INDUCIBLE EXPRESSION OF THE VACCINIA VIRUS A17L GENE PROVIDES A SYNCHRONIZED SYSTEM TO MONITOR SORTING OF VIRAL-PROTEINS DURING MORPHOGENESIS

The vaccinia virus (VV) A17L gene encodes a 21- to 23-kDa virion compo nent that forms a stable complex with the 14-kDa envelope protein (A27 L gene). In a previous report, we described the construction of a VV r ecombinant, VVindA17L, in which the expression of the A17L gene is ind ucibly regulated by isopropyl-beta-D-thiogalactoside (IPTG). We demons trated that shutoff of the A17L gene results in a blockade of virion m orphogenesis at a very early stage (D. Rodriguez, M. Esteban, and J. R . Rodriguez, J. Virol. 69:4630-4638, 1995). In the present study, we s how that virus growth is restored if the inducer is provided not later than 6 h postinfection. Immunofluorescence and immunoelectron microsc opy analysis of VVindA17L-infected cells revealed that in the absence of the 21- to 23-kDa protein, the 14-kDa protein is distributed throug hout the cyto-plasm. After IPTG addition, the 14-kDa protein can be de tected around viral factories and immature virions; at later times, it localizes in the external membranes of intracellular mature virions. Immunoelectron microscopy with anti-21 to 23-kDa antibodies showed tha t soon after induction, the protein accumulates in membranes of the ro ugh endoplasmic reticulum and in the nuclear envelope. With time, the protein localizes in viral crescents and subsequently associates to th e membranes of immature and intracellular mature virions. These result s are consistent with a model in which the 21- to 23-kDa protein would be synthesized at the endoplasmic reticulum, from where the protein c ould be translocated to the membranes of the intermediate compartment to generate the precursors of the viral membranes. Also, these results argue that 14-kDa envelope protein becomes posttranslationally associ ated to viral membranes through its interaction with the 21-kDa protei n.