Ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis C viruses in culture.
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.
Stuyver
Lieven J
LJ
Pharmasset Inc., Tucker, Georgia 30084, USA. lstuyver@pharmasset.com
Whitaker
Tony
T
McBrayer
Tamara R
TR
Hernandez-Santiago
Brenda I
BI
Lostia
Stefania
S
Tharnish
Phillip M
PM
Ramesh
Mangala
M
Chu
Chung K
CK
Jordan
Robert
R
Shi
Junxing
J
Rachakonda
Suguna
S
Watanabe
Kyoichi A
KA
Otto
Michael J
MJ
Schinazi
Raymond F
RF
D000818:Animals; D000998:Antiviral Agents / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D003562:Cytidine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000494:pharmacology*; D001908:Diarrhea Viruses, Bovine Viral / Q000187:drug effects* / Q000235:genetics; D005260:Female; D016174:Hepacivirus / Q000187:drug effects*; D051379:Mice; D012367:RNA, Viral / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects*
D000998:Antiviral Agents; D012367:RNA, Viral; D003562:Cytidine; C010737:N(4)-hydroxycytidine