Correlation between mutations in the interferon sensitivity-determining region of NS5A protein and viral load of hepatitis C virus subtypes 1b, 1c, and 2a.

In the present study, we analyzed the possible relationship between interferon (IFN) sensitivity-determining region (ISDR) sequence variation of various hepatitis C virus (HCV) subtypes and serum HCV titers in Indonesian patients without IFN treatment. The viremia titers (mean +/- standard deviation) of HCV subtype 1b (HCV-1b) isolates with low (three or fewer) and high (four or more) numbers of ISDR mutations were 5.4 +/- 0.6 and 4.2 +/- 0.9 log(10) RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Similarly, the viremia titers of HCV-1c isolates with low and high numbers of ISDR mutations were 5.3 +/- 0.6 and <3.0 +/- 0.0 log(10) RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Also, the virus titers of HCV-2a isolates with low and high numbers of ISDR mutations were 4.3 +/- 0.7 and 3.5 +/- 0.4 log(10) RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Thus, our results demonstrated that virus load in Indonesian patients infected with HCV-1b, HCV-1c, or HCV-2a correlated inversely with the number of mutations in the ISDR sequence, implying the possibility that the ISDR sequence plays an important role in determining the levels of HCV viremia.

Inhibition of p21/Waf1/Cip1/Sdi1 expression by hepatitis C virus core protein.

The possibility of interaction between hepatitis C virus (HCV) core protein and the cell cycle regulator protein p21/Waf1/Cip1/Sdi1 (p21/Waf1) in cultured cells was analyzed. Although colocalization of HCV core protein and p21/Waf1 was not clearly observed, p21/Waf1 expression was much weaker in HCV core protein-expressing cells than in the control. A Northern blot analysis showed nearly the same level of p21/Waf1 mRNA in both cells, suggesting that HCV core protein inhibited p21/Waf1 expression post-transcriptionally. The degradation patterns of p21/Waf1 did not differ significantly in HCV core protein-expressing cells and in the control, suggesting that the stability of p21/Waf1, once it was accumulated in the cell, was not significantly affected by HCV core protein. But this does not necessarily exclude the possibility that synthesis, maturation, and nuclear transport of p21/Waf1 is impaired, or that the degradation of newly synthesized, improperly processed p21/Waf1 is promoted by HCV core protein. The decrease in p21/Waf1 accumulation was partially inhibited by proteasome inhibitors and a calpain inhibitor in both HCV core protein-expressing cells and the control. In vitro kinase assay revealed that a p21/Waf1-mediated inhibition of cyclin-dependent kinase 2 activity was partially negated by HCV core protein. Taken together, the present results suggest that HCV core protein inhibits p21/Waf1 expression post-transcriptionally and impairs the function of p21/Waf1 in the cell.

Nuclear localization and intramolecular cleavage of N-terminally deleted NS5A protein of hepatitis C virus.

The full-size NS5A (NS5A-F) of hepatitis C virus is localized in the cytoplasm despite the presence of a functional nuclear localization signal (NLS) in its C-terminal region (amino acids (aa) 354-362). In the present study, we demonstrated that a short stretch of sequence near the N-terminus of NS5A (aa 27-38) masked the functional NLS, preventing NS5A from being transported to the nucleus. This sequence, referred to as an NLS-masking sequence, was distinct from a nuclear export signal, as it did not actively target a protein to the cytoplasm. We also found that other sequences located at either an N- (aa 1-21) or a C-terminal region (aa 353-447) were responsible for targeting NS5A to the cytoplasm. Western blot analysis of the transfected cells revealed that NS5A mutants that had been N-terminally deleted by 66 aa or more were cleaved at a certain cleavage site, generating a common fragment of ca. 40 kDa. This result implies the possible presence of a cleavage site in the NS5A sequence around aa 150, which is exposed through conformational alteration upon the N-terminal deletions.