The nucleocapsid protein (NC) of HIV-1 is 55 amino acids in length and possesses two CCHCtype zinc fingers. Finger one (N-terminal) contributes significantly more to helix destabilizing activity than finger two (C-terminal). Five amino acids differ between the two zinc fingers. To determine at the amino acid level the reason for the apparent distinction between the fingers, each different residue in finger one was incrementally replaced by the one at the corresponding location in finger two. Mutants were analyzed in annealing assays with unstructured and structured substrates. Three groupings emerged: (1) those similar to wild type levels (N17K, A25M), (2) those with diminished activity (I24Q, N27D), and (3) mutant F16W which had substantially greater helix destabilizing activity than wild type. Unlike I24Q and the other mutants, N27D was defective in DNA binding. Only I24Q and N27D showed reduced strand transfer in in vitro assays. Double and triple mutants F16W/I24Q, F16W/N27D, and F16W/I24Q/N27D all showed defects in DNA binding, strand transfer, and helix destabilization, suggesting that the I24Q and N27D mutations have a "dominant negative" effect and abolish the positive influence of F16W. Results show that amino acid differences at positions 24 and 27 contribute significantly to finger one's helix destabilizing activity.
The development of effective therapies for hepatitis C virus (HCV) must take into account genetic variation among HCV strains. Response rates to interferon-based treatments, including the current preferred treatment of pegylated alpha interferon administered with ribavirin, are genotype specific. Of the numerous HCV inhibitors currently in development as antiviral drugs, nucleoside analogs that target the conserved NS5B active site seem to be quite effective against diverse HCV strains. To test this hypothesis, we examined the effects of a panel of nucleotide analogs, including ribavirin triphosphate (RTP) and several chain-terminating nucleoside triphosphates, on the activities of purified HCV NS5B polymerases derived from genotype 1a, 1b, and 2a strains. Unlike the genotype-specific effects on NS5B activity reported previously for nonnucleoside inhibitors ( , only minor differences in inhibition were observed among the various genotypes; thus, nucleoside analogs that are current drug candidates may be more promising for treatment of a broader variety of HCV strains. We also examined the effects of RTP on the HCV NS3 helicase/ATPase. As with the polymerase, only minor differences were observed among 1a-, 1b-, and 2a-derived enzymes. RTP did not inhibit the rate of NS3 helicase-catalyzed DNA unwinding but served instead as a substrate to fuel unwinding. NS3 added to RNA synthesis reactions relieved inhibition of the polymerase by RTP, presumably due to RTP hydrolysis. These results suggest that NS3 can limit the incorporation of ribavirin into viral RNA, thus reducing its inhibitory or mutagenic effects.Hepatitis C virus (HCV) infects more than 170 million people worldwide, causing chronic hepatitis, cirrhosis, hepatocellular carcinoma, and/or liver failure (12). Clinical HCV isolates are categorized into different genotypes that display up to 30% sequence variation. Response rates to all interferon-based HCV therapies are genotype dependent, with patients infected with HCV genotype 1 having about one-half the chance of a sustained virological response as patients infected with other genotypes (37). Current HCV therapies combine pegylated alpha interferon and ribavirin (1--D-ribofuranosyl-1,2,4-triazole-3-carboxamide). A number of viral factors have been proposed to contribute to the different response rates to interferon (reviewed in reference 59), but less is known about the effects of viral variation on sensitivity to ribavirin. Here, we employ biochemical methods to examine the simple hypothesis that as a nucleoside analog, ribavirin may affect the enzymes encoded by HCV, and that variation among the enzymes from different genotypes could affect ribavirin response rates. We also examine whether HCV genetic variation influences the sensitivities of HCV enzymes to other nucleoside analogs similar to those currently in development as HCV-specific drugs.The ϳ9,600-nucleotide HCV genome harbors a single open reading frame that encodes a polyprotein, which is processed into 10 distinct proteins by both host and v...
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