Positively Charged Residues at the Five-Fold Symmetry Axis of Cell Culture-Adapted Foot-and-Mouth Disease Virus Permit Novel Receptor Interactions

Abstract

Field isolates of foot-and-mouth disease virus (FMDVF oot-and-mouth disease (FMD) is endemic in many regions of the world and is one of the most widespread, epizootic transboundary animal diseases, affecting many species of wildlife and livestock, such as cattle, sheep, goats, and pigs. The significant economic losses that result from FMD are due to the high morbidity of infected animals and stringent trade restrictions imposed on affected countries (1). Vaccination plays a major role in controlling FMD, either to lessen the effects of an outbreak in FMDfree countries or for control and eradication in regions where it is endemic. The etiological agent of FMD, foot-and-mouth disease virus (FMDV), exists as seven distinct serotypes (O, A, C, Asia-1, and the Southern African Territories [SAT] serotypes SAT-1, SAT-2, and SAT-3). Within each serotype, a large number of antigenic variants exist (2). Intraserotype diversity is driven by a high mutation rate during replication that is caused by an error-prone viral RNA-dependent RNA polymerase (3) and thus complicates efforts to control disease by vaccination due to incomplete protection between some antigenic variants (4). Hence, the most effective vaccines closely match the outbreak virus, which can necessitate the development of new vaccine strains. The current vaccines are inactivated virus preparations grown in large-scale cell culture. Therefore, the production of a new vaccine is critically dependent upon adaptation of viruses from the field for growth in cell culture, which can prove problematical for some viruses.Foot-and-mouth disease virus is the type species of the Aphthovirus genus of the Picornaviridae, a family of nonenveloped, single-stranded positive-sense RNA viruses. The viral capsid is formed by 60 copies each of four structural proteins (VP1 to VP4) arranged in icosahedral symmetry. The outer capsid surfaces are formed by VP1, which surrounds the five-fold symmetry axis, and VP2 and VP3, which alternate around the three-fold axis (5). VP4 is myristoylated and located inside the capsid and is thought to play an essential role in the final stage of assembly and in endosomal membrane penetration by the viral RNA (6, 7). In vivo, FMDV has a strong tropism for epithelial cells, which is in part due to the epithelial cell-restricted expression of integrin ␣v␤6, which is the principal receptor used by field viruses to initiate infection (8-12). Integrin binding is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) motif located at the apex of a structurally disordered loop (the GH loop of VP1). The integrin specificity of FMDV has been the subject of several studies, and three other RGD-dependent integrins (␣v␤1, ␣v␤3, and ␣v␤8) have also been reported to be receptors for field strains of the virus (13-15); however, the role of these integrins in pathogenesis is unclear, and we have found that ␣v␤3 is a poor receptor for FMDV in vitro (16). Furthermore, despite recognizing their ligands via the RGD motif, two other RGD-dependent integrins ...

Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions

“…In the coinfection competition assays, those cell-culture-adapted viruses swiftly outcompeted their parental competitors after only a limited number of passages (P 1-5 ), consistent with the presumed greater biological fitness the positively charged residues impart to the virus in cell culture (Table 4). Since CHO-K1 cells do not express any 'RGD'-binding integrin known to be used by FMDV, entry into these cells is postulated to be mediated by either HS or some non-integrin, non-HS receptors (Berryman et al, 2013;Jackson et al, 1996). While none of the viruses before gaining positively charged residues (P 0 or low passage populations) could replicate in CHO-K1 cells, the cell-culture-adapted variants manifested cytopathic effect, uniform small plaques (,1 mm diameter) and increase in virus titre between 1 and 24 h postinfection of CHO-K1 cells (Table 3).…”

“…Therefore, it is of interest to map critical amino acid substitutions on the capsid accounting for the plasticity in receptor interaction. More importantly, they could also be potential leads for the rational design of cell-culture-adaptable vaccine seed strains through a reverse genetic approach (Berryman et al, 2013;Maree et al, 2011). In this study, we explored the sequence hotspots of probable adaptive significance in cell culture by comparing the capsid coding (P1) sequences and threedimensional structures generated thereof for multiple strains of FMDV serotype A representing WT viruses derived directly from infected cattle tongue epithelium and after passaging in three cell lines originating from different animal species.…”

The Novel Oncolytic Adenoviral Mutant Ad5-3Δ-A20T Retargeted to αvβ6 Integrins Efficiently Eliminates Pancreatic Cancer Cells

“…The affinity to avb6 integrins was high, measured at K D ¼ 0.22 nmol/L, whereas binding to other integrins, including avb3, avb5, and avb1, was more than 80-fold lower (21,25). The selective binding to avb6 integrins was shown to be dependent on the DLXXL motif in the carboxy a-helical loop next to the RGD domain at the apex of the loop domain in the peptide (23,26,44,45).…”

“…We generated the replication-selective integrin-targeted Ad5-3D-A20T mutant expressing the small avb6-peptide ligand A20FMDV2. The A20FMDV2 peptide was derived from the GH-loop in VP1 in the capsid of the FMDV and is essential for infection in clovenhoofed animals (44). The affinity to avb6 integrins was high, measured at K D ¼ 0.22 nmol/L, whereas binding to other integrins, including avb3, avb5, and avb1, was more than 80-fold lower (21,25).…”

Challenges and prospects for the control of foot-and-mouth disease: an African perspective

“…112,129,[133][134][135][136][137][138] Furthermore, alternative cell entry pathways exist and can be applied in future to improve cell adaptation. 134,135,139 Continued reverse genetic approaches to rapidly adapt African FMDV to BHK-21 cell culture will greatly enhance the ability to produce region-specific vaccines tailored to current circulating strains.…”