Engineering Responses to Amino Acid Substitutions in the VP0- and VP3-Coding Regions of PanAsia-1 Strains of Foot-and-Mouth Disease Virus Serotype O

Abstract

The presence of sequence divergence through adaptive mutations in the major capsid protein VP1, and also in VP0 (VP4 and VP2) and VP3, of foot-and-mouth disease virus (FMDV) is relevant to a broad range of viral characteristics. To explore the potential role of isolate-specific residues in the VP0 and VP3 coding regions of PanAsia-1 strains in genetic and phenotypic properties of FMDV, a series of recombinant full-length genomic clones were constructed using Cathay topotype infectious cDNA as the original backbone. The deleterious and compensatory effects of individual amino acid substitutions at positions 4008 and 3060 and in several different domains of VP2 illustrated that the chain-based spatial interaction patterns of VP1, VP2, and VP3 (VP1-3), as well as between the internal VP4 and the three external capsid proteins of FMDV, might contribute to the assembly of eventually viable viruses. The Y2079H site-directed mutants dramatically induced a decrease in plaque size on BHK-21 cells and viral pathogenicity in suckling mice. Remarkably, the 2079H-encoding viruses displayed a moderate increase in acid sensitivity correlated with NH₄Cl resistance compared to the Y2079-encoding viruses. Interestingly, none of all the 16 rescued viruses were able to infect heparan sulfate-expressing CHO-K1 cells. However, viral infection in BHK-21 cells was facilitated by utilizing non-integrin-dependent, heparin-sensitive receptor(s) and replacements of four uncharged amino acids at position 3174 in VP3 of FMDV had no apparent influence on heparin affinity. These results provide particular insights into the correlation of evolutionary biology with genetic diversity in adapting populations of FMDV.IMPORTANCE The sequence variation within the capsid proteins occurs frequently in the infection of susceptible tissue cultures, reflecting the high levels of genetic diversity of FMDV. A systematic study for the functional significance of isolate-specific residues in VP0 and VP3 of FMDV PanAsia-1 strains suggested that the interaction of amino acid side chains between the N terminus of VP4 and several potential domains of VP1-3 had cascading effects on the viability and developmental characteristics of progeny viruses. Y2079H in VP0 of the indicated FMDVs could affect plaque size and pathogenicity, as well as acid sensitivity correlated with NH₄Cl resistance, whereas there was no inevitable correlation in viral plaque and acid-sensitive phenotypes. The high affinity of non-integrin-dependent FMDVs for heparin might be explained by the differences in structures of heparan sulfate proteoglycans on the surfaces of different cell lines. These results may contribute to our understanding of the distinct phenotypic properties of FMDV in vitro and in vivo.

Keywords: PanAsia-1 strain; deleterious and compensatory effects; foot-and-mouth disease virus; genetic and phenotypic properties; site-directed mutation.

FIG 1

Effects of amino acid difference at position 2079 in the VP0 coding region of PanAsia-1 strains on phenotypic properties of the indicated FMDVs. rHN/TAR6-VP0, rHN/FJ9-VP0, and their seven site-directed mutants were selected to be tested by three different plaque assays on BHK-21 cells and virulence comparison in suckling mice (means ± the SD are indicated; ANOVA results are indicated [*, P < 0.05; **, P < 0.01; ***, P < 0.001, if necessary]). (A) The plaques formed by the Y2079-encoding viruses (■) were distinctly larger than those formed by the 2079H-encoding viruses (□) on BHK-21 cells. At least 50 plaques were analyzed for each virus. (B) The Y2079-eoncoding viruses (●, ▲, ■, ◆, and ж) showed higher virulence in suckling mice than the 2079H-encoding viruses (□, ◇, ○, and △). A total of 20 animals were injected with each virus supernatant (about 100 PFU/200 μl per mouse). (C) The Y2079-encoding viruses (■) displayed a moderate decrease in acid-induced inactivation at acidic pH 6.9 compared to that of the 2079H-encoding viruses (□). The infectivity (%) of each diluted virus (0.5 MOI) at different pH values was calculated as the percentage of PFU by comparison with that obtained at neutral pH 7.4. (D) The infection efficiency of the Y2079-encoding viruses (●, ▲, ■, ◆, and ж) was more sensitive to NH₄Cl treatment than that of the 2079H-encoding viruses (□, ◇, ○, and △). The total yield per virus collections in 25 mM NH₄Cl (experimental group)- or DMSO (control group)-treated BHK-21 cells was determined by plaque-forming assay at 5 h postinfection.

FIG 2

Effect of amino acid replacements at position 3174 in the VP3 coding region of the specific FMDVs for heparin affinity in BHK-21 cells. The infection of BHK-21 cells with rHN, rHN/FJ9-VP0, rHN/FJ9-VP423, and their seven site-directed mutants was examined by the RGD-containing peptide VR-17 inhibition and heparin binding assays (see Materials and Methods). O/Fujian/CHA/5/99 tc and rHN/TAR6-VP0 were used as negative and positive controls separately. The results from these two plaque assays demonstrated that the remaining plaques of each virus on BHK-21 cells seemed to have been reduced by the addition of heparin (1 mg/ml, right) rather than the preincubation of the RGD-containing peptide VR-17 (1 mM, left). The high affinity of these ten rescued viruses for heparin was not affected by four uncharged amino acid replacements at position 3174 in VP3.

FIG 3

Location of isolate-specific amino acid residues in VP0 and VP3 of the icosahedral capsid of the selected PanAsia-1 strains of FMDV serotype O. The ribbon protein diagram represents 1 of the 60 protomeric subunits, plus one additional VP3 from a neighboring protomer. The structural proteins VP1 (missing 211 to 213 residues), VP2 (missing 1 to 4 residues), VP3, and VP4 (missing 1 to 14 and 41 to 64 residues) are highlighted as red, blue, green, and yellow (internal), respectively. The individual amino acid residues of VP1 to VP3 (listed in Table S1) are labeled as the space-filling atomic models. The target residues (left) of enlarged areas (right) are marked in pink font. The atomic-level interaction of isolate-specific amino acid residues was visualized, and the minimal side chain-side chain distances measured of critical amino acid residues were 4.71 Å between 2079H (N_τ) and R1145 (N_ω) of O/Fujian/CHA/9/99 tc, 4.68 Å between L2080Q (C_δ) and S1144A (C_β), 5.20 Å between E2136G (N) and L1192 (C_γ) of O/Tibet/CHA/6/99 tc, 5.63 Å between D2133N (C_β) and L1192 (C_δ), 5.78 Å between 3060G (C_α) and K2134 (C_ε) of the SPV [small plaque-cloned virus] of O/JPN/2000 strain, 4.42 Å between 2175R (C_ε) and 1142A (C_α), and 3.25 Å between 2214L (C_δ) and M3130 (C_ε) of O/Fujian/CHA/5/99 tc. The corresponding C_α-C_α distances were 7.07, 7.49, 7.33, 10.05, 9.90, 9.14, and 7.29 Å, respectively.