Ability of foot-and-mouth disease virus to form plaques in cell culture is associated with suppression of alpha/beta interferon.
(17/503)
A genetic variant of foot-and-mouth disease virus lacking the leader proteinase coding region (A12-LLV2) is attenuated in both cattle and swine and, in contrast to wild-type virus (A12-IC), does not spread from the initial site of infection after aerosol exposure of bovines. We have identified secondary cells from susceptible animals, i.e., bovine, ovine, and porcine animals, in which infection with A12-LLV2, in contrast to A12-IC infection, does not produce plaques; this result indicates that this virus cannot spread from the site of initial infection to neighboring cells. Nevertheless, A12-LLV2 can infect these cells, but cytopathic effects and virus yields are significantly reduced compared to those seen with A12-IC infection. Reverse transcription-PCR analysis demonstrates that both A12-LLV2 and A12-IC induce the production of alpha/beta interferon (IFN-alpha/beta) mRNA in host cells. However, only supernatants from A12-LLV2-infected cells have significant antiviral activity. The antiviral activity in supernatants from A12-LLV2-infected embryonic bovine kidney cells is IFN-alpha/beta specific, as assayed with mouse embryonic fibroblast cells with or without IFN-alpha/beta receptors. The results obtained with cell cultures demonstrate that the ability of A12-IC to form plaques is associated with the suppression of IFN-alpha/beta expression and suggest a role for this host factor in the inability of A12-LLV2 to spread and cause disease in susceptible animals. (+info)
Long-range RNA interactions between structural domains of the aphthovirus internal ribosome entry site (IRES).
(18/503)
Internal initiation of translation is promoted by internal ribosome entry site (IRES) cis-acting elements. Using transcripts that correspond to the structural domains of the foot-and-mouth disease virus (FMDV) IRES, we have identified RNA-RNA interactions between separated domains (1-2, 3, 4-5, or HH) of the IRES structure. All the assayed domains were able to interact with the full-length IRES as well as with domain 3, although to a different extent, with the most efficient interactions being those occurring between domains 3 and 4-5, and domains 3 and 1-2. RNA-RNA complexes were stable over 1 h of incubation at 37 degrees C, and depended on Mg2+ and RNA concentration. Neither the antisense domain 1-2 nor tRNA interacted with domain 3, providing experimental evidence of the specificity for the sense strand of the IRES sequence. Additionally, domain 1-2 did not interact with 4-5, leading to the suggestion that domain 3 acts as a scaffold structure where the other domains bind. The thermal disassociation profile of these complexes indicated different strength in these interactions. Whereas 50% of the complexes between domains 3 and 4-5 were destabilized at 45 degrees C, those formed by domain 1-2 and 3 required temperatures higher than 51 degrees C. Efficient self-dimerization of domains 3 and 4-5 was found in the absence of other transcripts. Formation of domain 3 homodimer competed with formation of heterocomplexes with other domains, and conversely, domain 3 homodimers were competed out by the presence of the other domains. RNA interactions were also observed at physiological concentrations of Mg2+ and K1+. The identification of the RNA-RNA complexes reported here provide direct experimental evidence of tertiary interactions within IRES elements. (+info)
Genetic determinants of altered virulence of Taiwanese foot-and-mouth disease virus.
(19/503)
In 1997, a devastating outbreak of foot-and-mouth disease (FMD) in Taiwan was caused by a serotype O virus (referred to here as OTai) with atypical virulence. It produced high morbidity and mortality in swine but did not affect cattle. We have defined the genetic basis of the species specificity of OTai by evaluating the properties of genetically engineered chimeric viruses created from OTai and a bovine-virulent FMD virus. These studies have shown that an altered nonstructural protein, 3A, is a primary determinant of restricted growth on bovine cells in vitro and significantly contributes to bovine attenuation of OTai in vivo. (+info)
Cell recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: flexibility in aphthovirus receptor usage.
(20/503)
Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-143-->Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin alpha(v)beta(3). In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type. (+info)
Engineering cowpea mosaic virus RNA-2 into a vector to express heterologous proteins in plants.
(21/503)
A series of new cowpea mosaic virus (CPMV) RNA-2-based expression vectors were designed. The jellyfish green fluorescent protein (GFP) was introduced between the movement protein (MP) and the large (L) coat protein or downstream of the small (S) coat protein. Release of the GFP inserted between the MP and L proteins was achieved by creating artificial processing sites each side of the insert, either by duplicating the MP-L cleavage site or by introducing a sequence encoding the foot-and-mouth disease virus (FMDV) 2A catalytic peptide. Eight amino acids derived from the C-terminus of the MP and 14-19 amino acids from the N-terminus of the L coat protein were necessary for efficient processing of the artificial Gln/Met sites. Insertion of the FMDV 2A sequence at the C-terminus of the GFP resulted in a genetically stable construct, which produced particles containing about 10 GFP-2A-L fusion proteins. Immunocapture experiments indicated that some of the GFP is present on the virion surface. Direct fusion of GFP to the C-terminus of the S coat protein resulted in a virus which was barely viable. However, when the sequence of GFP was linked to the C-terminus by an active FMDV 2A sequence, a highly infectious construct was obtained. (+info)
Memory in viral quasispecies.
(22/503)
Biological adaptive systems share some common features: variation among their constituent elements and continuity of core information. Some of them, such as the immune system, are endowed with memory of past events. In this study we provide direct evidence that evolving viral quasispecies possess a molecular memory in the form of minority components that populate their mutant spectra. The experiments have involved foot-and-mouth disease virus populations with known evolutionary histories. The composition and behavior of the viral population in response to a selective constraint were influenced by past evolutionary history in a way that could not be predicted from examination of consensus nucleotide sequences of the viral populations. The molecular memory of the viral quasispecies influenced both the nature and the intensity of the response of the virus to a selective constraint. (+info)
Foot-and-mouth disease virus is a ligand for the high-affinity binding conformation of integrin alpha5beta1: influence of the leucine residue within the RGDL motif on selectivity of integrin binding.
(23/503)
Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors for internalization, whereas strains that are adapted for growth in cultured cell lines appear to be able to use alternative receptors like heparan sulphate proteoglycans (HSPG). The ligand-binding potential of integrins is regulated by changes in the conformation of their ectodomains and the ligand-binding state would be expected to be an important determinant of tropism for viruses that use integrins as cellular receptors. Currently, alphavbeta3 is the only integrin that has been shown to act as a receptor for FMDV. In this study, a solid-phase receptor-binding assay has been used to characterize the binding of FMDV to purified preparations of the human integrin alpha5beta1, in the absence of HSPG and other RGD-binding integrins. In this assay, binding of FMDV resembled authentic ligand binding to alpha5beta1 in its dependence on divalent cations and specific inhibition by RGD peptides. Most importantly, binding was found to be critically dependent on the conformation of the integrin, as virus bound only after induction of the high-affinity ligand-binding state. In addition, the identity of the amino acid residue immediately following the RGD motif is shown to influence differentially the ability of FMDV to bind integrins alpha5beta1 and alphavbeta3 and evidence is provided that alpha5beta1 might be an important FMDV receptor in vivo. (+info)
Interspecies major histocompatibility complex-restricted Th cell epitope on foot-and-mouth disease virus capsid protein VP4.
(24/503)
T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4(+) CD8(+) cells-dominated by Th memory cells in the pig-and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines. (+info)