|De Los Santos, Teresa
|PICCONE, MARIA - UNIV OF CONNECTICUT
|DIAZ-SAN SEGUNDO, FAYNA - USDA, ARS ORISE FELLOW
|KRAMER, EDWARD - 1940-35-00 COLLABORATOR
Submitted to: American Society for Virology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2009
Publication Date: 7/10/2009
Citation: De Los Santos, T.B., Piccone, M.E., Diaz-San Segundo, F., Pauszek, S.J., Kramer, E.V., Rodriguez, L.L. 2009. Exploring the role of the lab protein of Foot-and-Mouth Disease Virus (FMDV) during viral Infection. American Society for Virology Meeting. Paper No. 32-7: p. 337.
Technical Abstract: The leader (L) protein of Foot-and-Mouth Disease Virus (FMDV) displays two forms, Lab and Lb, through initiation of translation at two in-frame AUG codons positioned 84 nucleotides apart. The short form (Lb) is the most abundant and functionally well characterized form of L. The presence of these two forms of the L protein is puzzling since both exhibit similar proteolytic activities on the viral polyprotein and the translation factor eIF-4G. . Recently, we have shown that introduction of a stable RNA stem-loop structure in the region of the genome located between the two functional AUGs, shifts start codon preference from the second to the first AUG (Lab form) and results in severe attenuation of viruses in cattle. To investigate the role of the Lab protein in virus pathogenesis, we have introduced peptide tag sequences within this region: Flag (DYKDDDK), Influenza-Hemagglutinin (HA) (YPYDVPDYA) and tetracysteine peptide (CCPGCC). Mutant viruses containing Flag or tetracysteine tag sequences were obtained. These viruses showed small plaque phenotype compared to the wild-type virus in BHK, LF-BK, and primary lamb kidney (LK) cells. Analysis of viral proteins on SDS-PAGE indicated initiation of translation upstream from the second AUG and showed that the presence of sequences coding for the tags did not interfere with the proteolytic processing of the viral polyprotein. However, expression of the Flag tag could not be detected by immunoprecipitation or by western blot analysis when an anti-flag antibody was used. Visualization of Lab protein with the tetracysteine tag may reveal different properties of the two forms of the L protein during infection.