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J Virol, February 1998, p. 1287-1296, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Vaccinia Virus 15-Kilodalton (A14L) Protein Is Essential for Assembly and Attachment of Viral Crescents to Virosomes

Juan Ramón Rodríguez,1 Cristina Risco,2 José L. Carrascosa,2 Mariano Esteban,1,* and Dolores Rodríguez1

Departments of Molecular and Cellular Biology1 and Macromolecular Structure,2 Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma, 28049 Madrid, Spain

Received 18 August 1997/Accepted 14 October 1997

Early stages in vaccinia virus (VV) assembly involve the recruitment of cellular membranes from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to virus factories (or virosomes). The key viral factors involved in this process are not yet known. We have previously identified and characterized two viral proteins, of 21 kDa (A17L gene) and 15 kDa (A14L gene), that associate with tubulovesicular elements related to the ERGIC and are localized in viral membranes at all stages of virion assembly. We showed that the 21-kDa protein is not responsible for the recruitment of membranes from the ERGIC to viral factories. However, it appears to be essential for the organization of viral membranes. In this investigation we have generated a VV recombinant, VVindA14L, in which the expression of the A14L gene is inducibly regulated by the Escherichia coli lacI operator-repressor system. Repression of 15-kDa protein synthesis has a dramatic effect on virus yields and severely impairs plaque formation. Compared to wild-type VV, reduced amounts of 15-kDa protein are produced in VVindA14L-infected cells in the presence of IPTG (isopropyl-beta -D-thiogalactoside), and this correlates with a small-plaque phenotype and reduced VVindA14L yields under these conditions. In the absence of the 15-kDa protein, early and late viral protein syntheses proceed normally; however, proteolytic cleavage of the major core precursors is inhibited. Electron microscopic examination of cells infected with VVindA14L under nonpermissive conditions reveals the presence of numerous membranous elements that look like unfinished or disassembled crescents interespersed between electron-dense masses. These abnormal membrane elements are usually well separated from the surfaces of the dense structures. These findings show that the 15-kDa protein is essential for VV morphogenesis and indicate that this polypeptide is necessary both for the correct assembly of viral crescents and for their stable attachment to the surfaces of viral factories.

* Corresponding author. Mailing address: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma, 28049 Madrid, Spain. Phone: 34-1-585-4503. Fax: 34-1-585-4506. E-mail: mesteban{at}cnb.uam.es.




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