Sequence-independent acylation of the vaccinia virus A-type inclusion protein.

Abstract

N-Terminal myristoylation of proteins typically occurs cotranslationally via an amide bond to the penultimate glycine residue within the canonical motif (M)GXXX(S/T/A) in a reaction catalyzed by N-myristoyltransferase. A second, less common myristoylation reaction occurs internally at dibasic amino acid doublets of proteins such as alpha-TNF. In this case, myristoylation occurs within a portion of the preprotein, which is subsequently removed by N-terminal proteolysis. The identity of the enzyme catalyzing internal myristoylation is unknown. Considering this information, the vaccinia virus (VV) A-type inclusion protein (ATI) presents a conundrum. Although this cytosolic protein is clearly myristoylated, the protein does not have the N-terminal myristoylation motif nor is it subject to proteolytic maturation. In the experiments reported here, we cleaved VV ATI with cyanogen bromide and determined that the myristoyl moiety was present in the C-terminal half of the protein. We also subjected a tryptic digest of VV ATI to liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry analyses, which indicated that ATI is randomly myristoylated at six different lysines or arginines. Analysis of the modification sites reveals no obvious conserved acceptor motifs or dibasic doublets. Mutation of these residues alone or in combination does not abrogate myristoylation of the protein, suggesting utilization of alternative modification sites. This information implies that the VV ATI protein is myristoylated in a sequence-independent manner. Because viral acylproteins typically utilize the host cell modification apparatus, this result suggests there may be an alternative type of myristoylation pathway in mammalian cells.