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Title: CHARACTERIZATION OF SYNTHETIC FOOT-AND-MOUTH DISEASE VIRUS PROVIRIONS SEPARATES ACID-MEDIATED DISASSEMBLY FROM INFECTIVITY

Author
item KNIPE, TINA - FORMER EMPLOYEE, PIADC
item RIEDER, ELIZABETH - FORMER EMPLOYEE, PIADC
item Baxt, Barry
item Ward Jr, Gordon
item Mason, Peter

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Foot-and-mouth disease virus (FMDV) causes a devastating disease of domestic animals which can be controlled, under some circumstances, with inactivated vaccines. However, these vaccines must be produced by chemical inactivation of large amounts of highly infectious virus, and the fact that commercial vaccines and vaccine production plants have been shown to be sources of outbreaks of the disease have contributed to the decision by the US to outlaw the production and use of this type of vaccine in the US. To overcome the safety problems associated with the use of an animal-infectious virus as the starting material for these vaccines, we are exploring the development of attenuated strains of the virus. To this end, we have generated genetically engineered viruses that specify new amino acids at the junction nbetween two virus proteins. This junction is normally cleaved by a viral enzyme. Viruses that have unnatural amino acid residues at this cleavage site are able to produce viruses that look "normal" but are non- infectious. This information will assist in future preparations of genetically-engineered vaccines for FMD.

Technical Abstract: One of the final steps in the maturation of foot-and-mouth disease virus (FMDV) is cleavage of the VP0 protein to produce VP4 and VP2. The mechanism of this cleavage is unknown, but is thought to function in stabilizing the virus particle and priming it for infecting cells. To investigate the cleavage process and to understand its role in virion maturation, we engineered synthetic FMDV RNAs with mutations at Ala 85 (A85) and Asp 86 (D86) of VP0, which border the cleavage site. Baby hamster kidney (BHK) cells transfected with synthetic RNAs containing substitutions at position 85 (A85N or A85H), or at position 86 (D86N) yielded particles indistinguishable from wild-type (WT) virus in sedimentation and electrophoretic profiles. Viruses derived from these transfected cells were infectious and maintained their mutant sequences upon passage. However, BHK cells transfected with synthetic RNAs encoding Phe and Lys at these positions (A85F/D86K) or a Cys at position 86 (D86C) produced non-infectious provirions with uncleaved VP0 molecules. Despite its lack of infectivity, the A85F/D86K provirions displayed cell binding and the acid sensitivity similar to WT virus. However, acid breakdown products of the A85F/D86K provirions differed in hydrophobicy from the comparable WT virion products. Taken together, these studies support a role for VP0 cleavage in the production of structural protein components that are capable of facilitating release of viral genome from the endosomal compartment of susceptable cells.