A DNA-launched reverse genetics system for rabbit hemorrhagic disease virus reveals that the VP2 protein is not essential for virus infectivity

Abstract

Rabbit hemorrhagic disease virus (RHDV), a member of the family Caliciviridae comprising positive-stranded RNA viruses, is a highly virulent pathogen of rabbits. Until recently, studies into the molecular mechanisms of RHDV replication and pathogenesis have been hindered by the lack of an in vitro culture system and reverse genetics. This study describes the generation of a DNAbased reverse genetics system for RHDV and the subsequent investigation of the biological role of the RHDV VP2 protein. The full-length RHDV genome was assembled as a single cDNA clone and placed under the control of the eukaryotic human cytomegalovirus promoter. Transfection of cells with the DNA clone resulted in a clear cytopathic effect and the generation of infectious progeny virions. The reconstituted virus was stable and grew to titres similar to that of the parental virus. Although previous reports have suggested that the minor structural protein (VP2) of other caliciviruses is essential for the production of infectious virions, using the DNA-launch-based RHDV reverse genetics system described here it was demonstrated that VP2 is not essential for RHDV infectivity. Transfection of cells with a cDNA clone of RHDV lacking VP2 resulted in the generation of infectious virions. These studies indicate that the presence of VP2 could reduce the replication of RHDV, suggesting that it may play a regulatory role in the life cycle of RHDV.

Functional Analysis of RNA Structures Present at the 3′ Extremity of the Murine Norovirus Genome: the Variable Polypyrimidine Tract Plays a Role in Viral Virulence

“…Although MNV, FCV, and GII/4 human noroviruses contain potential RNA structures within the VP2 coding region, secondary structure elements do not appear to be universally present in all members of the Caliciviridae; previous bioinformatic analysis provided no evidence for conserved RNA secondary structures in the homologous region for members of the Lagovirus genus (35). Furthermore, unlike the case with FCV, complete removal of the VP2 coding sequence from the genome of rabbit hemorrhagic disease virus (RHDV) had little effect on virus replication in tissue culture (23). Recent work has also highlighted that sequences within the 3Ј end of the Norwalk virus genome contain an RNA element or elements which stimulate the in vitro nucleotidylation of recombinant VPg in the presence of magnesium chloride (2).…”

Minor structural protein VP2 in rabbit hemorrhagic disease virus downregulates the expression of the viral capsid protein VP60

“…We then showed that VP2 is not essential for the production of infectious virons of RHDV (Liu et al, 2008), in contrast to results with the corresponding VP2 protein of feline calicivirus (FCV), a member of the genus Vesivirus (Sosnovtsev et al, 2005). In Norwalk virus (NV), VP2 was shown to increase the expression of capsid protein VP1 and to stabilize it (Bertolotti-Ciarlet et al, 2003) and we found that the presence of RHDV VP2 can increase virus replication (Liu et al, 2008). We now report experiments investigating the effect of RHDV VP2 on the expression of VP60 in vitro and in vivo, showing that VP2 is capable of downregulating the expression of VP60.…”

“…In 2006, we successfully constructed an infectious cDNA clone of RHDV (Liu et al, 2006). We then showed that VP2 is not essential for the production of infectious virons of RHDV (Liu et al, 2008), in contrast to results with the corresponding VP2 protein of feline …”

“…In 2006, we successfully constructed an infectious cDNA clone of RHDV (Liu et al, 2006). We then showed that VP2 is not essential for the production of infectious virons of RHDV (Liu et al, 2008), in contrast to results with the corresponding VP2 protein of feline calicivirus (FCV), a member of the genus Vesivirus (Sosnovtsev et al, 2005). In Norwalk virus (NV), VP2 was shown to increase the expression of capsid protein VP1 and to stabilize it (Bertolotti-Ciarlet et al, 2003) and we found that the presence of RHDV VP2 can increase virus replication (Liu et al, 2008).…”

Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA

“…DNA-based systems using the pol II promoter to drive expression of viral cDNA directly or by baculovirus delivery have produced MNV (16), and a CMV promoter system recovered rabbit hemorrhagic disease virus (RHDV) (28). In vitro-transcribed RNA-based systems have recently been successful for several animal NoVs and caliciviruses, with variation in the requirement for capped or uncapped RNA [FCV (29,30), PEC (26), MNV (15,31), RHDV (32), and Tulane virus (33)].…”