Foot-and-mouth disease virus (FMDV) utilizes four integrins (αvβ1, αvβ3, αvβ6, and αvβ8) as its primary cell receptor. During cell culture propagation, FMDV frequently adapts to use heparan sulfate (HS), and rarely utilizes an unidentified third receptor. Capsid mutations acquired by a soluble integrin resistant FMDV cause (i) adaptation to CHO-677 cells (ii) increased affinity to membrane-bound Jumonji C-domain containing protein 6 (JMJD6) (iii) induced JMJD6 re-localization from the cell surface and cytoplasm to the nucleus. Interestingly, pre-treatment of cells with N- and C-terminal JMJD6 antibodies or by simultaneous incubation of mutant virus with soluble JMJD6 (but not by treatment with HS or αvβ6) impaired virus infectivity in cultured cells. JMJD6 and mutant virus co-purified by reciprocal co-immunoprecipitation. Molecular docking predictions suggested JMJD6 C-terminus interacts with mutated VP1 capsid protein. We conclude when specific VP1 mutations are displayed, JMJD6 contributes to FMDV infectivity and may be a previously unidentified FMDV receptor.
The foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) is a papain like protease that cleaves the viral polyprotein and several host factors affecting host cell translation and induction of innate immunity. Introduction of Lpro mutations ablating catalytic activity is not tolerated by the virus, however, complete coding sequence deletion or introduction of targeted amino acid substitutions can render viable progeny. In proof-of-concept studies, we have previously identified and characterized FMDV Lpro mutants that are attenuated in cell culture and in animals, while retaining their capacity for inducing a strong adaptive immunity. By using molecular modeling, we have now identified a His residue (H138), that resides outside the substrate binding and catalytic domain, and is highly conserved across serotypes. Mutation of H138 renders possible FMDV variants of reduced virulence in vitro and in vivo. Kinetics studies showed that FMDV A12-LH138L mutant replicates similarly to FMDV A12-wild type (WT) virus in cells that do not offer immune selective pressure, but attenuation is observed upon infection of primary or low passage porcine epithelial cells. Western blot analysis on protein extracts from these cells, revealed that while processing of translation initiation factor eIF-4G was slightly delayed, no degradation of innate sensors or effector molecules such as NF-κB or G3BP2 was observed, and higher levels of interferon (IFN) and IFN-stimulated genes (ISGs) were induced after infection with A12-LH138L as compared to WT FMDV. Consistent with the results in porcine cells, inoculation of swine with this mutant resulted in a mild, or in some cases, no clinical disease but induction of a strong serological adaptive immune response. These results further support previous evidence that Lpro is a reliable target to derive numerous viable FMDV strains that alone or in combination could be exploited for the development of novel FMD vaccine platforms.
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