Foot-and-mouth disease virus (FMDV), which is a member of the Aphthovirus genus of Picornaviridae, is an economically important animal virus that infects cloven-hoofed animals. At least two families of cell-surface receptors have been identified that mediate binding of FMDV to susceptible cells, i.e. integrins and heparan sulfate proteoglycans (HSPG). Whereas several tissue culture-adapted FMD viruses use heparan sulfate proteoglycans for internalization, field isolates of FMDV use integrins as receptors. A conserved amino acid sequence Arg-Gly-Asp (RGD), located in the surface-exposed flexible ƒÒG-ƒÒH loop of the viral outer capsid protein 1D, participates in binding FMDV to integrin receptors on the surface of susceptible cells. Sequence analyses of the 1D-encoding genome region of a SAT1 type FMDV field isolate, NAM/307/98, indicated the presence of a second RGD sequence upstream of the conserved G-H loop RGD sequence. Since FMDV is capable of binding to RGD-binding integrin receptors, the aim of this study was to investigate whether the second RGD motif in capsid protein 1D of NAM/307/98 may function as a ligand for receptor-binding in baby hamster kidney (BHK) cells. Towards this end, a cDNA copy of the genomic region encoding the external capsid proteins (1B-1D) of NAM/307/98 was cloned into pSAT2, a genome-length cDNA clone derived from the SAT2 strain ZIM/7/83. Transfection of BHK-21 cells with in vitro-transcribed RNA derived from the chimeric pNAM/SAT2 clone resulted in the recovery of infectious chimeric virus particles. The availability of such an infectious chimeric cDNA clone greatly facilitated the introduction of specific, targeted mutations in the 1D capsid-encoding region of NAM/307/98 in order to investigate the functional role of the second RGD sequence. Using the chimeric SAT1/SAT2 cDNA clone as template, the RGD codons in the G-H loop of NAM/307/98 were replaced with codons specifying a KGE sequence by a polymerase chain reaction (PCR)-based method of site-directed mutagenesis. The mutated DNA fragment was introduced into the pSAT2 infectious cDNA clone, transcribed in vitro, and the resulting RNA transfected into BHK-21 cells. The transfected cells were analyzed for cytopathic effect (CPE). In contrast to cells transfected with non-mutated RNA transcripts, from which infectious virus could be recovered, cells transfected with the mutant RNA transcripts showed cell lysis, but no CPE could be observed upon subsequent passaging of the resultant viruses on BHK cells. Notably, subsequent replacement of the KGE sequence with an RGD sequence in the mutant clones led to recovery of infectious viruses. Furthermore, RNA replication could be demonstrated with the mutant and non-mutant chimeric viruses, suggesting that virus particles were indeed present in the tissue culture supernatants of all transfected cells. Based on the results obtained during the course of this investigation, it was therefore concluded that the second RGD motif, situated upstream of the conserved RGD motif in the G-H loop of capsid protein 1D of NAM/307/98, does not function as a ligand for receptor-binding in BHK-21 cells.
Dissertation (MSc (Microbiology))--University of Pretoria, 2007.