Effects of a temperature sensitivity mutation in the J1R protein component of a complex required for vaccinia virus assembly.
Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol. 76:9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus Cts45. The mutant Cts45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39 degrees C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with Cts45 at 39 degrees C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.
Chiu
Wen-Ling
WL
Institute of Molecular Biology, Academia Sinica, 128, Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan, Republic of China.
Szajner
Patricia
P
Moss
Bernard
B
Chang
Wen
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D005816:Genetic Complementation Test; D005819:Genetic Markers; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009024:Morphogenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D014616:Vaccinia virus / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014771:Virion / Q000235:genetics
D005819:Genetic Markers; D014764:Viral Proteins