Conditional lethal expression of the vaccinia virus L1R myristylated protein reveals a role in virion assembly

Abstract

Within vaccinia virus-infected cells, the product of the L1R open reading frame is covalently modified by myristic acid at the penultimate NH2-terminal glycine residue. Previously we have shown that while the L1R protein is a constituent of both intracellular mature virus particles and extracellular enveloped virions which are released from the infected cell, it is associated exclusively with the primary membranes surrounding the virion core. Given this rather specific localization, it was of interest to study the potential role of this essential gene in virus replication and morphogenesis. To this end, we have constructed a recombinant vaccinia virus in which expression of the L1R gene can be transcriptionally repressed. Without the inducer isopropylthiogalactopyranoside (IPTG), synthesis of the L1R protein was blocked, resulting in a total inhibition of plaque formation. Velocity sedimentation of viral particles labeled in the presence of [3H]thymidine, grown in the absence of IPTG, revealed a substantial reduction in viral DNA incorporation into virions. Likewise, proteolysis of the major core proteins p4a, p4b, and p25K, believed to occur during the final stages of virion maturation, was severely impaired. In the absence of L1R expression, only immature virions could be detected by electron microscopy. Transient expression of a plasmid containing the full-length L1R gene driven by its own promoter was able to complement and rescue the defective phenotype. However, a plasmid bearing a mutation in the myristyl acceptor glycine residue was unable to biologically rescue the recombinant, and the protein was not detected in purified virions.trans complementation using a truncated, myristylated form of the L1R protein partially rescued the defective mutant. Collectively, these data suggest that myristic acid mediates essential interactions of the L1R protein with viral membranes and/or other virion components that lead to the productive assembly, maturation, and release of particles.