Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication.
Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3D(pol), in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3D(pol)-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3D(pol) was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3D(pol) could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3D(pol) to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu(6) to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3D(pol) in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.
Cheney
I Wayne
IW
Ribapharm, Inc., Costa Mesa, California 92626, USA.
Naim
Suhaila
S
Shim
Jae Hoon
JH
Reinhardt
Meghan
M
Pai
Bharati
B
Wu
Jim Z
JZ
Hong
Zhi
Z
Zhong
Weidong
W
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002678:Chimera; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017955:Poliovirus / Q000235:genetics / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis*; D012229:Rhinovirus / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014758:Viral Core Proteins / Q000737:chemistry / Q000502:physiology*
D017931:DNA Primers; D012367:RNA, Viral; D014758:Viral Core Proteins