Hantaviruses represent an important and growing source of disease emergence in both established and developing countries (27
). Hantaviruses are enveloped, negative-stranded RNA viruses with a tripartite, segmented genome (28
). The genome consists of S (small), M (medium), and L (large) segments of RNA, which encode the nucleocapsid protein (N), glycoproteins (Gn and Gc), and L protein (RNA-dependent RNA polymerase [RdRp]), respectively. When virus is transmitted to humans through inhalation of aerosols of virus shed in rodent excreta, hantaviruses may cause either of two serious illnesses: hemorrhagic fever renal syndrome (HFRS) (21
) or hantavirus pulmonary syndrome (HPS) (18
). Hantaan virus (HTNV), an Old World hantavirus, is the primary agent of HFRS, while Sin Nombre and Andes viruses are the primary agents in New World cases of HPS in North and South America, respectively.
At present, there are no vaccines or antivirals approved by the U.S. Food and Drug Administration for treatment of any of the hemorrhagic fever viruses or HPS. A limited number of antivirals have been tested, and few have been effective against viruses within the family Bunyaviridae
. Ribavirin (1-β-d
-ribofuranosyl-1,2,4-triazole-3-carboamide) (RBV), a broad-spectrum antiviral agent, and related C-nucleoside analogues ribamidine, selenazofurin (SEL), and tiazofurin (TIA) show potent antiviral activity in vitro against HTNV (17
). RBV was proven effective in the treatment of lethal encephalitic suckling mice infected with HTNV (16
). Moreover, studies performed in China with HFRS patients suggest that RBV provides an improved prognosis when given early in the course of disease (15
). As no other antiviral drugs with this level of activity for the hantaviruses have been reported, RBV remains an important lead for the development of new and better drugs for HTNV and perhaps other members of the Bunyaviridae
. However, RBV's mechanism of action has remained elusive; therefore, efforts toward understanding its mechanism should bring new insights into the design of new drugs. For example, one could use the mechanistic information to explore chemical modifications that would increase its selectivity toward RdRps and decrease selectivity for IMP dehydrogenase (IMPDH).
A cursory review of the literature may suggest that RBV's mechanism of action is understood, but an intensive review of the literature reveals a complex profile of activity and perhaps a pleiotropic mechanism for this compound. These seemingly diverse mechanisms of action may derive from RBV's unique interaction with the polymerase machinery for each virus family and the dependence or requirements of that virus family on the nucleotide pool levels, specifically those of GTP. RBV decreases cellular GTP pools by competitive inhibition with IMP for the active site of IMPDH (2
). This has been reported to be the mechanism of action for RBV for several viruses (22
). However, viral enzymatic activities are also targeted, as suggested by several reports that show RBV's effect on capping (11
), translation efficiency of viral mRNA (41
), and viral polymerase activity (9
). These activities may be due to direct incorporation of RBV 5′-monophosphate into mRNA or viral genomic RNAs since it resembles GMP, although it can pseudo-base pair with C and U. Recently, error-prone replication mediated by RBV has been reported for several RNA viruses (reviewed in reference 42
), which would also require direct incorporation of RBV. We have also reported that RBV induced error-prone replication in HTNV (31
). RBV can act as a potent RNA virus mutagen in poliovirus-infected cells, and it has been proposed that its incorporation into viral RNAs (vRNAs) causes the virus to enter error catastrophe (5
). It is apparent that RBV acts as a mutagen for several viruses, including hantaviruses, but from the studies reported to date, it is not clear for most viruses how or if GTP repression via IMPDH contributes to the observed antiviral effect. Recently we have reported that, at least for HTNV, RBV's activity did not correlate with inhibition of IMPDH but rather with production of RBV triphosphate (RTP) (38
). This suggests the interaction of RTP with the hantaviral RdRp, and while consistent with the observed increase in mutation frequency reported earlier for HTNV (31
), it does not provide additional insight into the operational mechanism of RBV.
Recently, alternative theoretical models to error catastrophe have been developed for drug-induced mutation frequency in viruses (3
). One of the most recent theoretical models, lethal mutagenesis, differs from error catastrophe conceptually. In essence, the theory of lethal mutagenesis is based on a demographic process reflected by an apparent decline in the absolute abundance of the quasispecies population rather than an evolutionary shift in genotype space (3
). Furthermore, lethal mutagenesis mandates the extinction of a population, while error catastrophe theory can shift the population toward higher fitness as well as driving what is often referred to as the mutational meltdown of the genome. Clearly, the design of effective antivirals and treatment strategies should be guided by these theoretical considerations. With that in mind, we have designed experiments to reveal which model, if any, was operational for RBV's inhibitory activity against HTNV. We undertook a comprehensive analysis of the mutation frequency over a wide range of RBV concentrations. We also examined the antiviral effects of several additional IMPDH inhibitors, including mycophenolic acid (MPA), selenazofurin (SEL), and tiazofurin (TIA), of which SEL and TIA are C-nucleoside analogues of RBV. This enhanced mutation frequency was observed only with RBV and not with other IMPDH inhibitors. Importantly, we observed that a relatively narrow lethal threshold was sufficient for extinction of HTNV. In essence, this threshold reflects a point in the drug concentration at which additional mutations are not tolerated and hence the virus is unable to replicate. However, this point did not lead to a complete loss of the genomic information, as predicted by the alternative hypothesis of error catastrophe.