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. 2003 Jul;77(13):7434-43.
doi: 10.1128/jvi.77.13.7434-7443.2003.

Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication

I Wayne Cheney  1 , Suhaila Naim, Jae Hoon Shim, Meghan Reinhardt, Bharati Pai, Jim Z Wu, Zhi Hong, Weidong Zhong
Affiliations

Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication

I Wayne Cheney et al. J Virol. 2003 Jul.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Purified HRV16 3Dpol is functionally active. (A) HRV16 3Dpol expressed from BL21(DE3) cells was purified on sequential nickel(II) and heparin chromatography columns prior to visualization on a sodium dodecyl sulfate-4 to 20% PAGE gel. Proteins were purified from BL21 cells transformed either with pET26Ub-3Dpol alone, which produced an ubiquitin-3Dpol fusion protein (lane 2), or together with the ubiquitinase-carrying pCG1 plasmid, which produced the fully cleaved HRV16 3Dpol (lane 3). Lane 4 contained the purified PV 3Dpol. (B) Nucleotide incorporation catalyzed by the purified HRV16 3Dpol with a duplexed symmetrical RNA template (sym/sub). Each reaction mixture contained 2 μM end-labeled, preannealed sym/sub RNA, 2 μg of HRV16 3Dpol (R), and 100 μM ATP (lanes 2 to 5) or 100 μM ATP and UTP (lanes 6 to 9). Purified PV 3Dpol (P) was also shown to be active in this assay (lanes 10 and 11). (C) Uridylylation of VPg peptide by HRV16 3Dpol. A 21-mer synthetic peptide corresponding to the wild-type HRV16 VPg was uridylylated (to become VPg-pU) by increasing amounts of VPg in the presence of HRV16 3Dpol, poly(A) RNA, and [α-33P]UTP (lanes 1 to 4). The addition of unlabeled UTP (100 μM) to the reaction mixture enabled elongation of the uridylylated UTP primer and produced elongated VPg-poly(U) (lanes 5 to 8).
FIG. 1.
FIG. 1.
Purified HRV16 3Dpol is functionally active. (A) HRV16 3Dpol expressed from BL21(DE3) cells was purified on sequential nickel(II) and heparin chromatography columns prior to visualization on a sodium dodecyl sulfate-4 to 20% PAGE gel. Proteins were purified from BL21 cells transformed either with pET26Ub-3Dpol alone, which produced an ubiquitin-3Dpol fusion protein (lane 2), or together with the ubiquitinase-carrying pCG1 plasmid, which produced the fully cleaved HRV16 3Dpol (lane 3). Lane 4 contained the purified PV 3Dpol. (B) Nucleotide incorporation catalyzed by the purified HRV16 3Dpol with a duplexed symmetrical RNA template (sym/sub). Each reaction mixture contained 2 μM end-labeled, preannealed sym/sub RNA, 2 μg of HRV16 3Dpol (R), and 100 μM ATP (lanes 2 to 5) or 100 μM ATP and UTP (lanes 6 to 9). Purified PV 3Dpol (P) was also shown to be active in this assay (lanes 10 and 11). (C) Uridylylation of VPg peptide by HRV16 3Dpol. A 21-mer synthetic peptide corresponding to the wild-type HRV16 VPg was uridylylated (to become VPg-pU) by increasing amounts of VPg in the presence of HRV16 3Dpol, poly(A) RNA, and [α-33P]UTP (lanes 1 to 4). The addition of unlabeled UTP (100 μM) to the reaction mixture enabled elongation of the uridylylated UTP primer and produced elongated VPg-poly(U) (lanes 5 to 8).
FIG. 2.
FIG. 2.
Uridylylation potential of HRV16 and PV 3Dpol toward heterologous VPg peptides. HRV16 3Dpol successfully uridylylated, at similar efficiencies, HRV16 VPg (lanes 1 and 2) and PV VPg peptides (lanes 5 and 6). PV 3Dpol uridylylated PV VPg peptide (lanes 7 and 8) but was unable to utilize HRV16 VPg peptide (lanes 9 and 10). In addition, the mutant VPg (Y3F) of HRV16, in which the tyrosine residue at position 3 had been changed to a phenylalanine residue, was inactive for uridylylation by HRV16 3Dpol (lanes 3 and 4). Odd lanes contained [α-33P]UTP alone; even lanes contained both [α-33P]UTP and 100 μM unlabeled UTP for further elongation.
FIG. 3.
FIG. 3.
Schematic representation of VPg (3B) chimeric virus constructs in a PV backbone (PV/R16-VPg) (left) or an HRV16 backbone (R16/PV-VPg) (right).
FIG. 4.
FIG. 4.
Viability of chimeric viral RNAs in transfected H1-HeLa cells. (A) PV/R16-VPg and R16/PV-VPg chimeric RNA, as well as wild-type (wt) PV and HRV16 RNA, were electroporated into H1-HeLa cells. Total cellular RNA was extracted from the transfected cells at various time intervals posttransfection and subjected to Northern blot analysis. Virus-specific probes from either HRV16 3Dpol (left panel) or PV 3Dpol (right panel) and GAPDH were used in the hybridization. (B) Production kinetics of infectious virus particles released from transfected cells as determined by plaque assay. Medium from the cells was collected over time and assayed for PFU as described in Materials and Methods. Infectious virions derived from the PV/R16-VPg chimera first appeared in the electroporated cultures between 6 to 18 h. No infectious virus was detected for the R16/PV-VPg chimera even after 120 h of incubation.
FIG. 5.
FIG. 5.
Plaque morphology of serially passaged PV/R16-VPg chimeric viruses and sequence alteration in the VPg region. (A) Virus particles generated from H1-HeLa cells transfected with PV/R16-VPg chimeric (P0) were further passaged 10 times (P1 to P10). A plaque assay was then performed to determine changes in plaque phenotype. Shown are typical plaques from P0, P1, and P10 of the chimeric virus and from wild-type PV1(M) for comparison. All plaques were developed over a period of 72 h at 37°C. (B) Sequence analysis of the VPg gene in the chimeric viral RNA at various passages. A consensus mutation was identified at position 6 (Glu to Val or Ala) of the VPg gene. This mutation became fixed on E6V after the P0 generation. No other sequence changes were noted in the chimeric backbone.
FIG. 6.
FIG. 6.
The E6V mutation within VPg of PV/R16-VPg chimeric RNA significantly enhanced its replication capability in transfected cells. (A) Growth kinetics of wild-type (wt) or chimeric viral RNA upon transfection of H1-HeLa cells. Supernatants from the electroporated cells were plaque assayed to determine virus titers. (B) Plaque morphologies from supernatants collected 72 h posttransfection.
FIG. 7.
FIG. 7.
PV 3Dpol requires a hydrophobic residue at position 6 of VPg peptide for in vitro uridylylation. Wild-type (WT) PV VPg (lanes 1 and 2), mutant PV VPg/L6E (lanes 3 and 4), WT HRV16 VPg (lanes 5 and 6), and mutant HRV16 VPg/E6V (lanes 7 and 8) were tested for in vitro uridylylation by PV 3Dpol. The E6V mutation in HRV16 VPg rendered partial recovery in uridylylation activity by PV 3Dpol.
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