Mutagenesis of tyrosine 24 in the VPg protein is lethal for feline calicivirus

Abstract

The genome of feline calicivirus (FCV) is an approximately 7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3' end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5' end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.

FIG. 1.

Summary of mutations in the FCV VPg protein and their effects on recovery of virus. The genome organization and coding assignments of the Urbana strain of FCV (GenBank accession no. L40021) are shown. Dipeptide cleavage sites recognized by the virus-encoded cysteine proteinase are indicated. The four tyrosines (numbered according to the VPg protein amino acid sequence) at positions 12, 24, 76, and 104 were changed to alanine in the infectious cDNA clone pQ14. In addition, the tyrosine at position 24 was changed to serine, threonine, or phenylalanine. For each construct, the ability to recover viable virus is indicated by a plus sign and a lethal mutation is indicated by a minus sign. wt, wild type.

FIG. 2.

Analysis of RNA and proteins derived from plasmids containing lethal mutations at VPg residue 24. (A) Capped RNA transcripts were synthesized as previously described (27) from the NotI-linearized plasmid DNA of constructs pQ14 (lane 2), Y24A (lane 3), Y24S (lane 4), Y24T (lane 5), and Y24F (lane 6) and analyzed with a 1% agarose gel (Ambion). The RNA was visualized by ethidium bromide staining. An RNA marker (Invitrogen) was included in lane 1. The arrow indicates the full-length RNA. (B) The capped RNA transcripts were translated in a rabbit reticulocyte lysate in the presence of radiolabeled methionine, and the proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 10 to 20% polyacrylamide gel. The proteins correspond to RNAs derived from pQ14 (lane 2), Y24A (lane 3), Y24S (lane 4), Y24T (lane 5), and Y24F (lane 6). The previously characterized proteins derived by coupled transcription and translation (TNT) of the FCV ORF1 clone pTMF-1 (32) are shown in lane 1 for comparison. (C) CRFK cells were infected with MVA/T7 and transfected with wild-type pQ14 or mutagenized plasmids. After 5 h, the proteins were radiolabeled with [³⁵S]methionine for 12 h. Cell lysates were prepared and incubated with p39-specific serum, followed by precipitation of antigen-antibody complexes with Sepharose protein A beads. The precipitated proteins were resolved in a 10% Tris-glycine polyacrylamide gel and visualized by autoradiography. Immunoprecipitation of the p39 protein (indicated by an arrow) from MVA/T7-infected cells transfected with plasmids pQ14 (lane 2), Y24A (lane 3), Y24S (lane 4), Y24T (lane 5), and Y24F (lane 6) is shown. Lane 1 contains mock-transfected MVA/T7 cells incubated with p39-specific antibodies, and lane 7 contains radiolabeled TNT products derived from pTMF-1 as shown for panel B.

FIG. 3.

Comparison of the plaque phenotypes of recovered viruses to that of the wild-type (wt) virus. Recovered viruses were assayed for their ability to form plaques in CRFK cells. Serial dilutions of viruses (10⁻² to 10⁻⁸) were used to infect monolayers of CRFK cells seeded in six-well plates. After 1 h of incubation of the virus inoculum at 37°C, cells were washed and an agarose overlay was added. Cells were then incubated at 37°C for 24 h in a humidified CO₂ incubator. The monolayers were fixed with formalin and stained with crystal violet to visualize viral plaques (23).

FIG. 4.

Growth characteristics of mutant viruses in comparison with those of the wild-type (wt) virus. Confluent monolayers of CRFK cells were infected with wild-type or mutant viruses at a multiplicity of infection of 0.1. Cell culture fluid was harvested 2, 4, 6, 8, 10, and 25 h postinfection. Virus titer was determined by end point titration in a plaque assay as described in the legend to Fig. 3.

FIG. 5.

Effects of mutations on the proteolytic processing of VPg and its precursors. CRFK cells were infected with wild-type (WT) or mutant viruses and radiolabeled with [³⁵S]methionine. Cell lysates were prepared and incubated with VPg-specific serum, followed by precipitation of antigen-antibody complexes and analysis with a 10 to 20% polyacrylamide gel. Lane 1, wild-type virus-infected cell lysate incubated with preimmunization guinea pig serum. Lanes 2 to 6, the VPg-specific postimmunization guinea pig serum was incubated with infected cell lysates prepared from wild-type virus (lane 2), Y12A (lane 3), Y76A (lane 4), Y104A (lane 5), and MVA/T7 (lane 6). Lane 7, mock-infected CRFK cell lysate control incubated with VPg-specific serum. The known precursor proteins containing VPg, as well as free and modified forms of the mature VPg (29), are indicated. The 33-kDa protein is indicated by an unmarked arrow.