VACCINIA VIRUS 15-KILODALTON (A14L) PROTEIN IS ESSENTIAL FOR ASSEMBLYAND ATTACHMENT OF VIRAL CRESCENTS TO VIROSOMES

Early stages in vaccinia virus (VV) assembly involve the recruitment o f 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 stag es of virion assembly. We showed that the 21-kDa protein is not respon sible for the recruitment of membranes from the ERGIC to viral factori es. However, it appears to be essential for the organization of viral membranes. In this investigation we have generated a VV recombinant, V VindA14L, in which the expression of the A14L gene is inducibly regula ted 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 corr elates with a small-plaque phenotype and reduced VVindA14L yields unde r these conditions. In the absence of the 15-kDa protein, early and la te viral protein syntheses proceed normally; however, proteolytic clea vage of the major core precursors is inhibited. Electron microscopic e xamination of cells infected with VVindA14L under nonpermissive condit ions reveals the presence of numerous membranous elements that look li ke unfinished or disassembled crescents interspersed between electron- dense masses. These abnormal membrane elements are usually well separa ted from the surfaces of the dense structures. These findings show tha t the 15-kDa protein is essential for VV morphogenesis and indicate th at this polypeptide is necessary both for the correct assembly of vira l crescents and for their stable attachment to the surfaces of viral f actories.