Analysis of the site occupancy constraints of primary amino acid sequences in the motif directing palmitylation of the vaccinia virus 37-kDa envelope protein

Abstract

Vaccinia virus (VV) encodes a 37-kDa envelope protein (p37) that is palmitylated on cysteine residues 185 and 186 of the 372-amino acid protein. We have previously reported on a loosely conserved consensus motif. Further analysis has identified a conserved consensus sequence, Hydro*AAC(C)A (Hydro* represents a hydrophobic portion of a protein determined by any one of the following: a hydrophobic sequence, a transmembrane domain 1-12 amino acids away from the modification site, or the prior addition of a hydrophobic molecule; C, palmitate acceptor cysteines; A, aliphatic residue) that is responsible for directing palmitylation of certain classes of palmitylproteins. We have analyzed the amino acid site occupancy upstream and downstream of the palmitate acceptor residues in p37 by site-directed mutagenesis and transient expression of mutated proteins in VV-infected cells. The two aliphatic alanines naturally found at positions 183 and 184 of the wild-type p37 allow for efficient palmitylation. In contrast, the replacement of leucine at position 187 with glycine increases palmitylation efficiency. The 10 amino acids immediately upstream of the palmitate acceptor site are absolutely necessary while the downstream 10 amino acids are dispensable. These results together with previous data suggests that the Hydro*AAC(C)A motif is required for efficient palmitylation of p37.