Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase.
ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.
Shibata
S
S
Laboratory of Virology, Nagoya University School of Medicine, Japan.
Yamamoto-Goshima
F
F
Maeno
K
K
Hanaichi
T
T
Fujita
Y
Y
Nakajima
K
K
Imai
M
M
Komatsu
T
T
Sugiura
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000648:ultrastructure; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics*; D006358:Hot Temperature; D009981:Influenza B virus / Q000235:genetics* / Q000254:growth & development* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics*
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D008565:Membrane Proteins; D014764:Viral Proteins; D009439:Neuraminidase; D014357:Trypsin