Influenza C virus CM2 protein is produced from a 374-amino-acid protein (P42) by signal peptidase cleavage

Although unspliced mRNA from influenza C virus RNA segment 6 (M gene) has a single open reading frame capable of encoding a 374-amino-acid protein (M- r, 42,000), the major polypeptide synthesized from this mRNA species is the CM2 protein, with an M-r of 18,000, The present study was performed to inv estigate the mechanism by which CM2 is generated from the unspliced mRNA. I t was reported previously that the 374-amino-acid protein (P42) is an integ ral membrane protein having two internal hydrophobic domains, one of which (residues 241 to 252) is followed by two sequences (252 Ile-Thr-Ser and 257 Ala-Ser-Ala) favorable for cleavage by signal peptidase, To examine the po ssibility that P42 is cleaved by signal peptidase after Ser residue 254 or Ala residue 259 to yield CM2, we constructed three mutated M gene cDNAs in which either or both of the two sequences were eliminated and tested their ability to synthesize CM2 in the transfected COS cells. The results showed that CM2 synthesis was blocked completely when the second recognition motif for signal peptidase was removed. It was also found that when the mRNA tra nscript of the wild-type M gene was translated in vitro, P42, but not CM2, was synthesized in the absence of dog pancreas microsomal membranes, wherea s CM2, in addition to a polypeptide (designated M1') slightly larger than m atrix protein (M1), was synthesized in the presence of microsomes. When the same experiment was done with the transcript of the mutated M gene in whic h the second recognition motif was removed, synthesis of CM2 could not be s een, even in the presence of microsomes, From these results, we conclude th at cleavage of P42 by signal peptidase after Ala residue 259 produces CM2, composed of the C-terminal 115 amino acids, in addition to M1', composed of the N-terminal 259 amino acids.