family currently comprises three genera of single-stranded positive-sense RNA viruses: flaviviruses, pestiviruses, and hepaciviruses (36
). Bovine viral diarrhea virus
(BVDV) is a prototype virus in the genus Pestivirus
, which also includes Classical swine fever virus
(CSFV) and Border disease virus
. The RNA genome of BVDV is one of the largest (12.5 kb) among members of the Flaviviridae
). Similar to hepatitis C virus (HCV), it consists of a long 5′ untranslated region (UTR) which contains an internal ribosomal entry site (IRES) for the translation of viral proteins (6
). The single large open reading frame encodes a polyprotein of approximately 3,900 amino acids (8
) that is processed into at least 12 functional proteins (Npro-C-Erns
/p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B) by both host and viral proteases (10
). The first virally encoded protein is a unique protease (Npro for N-terminal protease), responsible for the cleavage between Npro and the core protein (C) (38
). A study by Rümenapf et al. showed that Npro is a novel type of cysteine proteinase which required cysteine69
for proteolytic activity (38
). Interestingly, partial and complete replacement of the Npro protein by a ubiquitin or fusion with a chloramphenicol acetyltransferase in pestivirus genomes had been shown to produce viable viruses (32
). The resulting chimeric viruses were demonstrated to have growth properties similar to the wild-type viruses.
As one of the most characterized members of the Flaviviridae
family, BVDV provides a good model system for HCV, a major etiologic agent for non-A non-B hepatitis (1
). It shares many important features with HCV. Both viruses utilize an IRES within the 5′ UTR, for the translation of the viral polyprotein (6
). Furthermore, the viral NS3 proteases of both viruses require NS4A as a cofactor for polyprotein processing (11
). The cytopathic and plaque-forming properties of BVDV in cell cultures allow rapid and quantitative analysis of viral replication and growth. The availability of infectious clones (28
) provides opportunities for genetic manipulation to alter viral functions and to construct chimeric viruses. Indeed, a recent report by Frolov et al. found that the entire BVDV IRES could be replaced by HCV IRES. The resulting chimeric viruses relied on the HCV IRES for growth (15
), which should allow the in vitro efficacy evaluation of HCV IRES inhibitors.
HCV infection is prevalent and a major global health issue. A recently completed population-based survey revealed that in the United States alone the overall prevalence of anti-HCV was 1.8%, corresponding to an estimated 3.9 million individuals infected by HCV nationwide. A total of 74% of these seropositive individuals tested positive for HCV RNA, indicating that an estimated 2.7 million persons were chronically infected (2
). Currently, the combination of alpha 2b interferon and ribavirin (Rebetron; Schering Plough, Kenilworth, N.J.) has been shown to have clinical efficacy in only a proportion (<50%) of patients with chronic HCV infection (9
). Vaccine development has been hampered by the high immune evasion rate with poor or no protection against reinfection with a heterologous or homologous inoculum in chimpanzees (12
). Development of small molecule inhibitors directed against specific viral targets has thus become the major focus of anti-HCV drug development.
Extensive characterization of the HCV NS3 serine protease (3
) has shed light in developing assays and identifying inhibitors of HCV. Major advances in the determination of crystal structures for NS3 protease have begun to delineate important features for the development of potent and specific anti-HCV inhibitors (23
). Many high-throughput enzyme-based screening assays, targeting HCV NS3 serine protease, have been developed. Further development of potential inhibitors has to rely on a convenient and reliable cell-based assay system to demonstrate their antiviral efficacy. The lack of a bona fide cell culture system that permits HCV infection makes it a daunting task to evaluate the antiviral efficacy of candidate inhibitors prior to in vivo studies in animals and humans.
Several HCV NS3 protease-dependent chimeric viruses using the genetic backbones of Sindbis virus and poliovirus have been created, providing potential cell-based antiviral assays to evaluate the efficacy of candidate inhibitors against HCV protease (4
). Similar schemes were adopted to create these chimeric viruses in which HCV NS3 protease-containing genes were inserted and fused in frame to an essential viral protein through an HCV junction site cleavable by HCV NS3 protease. Failure to cleave the junction by a mutant protease or in the presence of any potent HCV NS3 protease inhibitors would render the unprocessed viral proteins unable to perform their designated functions for viral growth (4
). However, genetic stability of such chimeric viruses with foreign gene inserts was a major issue since RNA viruses recombined at a high frequency (29
). Indeed, the HCV NS3 genes inserted in the Sindbis viral genome were quickly deleted during initial viral passages and the revertant viruses appeared rapidly and exhibited similar advantageous growth properties as the wild-type viruses (13
). Although a second generation of chimeric Sindbis viruses was generated in which a second HCV NS3 cleavage site was created, these viruses were rather defective and unable to replicate at the normal physiological temperature (13
). This would limit the development of animal models for in vivo testing of the protease inhibitors.
We describe here the generation of a chimeric BVDV in which the Npro coding region is replaced by that of an NS4A cofactor-tethered HCV NS3 protease. This tethered HCV protease domain is fused in frame with the BVDV core protein via an HCV NS5A-NS5B junction site. In this chimeric design, the normal proteolytic function of the Npro is substituted by that of the HCV NS3 serine protease. We demonstrated that viable and cytopathic chimeric viruses were produced. They had growth kinetics comparable to that of the wild-type BVDV and were stable during subsequent serial passages. Our results suggest that the development of a cell-based antiviral assay is feasible using the HCV NS3 protease-dependent BVDV chimeric virus for in vitro testing of potential HCV NS3 protease inhibitors.