A base-modified
nucleoside analogue, beta-D-N(4)-hydroxycytidine (NHC), was found to have antipestivirus and antihepacivirus activities. This compound inhibited the production of cytopathic bovine viral
diarrhea virus (BVDV)
RNA in a dose-dependant manner with a 90% effective concentration (EC(90)) of 5.4 microM, an observation that was confirmed by virus yield assays (EC(90) = 2 microM). When tested for hepatitis C virus (HCV) replicon
RNA reduction in Huh7 cells, NHC had an EC(90) of 5 microM on day 4. The HCV
RNA reduction was incubation time and
nucleoside concentration dependent. The in vitro
antiviral effect of NHC was additive with recombinant alpha interferon-2a and could be prevented by the addition of exogenous
cytidine and
uridine but not of other natural ribo- or 2'-deoxynucleosides. When HCV
RNA replicon cells were cultured in the presence of increasing concentrations of NHC (up to 40 micro M) for up to 45 cell passages, no resistant replicon was selected. Similarly, resistant BVDV could not be selected after 20 passages. NHC was phosphorylated to the
triphosphate form in Huh7 cells, but in cell-free HCV NS5B assays, synthetic NHC-
triphosphate (NHC-TP) did not inhibit the polymerization reaction. Instead, NHC-TP appeared to serve as a weak alternative substrate for the viral polymerase, thereby changing the mobility of the product in
polyacrylamide electrophoresis
gels. We speculate that incorporated
nucleoside analogues with the capacity of changing the thermodynamics of regulatory secondary structures (with or without introducing mutations) may represent an important class of new
antiviral agents for the treatment of
RNA virus infections, especially HCV.