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Journal of Virology, October 2004, p. 10238-10248, Vol. 78, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.19.10238-10248.2004

Vaccinia Virus Mutants with Alanine Substitutions in the Conserved G5R Gene Fail To Initiate Morphogenesis at the Nonpermissive Temperature

Flavio G. da Fonseca,{dagger} Andrea S. Weisberg, Maria F. Caeiro,{ddagger} and Bernard Moss*

Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

Received 1 May 2004/ Accepted 25 May 2004

The initial characterization of the product of the vaccinia virus G5R gene, which is conserved in all poxviruses sequenced to date, is described. The G5 protein was detected in the core fraction of purified virions, and transcription and translation of the G5R open reading frame occurred early in infection, independently of DNA replication. Attempts to delete the G5R gene and isolate a replication-competent virus were unsuccessful, suggesting that G5R encodes an essential function. We engineered vaccinia virus mutants with clusters of charged amino acids changed to alanines and determined that several were unable to replicate at 40°C but grew well at 37°C. At the nonpermissive temperature, viral gene expression and DNA replication and processing were unperturbed. However, tyrosine phosphorylation and proteolytic cleavage of the A17 membrane protein and proteolytic cleavage of core proteins were inhibited at 40°C, suggesting an assembly defect. The cytoplasm of cells that had been infected at the nonpermissive temperature contained large granular areas devoid of cellular organelles or virus structures except for occasional short crescent-shaped membranes and electron-dense lacy structures. The temperature-sensitive phenotype of the G5R mutants closely resembled the phenotypes of vaccinia virus mutants carrying conditionally lethal F10R protein kinase and H5R mutations. F10, although required for phosphorylation of A17 and viral membrane formation, was synthesized by the G5R mutants under nonpermissive conditions. An intriguing possibility is that G5 participates in the formation of viral membranes, a poorly understood event in poxvirus assembly.

* Corresponding author. Mailing address: Laboratory of Viral Diseases, National Institutes of Health, 4 Center Dr., MSC 0445, Bethesda, MD 20892-0445. Phone: (301) 496-9869. Fax: (301) 480-1147. E-mail: bmoss{at}nih.gov.

{dagger} Present address: Laboratório de Imunologia Celular e Molecular, Centro de Pesquisas René Rachou-FIOCRUZ, Bairro Barro Preto, Belo Horizonte, MG, Brazil.

{ddagger} Present address: Department of Vegetal Biology, Faculty of Sciences, University of Lisbon, Lisbon, Portugal.

Journal of Virology, October 2004, p. 10238-10248, Vol. 78, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.19.10238-10248.2004




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