Limits...
Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor.

Bai X, Bao H, Li P, Wei W, Zhang M, Sun P, Cao Y, Lu Z, Fu Y, Xie B, Chen Y, Li D, Luo J, Liu Z - Virol. J. (2014)

Bottom Line: However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O.Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells.The results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Veterinary Etiological Biology, OIE/National Foot-and-Mouth Disease Reference Laboratory, Engineering Research Center of Biological Detection of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730046, China. baixingwen@caas.cn.

ABSTRACT

Background: Some cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O. To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells.

Results: Only two of the rescued viruses encoding VP0 of O/Tibet/CHA/6/99tc or VP1 of O/Fujian/CHA/9/99tc formed plaques on wild-type Chinese hamster ovary (WT-CHO; HS+) cells, but not on HS-negative pgsD-677 cells. The formation of plaques by these two chimeric viruses on WT-CHO cells could be abolished by the introduction of single amino acid mutations Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc and Lys-1083 → Glu in VP1 of O/Fujian/CHA/9/99tc, respectively. Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells. Significant differences in the inhibition of the infectivity of four HS-utilizing viruses by heparin and RGD-containing peptide were observed in BHK-21 cells. Interestingly, the chimeric virus encoding VP0 of O/Fujian/CHA/9/99tc, and the site-directed mutant encoding Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc could bind to HS, but there was no expression of the 3A protein of these two viruses in WT-CHO cells.

Conclusion: The results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction.

Show MeSH

Related in: MedlinePlus

Locations of the distinct amino acid residues in the FMDV capsid of (A) rHN/TAR6-VP0 and (B) rHN/FJ9-VP1. The crystallographic coordinates of O1BFS (missing the first 15 amino acid residues at the N-terminus of VP4) were used as templates[35], and 3D structures of one of the twelve pentamers were optimized using the SWISS-MODEL program[36-38]. The respective capsid proteins VP1–4 are represented as green, cyan, magenta, and blue (internal). The G-H loop of VP1 is highlighted in yellow. The RGD motif (residues 1145–1147, colored green), residues 2079, 2080 (colored red) and 2136 in VP2 of O/Tibet/CHA/6/99tc, as well as residues 1041, 1045, 1083 (colored red), 1095 and 1139 in VP1 of O/Fujian/CHA/9/99tc are displayed in the space-filling models.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4118260&req=5

Figure 5: Locations of the distinct amino acid residues in the FMDV capsid of (A) rHN/TAR6-VP0 and (B) rHN/FJ9-VP1. The crystallographic coordinates of O1BFS (missing the first 15 amino acid residues at the N-terminus of VP4) were used as templates[35], and 3D structures of one of the twelve pentamers were optimized using the SWISS-MODEL program[36-38]. The respective capsid proteins VP1–4 are represented as green, cyan, magenta, and blue (internal). The G-H loop of VP1 is highlighted in yellow. The RGD motif (residues 1145–1147, colored green), residues 2079, 2080 (colored red) and 2136 in VP2 of O/Tibet/CHA/6/99tc, as well as residues 1041, 1045, 1083 (colored red), 1095 and 1139 in VP1 of O/Fujian/CHA/9/99tc are displayed in the space-filling models.

Mentions: Nonetheless, the 3D structural models of rHN/TAR6-VP0 and rHN/FJ9-VP1 indicated that Gln-2080 in the B-C loop of VP2 of O/Tibet/CHA/6/99tc and Lys-1083 within the D-E loop of VP1 of O/Fujian/CHA/9/99tc surround the G-H loop of VP1 and the pore at the fivefold axis of symmetry, respectively (Figure 5A, 5B)[35-38]. The difference in phenotypic properties of the two chimeric viruses (rHN/TAR6-VP0 and rHN/FJ9-VP1) and their site-directed mutants (rHN/TAR6-VP0Q2080L and rHN/FJ9-VP1K1083E) on WT-CHO cells were consistent with our expectations (Figure 1). Yet, the introduction of a Leu-2080 → Gln mutation in VP2 of O/Fujian/CHA/9/99tc (pOFS/FJ9-VP0L2080Q) was particularly deleterious for the generation of progeny virus in BHK-21 cells, and a Glu-1083 → Lys mutation in VP1 of O/Tibet/CHA/6/99tc did not result in the acquisition of the HS-utilizing ability of rHN/TAR6-VP1E1083K to infect WT-CHO cells (Figure 1). Moreover, a second-site mutation (Glu-2136 → Gly) in VP2 of O/Fujian/CHA/9/99tc was unable to compensate for the lethal effect of the primary mutation Leu-2080 → Gln in the VP2 coding region of pOFS/FJ9-VP0L2080Q and the site-directed mutant encoding Glu-1083 → Lys and Ser-1139 → Arg in VP1 of O/Tibet/CHA/6/99tc also could not utilize HS as a receptor to establish an efficient infection in WT-CHO cells (results not shown). There are a limited number of amino acid differences fixed in the VP2 and VP1 coding regions of the mutated plasmids as compared to rHN/TAR6-VP0 and rHN/FJ9-VP1, respectively (Table 1). In particular, the distinct amino acid residues at 2079 (Tyr) of VP2 lies adjacent to the RGD motif (Figure 5A), and (ii) Thr-1041 and Gln-1045 of VP1 are clustered around the fivefold axis (Figure 5B). The outcome of the substitution at residue 2079 of VP2 could potentially dislocate both antigenic sites 1 (1141–1160 residues of the VP1 G-H loop,[39]) and 2 (2070–2078 residues of the VP2 B-C loop,[40]), with a structural and/or functional change[33]. One of the amino acid mutations, Lys-1041 → Glu in VP1 was fixed in two heparin-binding derivatives of C-S8c1 (C-S8c1p100c10 and the MARLS mutant)[20,22]. Accordingly, His-2079 → Tyr in VP2 of O/Fujian/CHA/9/99tc and Lys-1141 → Thr and/or Lys-1145 → Gln in VP1 of O/Tibet/CHA/6/99tc may potentially be able to restore infectious viral phenotypes in BHK-21 cells, and be helpful in gaining the HS-utilizing ability of the corresponding site-directed mutants for viral infection in WT-CHO cells. In other words, the specific amino acid residues at positions 1041 (Lys) and 1045 (Lys) in VP1 of O/Tibet/CHA/6/99tc and 2079 (His) in VP2 of O/Fujian/CHA/9/99tc (Table 1), could be detrimental to the genetic response of the indicated viruses to FMDV-HS interaction.


Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor.

Bai X, Bao H, Li P, Wei W, Zhang M, Sun P, Cao Y, Lu Z, Fu Y, Xie B, Chen Y, Li D, Luo J, Liu Z - Virol. J. (2014)

Locations of the distinct amino acid residues in the FMDV capsid of (A) rHN/TAR6-VP0 and (B) rHN/FJ9-VP1. The crystallographic coordinates of O1BFS (missing the first 15 amino acid residues at the N-terminus of VP4) were used as templates[35], and 3D structures of one of the twelve pentamers were optimized using the SWISS-MODEL program[36-38]. The respective capsid proteins VP1–4 are represented as green, cyan, magenta, and blue (internal). The G-H loop of VP1 is highlighted in yellow. The RGD motif (residues 1145–1147, colored green), residues 2079, 2080 (colored red) and 2136 in VP2 of O/Tibet/CHA/6/99tc, as well as residues 1041, 1045, 1083 (colored red), 1095 and 1139 in VP1 of O/Fujian/CHA/9/99tc are displayed in the space-filling models.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4118260&req=5

Figure 5: Locations of the distinct amino acid residues in the FMDV capsid of (A) rHN/TAR6-VP0 and (B) rHN/FJ9-VP1. The crystallographic coordinates of O1BFS (missing the first 15 amino acid residues at the N-terminus of VP4) were used as templates[35], and 3D structures of one of the twelve pentamers were optimized using the SWISS-MODEL program[36-38]. The respective capsid proteins VP1–4 are represented as green, cyan, magenta, and blue (internal). The G-H loop of VP1 is highlighted in yellow. The RGD motif (residues 1145–1147, colored green), residues 2079, 2080 (colored red) and 2136 in VP2 of O/Tibet/CHA/6/99tc, as well as residues 1041, 1045, 1083 (colored red), 1095 and 1139 in VP1 of O/Fujian/CHA/9/99tc are displayed in the space-filling models.
Mentions: Nonetheless, the 3D structural models of rHN/TAR6-VP0 and rHN/FJ9-VP1 indicated that Gln-2080 in the B-C loop of VP2 of O/Tibet/CHA/6/99tc and Lys-1083 within the D-E loop of VP1 of O/Fujian/CHA/9/99tc surround the G-H loop of VP1 and the pore at the fivefold axis of symmetry, respectively (Figure 5A, 5B)[35-38]. The difference in phenotypic properties of the two chimeric viruses (rHN/TAR6-VP0 and rHN/FJ9-VP1) and their site-directed mutants (rHN/TAR6-VP0Q2080L and rHN/FJ9-VP1K1083E) on WT-CHO cells were consistent with our expectations (Figure 1). Yet, the introduction of a Leu-2080 → Gln mutation in VP2 of O/Fujian/CHA/9/99tc (pOFS/FJ9-VP0L2080Q) was particularly deleterious for the generation of progeny virus in BHK-21 cells, and a Glu-1083 → Lys mutation in VP1 of O/Tibet/CHA/6/99tc did not result in the acquisition of the HS-utilizing ability of rHN/TAR6-VP1E1083K to infect WT-CHO cells (Figure 1). Moreover, a second-site mutation (Glu-2136 → Gly) in VP2 of O/Fujian/CHA/9/99tc was unable to compensate for the lethal effect of the primary mutation Leu-2080 → Gln in the VP2 coding region of pOFS/FJ9-VP0L2080Q and the site-directed mutant encoding Glu-1083 → Lys and Ser-1139 → Arg in VP1 of O/Tibet/CHA/6/99tc also could not utilize HS as a receptor to establish an efficient infection in WT-CHO cells (results not shown). There are a limited number of amino acid differences fixed in the VP2 and VP1 coding regions of the mutated plasmids as compared to rHN/TAR6-VP0 and rHN/FJ9-VP1, respectively (Table 1). In particular, the distinct amino acid residues at 2079 (Tyr) of VP2 lies adjacent to the RGD motif (Figure 5A), and (ii) Thr-1041 and Gln-1045 of VP1 are clustered around the fivefold axis (Figure 5B). The outcome of the substitution at residue 2079 of VP2 could potentially dislocate both antigenic sites 1 (1141–1160 residues of the VP1 G-H loop,[39]) and 2 (2070–2078 residues of the VP2 B-C loop,[40]), with a structural and/or functional change[33]. One of the amino acid mutations, Lys-1041 → Glu in VP1 was fixed in two heparin-binding derivatives of C-S8c1 (C-S8c1p100c10 and the MARLS mutant)[20,22]. Accordingly, His-2079 → Tyr in VP2 of O/Fujian/CHA/9/99tc and Lys-1141 → Thr and/or Lys-1145 → Gln in VP1 of O/Tibet/CHA/6/99tc may potentially be able to restore infectious viral phenotypes in BHK-21 cells, and be helpful in gaining the HS-utilizing ability of the corresponding site-directed mutants for viral infection in WT-CHO cells. In other words, the specific amino acid residues at positions 1041 (Lys) and 1045 (Lys) in VP1 of O/Tibet/CHA/6/99tc and 2079 (His) in VP2 of O/Fujian/CHA/9/99tc (Table 1), could be detrimental to the genetic response of the indicated viruses to FMDV-HS interaction.

Bottom Line: However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O.Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells.The results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Veterinary Etiological Biology, OIE/National Foot-and-Mouth Disease Reference Laboratory, Engineering Research Center of Biological Detection of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730046, China. baixingwen@caas.cn.

ABSTRACT

Background: Some cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O. To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells.

Results: Only two of the rescued viruses encoding VP0 of O/Tibet/CHA/6/99tc or VP1 of O/Fujian/CHA/9/99tc formed plaques on wild-type Chinese hamster ovary (WT-CHO; HS+) cells, but not on HS-negative pgsD-677 cells. The formation of plaques by these two chimeric viruses on WT-CHO cells could be abolished by the introduction of single amino acid mutations Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc and Lys-1083 → Glu in VP1 of O/Fujian/CHA/9/99tc, respectively. Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells. Significant differences in the inhibition of the infectivity of four HS-utilizing viruses by heparin and RGD-containing peptide were observed in BHK-21 cells. Interestingly, the chimeric virus encoding VP0 of O/Fujian/CHA/9/99tc, and the site-directed mutant encoding Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc could bind to HS, but there was no expression of the 3A protein of these two viruses in WT-CHO cells.

Conclusion: The results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction.

Show MeSH
Related in: MedlinePlus