title: Active influenza virus neuraminidase is expressed in monkey cells from cDNA cloned in simian virus 40 vectors.
abstract: We have replaced the late genes of simian virus 40 (SV40) with a cloned cDNA copy of the neuraminidase (NA; EC 3.2.1.18) gene of the WSN (H1N1) strain of human influenza virus. When the SV40-NA recombinant virus was complemented in a lytic infection of monkey cells with a helper virus containing an early region deletion mutant, influenza NA was expressed and readily detected by immunofluorescence as well as by immunoprecipitation of in vivo labeled proteins with monoclonal antibodies against NA. In addition, the expressed NA exhibited enzymatic activity by cleaving the sialic acid residue from alpha-2,3-sialyllactitol. The expressed protein was glycosylated and transported to the cell surface, and it possessed the same molecular weight as the NA of WSN virus grown in monkey cells. Because the structure of NA is quite different from that of other integral membrane proteins and includes an anchoring region at the NH2 terminus consisting of hydrophobic amino acids, we also constructed deletion mutants of NA in this region. Replacement of DNA coding for the first 10 NH2-terminal amino acids with SV40 and linker sequences had no apparent effect on NA expression, glycosylation, transport to the cell surface, or enzymatic activity. However, further deletion of NA DNA encoding the first 26 amino acids abolished NA expression. These data suggest that the hydrophobic NH2-terminal region is multifunctional and is important in biosynthesis and translocation of NA across the membrane as well as in anchoring the protein.
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D009439:Neuraminidase / Q000235:genetics*; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D004247:DNA; D004262:DNA Restriction Enzymes; D009439:Neuraminidase