesting that Arg72 participates in the reaction after the formation of the first phosphodiester bond. We propose this step to be the pyrophosphate binding and removal step following the nucleotidyltransferase reaction. Support for this proposal is obtained from the observation that the R72A mutant (i) exhibited a pronounced translocation defect in the processivity analysis, (ii) lacked the ability to catalyze pyrophosphorolysis, and (iii) showed complete resistance to phosphonoformate, an analog of PPi.Arg72 is the first residue of HIV-1 RT proposed to be involved in the pyrophosphate binding/removal function of RT.