MEMBRANE-BINDING DOMAINS AND CYTOPATHOGENESIS OF THE MATRIX PROTEIN OF VESICULAR STOMATITIS-VIRUS

The membrane-binding affinity of the matrix (M) protein of vesicular s tomatitis virus (VSV) was examined by comparing the cellular distribut ion of wild-type (wt) virus M protein with that of temperature-sensiti ve (ts) and deletion mutants probed by indirect fluorescent-antibody s taining and fractionation of infected or plasmid-transfected CV1 cells . The M-gene mutant tsO23 caused cytopathic rounding of cells infected at permissive temperature but not of cells at the nonpermissive tempe rature; wt VSV also causes rounding, which prohibits study of M protei n distribution by fluorescent-antibody staining. Little or no M protei n can be detected in the plasma membrane of cells infected with tsO23 at the permissive temperature, whereas similar to 20% of the M protein colocalized with the membrane fraction of cells infected with tsO23 a t the permissive temperature. Cells transfected with a plasmid express ing intact 220-amino-acid wt M protein (M1-229) exhibited cytopathic c ell rounding and actin filament dissolution, whereas cells retained no rmal polygonal morphology and actin filaments when transfected with pl asmids expressing M proteins truncated to the first 74 N-terminal amin o acids (M1-74) or deleted of the first 50 amino acids 1 to 50 and 75 to 106 (M51-74/107-229). Truncated proteins M1-74 and M51-229 were rea dily detectable in the plasma membrane and cytosol of transfected cell s as determined by both fluorescent-antibody staining cell fractionati on, as was the plasmid-expressed intact we M protein. However, the exp ressed double deleted protein M51-74/107-229 could not be detected in plasma membrane by fluorescent-antibody staining or by cell fractionat ion, suggesting the presence of two membrane-binding sites spanning th e region of amino acids 1 to 50 and amino acids 75 to 106 of the VSV M protein. These in vivo data were confirmed by an in invitro binding a ssay in which intact M protein and its deletion mutants were reconstit uted in high- or low-ionic-strength buffers with synthetic membranes i n the form of sonicated unilammelar vesicles. The results of these exp eriments appear to confirm the presence of two membrane-binding sites on the VSV M protein, one binding peripherally by electrostatic forces at the highly charged NH2 terminus and the other stably binding membr ane integration of hydrophobic amino acids and located by a hydropathy plot between amino acids 88 and 119.