Cytoskeletal association of an aphthovirus-induced polypeptide derived from the P3ABC region of the viral polyprotein.

Monolayers of BHK cells infected with aphthovirus (FMDV) were labeled for short times with [35S]methionine at 2 hr p.i. and fractionated by detergent treatment and low speed centrifugation. Polyacrylamide gel analysis showed an asymmetric distribution of the FMDV-induced polypeptides among the three different subcellular fractions obtained. Polypeptide P88-1, the viral capsid protein precursor, is mainly found in the soluble cytoplasmic extract while polypeptides P100-3, P52-2AC, P34-2C, and P14-2A are the major viral components of a detergent soluble extract of the crude nuclear pellet. Analysis of the detergent resistant fraction (DRF), which is mainly composed of cell nuclear chromatin and insoluble cytoskeletal elements, shows a clear enrichment in an incompletely characterized polypeptide which is tentatively designated P54. Variable amounts of polypeptides P100-3 and those of the P72 complex are also detected in this fraction. The preferential location of P54 in an equivalent subcellular fraction obtained by mild detergent treatment of infected monolayers in situ, and also in a high-salt resistant subfraction of the DRF, strongly suggests a close association of this polypeptide with vimentin-actin containing components of the cell. Polypeptide P54 is immunoprecipitated by viral specific antiserum from convalescent guinea pigs but not by serum against FMDV capsid proteins, indicating that it does not share common antigenic determinants with polypeptides processed from the viral capsid precursors. On the other hand, protease V8 mapping of polypeptides P100-3, P54, P88-1, and VP1-3 shows that P54 derived from the 3' end coding region of the viral genome. Further analysis by limited protease digestion also demonstrates that P54 has partial overlap with P72-3CD while it does not share any common peptide with P56a-3D, indicating that P54 contains the sequences coded in the 3ABC region of the FMDV RNA. This assumption is reinforced by the basic behavior shown by P54 in two-dimensional gels. The results support the hypothesis of a close intracellular interaction of a short-lived polypeptide, containing the viral protease and VPg sequences, with the host cytoskeleton, during infection of BHK cells with FMDV.