A single amino acid mutation in VP1 of coxsackievirus A6 determining efficiency of VP0 cleavage and proliferation

Abstract

Coxsackievirus A6 (CV-A6) has emerged as a major pathogen associated with hand, foot, and mouth disease (HFMD), capable of infecting both children and adults. However, currently, there is no effective vaccine to prevent HFMD caused by non-EV-A71 enteroviruses. In this study, a pair of CV-A6 strains was selected from a rhabdomyosarcoma (RD)-isolated and Vero-adapted stock with a difference of 7 nucleotides in their genomes, resulting in three amino acid mutations in the structural proteins. Distinct differences in propagation, virulence in cells, and plaque size were observed. A series of single-site mutants was constructed, and a single mutation in VP1-143 was mapped to associate with phenotype changes. The mutation from glycine to arginine at VP1-143 dramatically increased infectivity but decreased virulence, growth rate, and plaque size. Furthermore, the experiments using both purified whole virus and full particle (FP) demonstrated that glycine-to-arginine mutation increased VP0 cleavage efficiency because of decreased VP0/VP2 ratio. The decrease in VP0 cleavage efficiency led to the accumulation of non-infectious provirion. The efficiency of virus transmission between cells determined the rates of viral RNA (vRNA) and protein synthesis and was related to fast-slow growth and virulence phenotypes. In addition, the data indicated that the mutation did not affect the encapsidation of the genomic RNA, and the ratio of empty and full particles was unchanged. The results are important for understanding the mechanism of VP0 cleavage regulation and are relevant to developing vaccines and therapeutic reagents against CV-A6 infection and diseases.

Importance: CV-A6 is a major pathogen in the context of HFMD. The cost of treatment and hospitalization of children with HFMD may have a considerable financial impact on the families of patients. CV-A6 is a member of picornaviruses and forms infectious virion through maturation cleavage of VP0 into VP4 and VP2. Although it is well accepted that the autocatalytic process involves viral RNA, the detailed mechanism remains unclear. In this study, residues in VP1-143 were demonstrated to regulate the efficiency of VP0 cleavage and affect the ratio of provirion and virion. Glycine-to-arginine mutation was tolerant, not abolished, but affected the efficiency of VP0 cleavage. The results support a theory that residue mutations on a structural protein of a serotype/genotype within enteroviruses, not well-conserved across picornaviruses and far away from the VP0 cleavage site on the outside surface, regulate the efficiency of VP0 cleavage and render phenotypically different strains.

Keywords: CV-A6; VP0 cleavage regulation; proliferation; virulence.

Fig 1

Proliferation characteristics of CVA6-c45 and CVA6-c61. (A) Growth ability of CVA6-c45 and CVA6-c61 in Vero cells. The viruses were inoculated in Vero cells at an MOI of 0.001, and the supernatants were collected at 12, 24, 36, and 48 h and titrated in Vero cells. The titers were averaged over three experiments, and the error bars indicated the standard deviation. (B) The CPE of CVA6-c45- and CVA6-c61-infected Vero cells at an MOI of 0.001 was observed at 24, 48, and 72 h post-infection. ns, not significant, P > 0.05; ****, P < 0.0001.

Fig 2

Critical role of amino acid 143 of VP1 in infectivity of CV-A6. (A) Schematic representation of the site-directed mutagenesis of CVA6-c45 and CVA6-c61. (B) Infectivity of the rescued recombinants. Vero cells were infected with each rescued strain at an MOI of 0.001, and the cells were fixed 12 and 24 h post-infection. The indirect immunofluorescence assay (IFA) was then performed using anti-CV-A6 and fluorescent secondary antibodies, and the nuclei were labeled with DAPI. (C and D) Growth curves of parental r45, r61, and their single-amino-acid mutants. Each strain was inoculated in Vero cells at an MOI of 0.001, and supernatants were collected at 6, 12, 24, 36, 48, 60, and 72 h post-infection and titrated in Vero cells. The titers represent the mean of three experiments, and the error bars indicate the standard deviation. ns, not significant, P > 0.05; ***, P < 0.001 and ****, P < 0.0001.

Fig 3

Proliferation characteristics of r45-1143R, r45-R1143G, r61-1143G, and r61-G1143R. (A) Representative images of r45-1143R, r45-R1143G, r61-1143G, r61-G1143R plaques, and Mock. Four days post-infection, Vero cells were fixed and stained with 1% crystal violet. (B) A comparison of the average plaque (n = 20) sizes of r45-1143R, r45-R1143G, r61-1143G, and r61-G1143R. The area of plaque was quantified using ImageJ software. (C) The survival of Vero cells infected with r45-1143R, r45-R1143G, r61-1143G, and r61-G1143R. (D and E) The relative expression levels of viral negative-strand RNAs and viral proteins at 24 h post-infection. Vero cells were infected with r45-1143R, r45-R1143G, r61-1143G, and r61-G1143R at an MOI of 0.001 or mock-infected. The cells were harvested at 6, 12, and 24 h, and RT-PCR and western blotting were conducted. Antibody against the viral capsid protein VP1 was used in western blotting. Anti-β-tubulin antibody was used as a loading control. Cell, mock-infected Vero cells. ns, not significant, P > 0.05; *, 0.01 ≤ P < .05; **, P < 0.01; ***, P < 0.001 and ****, P < 0.0001.

Fig 4

Amino acid 143 of VP1 regulating the cleavage efficiency of VP0. (A) Propagation of r45-1143R, r45-R1143G, r61-1143G, and r61-G1143R. Vero cells in 10-layer cell factories were infected with each of the four viruses at an MOI of 0.001. Viruses were harvested when CPEs were reached to 95%. Total viral particles from each strain were obtained by ultracentrifugation through a 20% sucrose cushion. Particles were adjusted to a concentration of 200 ng/100 µL and titrated in Vero cells. The titers were averaged across three independent replicates with error bars representing the standard deviation. (B) Protein profiles of total virus particles of r45-1143R, r45-R1143G, r61-1143G, and r61-G1143R. Equal volumes (1 µL) of the samples were subjected to immunoblotting using anti-CV-A6 VP0/VP2 and VP1 rabbit antibodies. (C) Relative expression levels of VP1 in r45-1143R, r45-R1143G, r61-1143G, and r61-G1143R. Grayscale values of each band were quantified using ImageJ software and normalized relatively to r45-1143R. (D) Effect of amino acid 143 of VP1 on the cleavage efficiency of VP0. Grayscale values of each band were quantified using ImageJ software. The cleavage efficiency of VP0 was expressed as the ratio of VP0 to VP2. (E) The impact of amino acid 143 of VP1 on vRNA packaging efficiency. The vRNA copy numbers were quantified by RT-PCR, and capsid protein content was measured using the bicinchoninic acid (BCA) assay. The packaging efficiency was calculated as the ratio of RNA copy number to the amount of capsid protein, and the ratio was normalized relative to that of r45-1143R. ns, not significant, P > 0.05; *, 0.01 ≤ P < .05; **, P < 0.01; ***, P < 0.001 and ****, P < 0.0001.

Fig 5

Amino acid 143 of VP1 regulating cleavage of the VP0 in provirion. (A) A representative CsCl gradient ultracentrifugation tube and virus fractions. (B) Western blotting analysis of EP, FP, and AP. The EP, FP, and AP of r45-1143R, r45-R1143G, r61-1143G, and r61-G1143R purified through CsCl density gradient centrifugation were subjected to immunoblotting with anti-CV-A6 VP0/VP2 and VP1 rabbit antibodies. (C) Excess FP samples were subjected to immunoblotting with anti-CV-A6 VP0/VP2 and VP1 rabbit antibodies to detect traces of VP0 in FP fractions. (D) Gray-scale values of each band in (C) were determined using ImageJ, and VP0 cleavage efficiency was expressed as the ratio of VP0 to VP2. The relative ratio was normalized to that of r45-1143R. (E) The capsid protein content of all EP and FP fractions was quantified using the BCA assay, and the ratio of EP to FP was subsequently calculated. (F) Infectivity of the EP, FP, and AP of the recombinants. Each fraction of r45-1143R, r45-R1143G, r61-1143G, and r61-G1143R was diluted to 200 ng/100 µL and titrated in Vero cells. The titers are averaged over three replicates, and the standard deviations are indicated by error bars. ns, not significant, P > 0.05; **, P < 0.01 and ****, P < 0.0001.