The amino-conserved domain of human cytomegalovirus UL80a proteins is required for key interactions during early stages of capsid formation and virus production.
Assembly of many spherical virus capsids is guided by an internal scaffolding protein or group of proteins that are often cleaved and eliminated in connection with maturation and incorporation of the genome. In cytomegalovirus there are at least two proteins that contribute to this scaffolding function; one is the maturational protease precursor (pUL80a), and the other is the assembly protein precursor (pUL80.5) encoded by a shorter genetic element within UL80a. Yeast GAL4 two-hybrid assays established that both proteins contain a carboxyl-conserved domain that is required for their interaction with the major capsid protein (pUL86) and an amino-conserved domain (ACD) that is required for their self-interaction and for their interaction with each other. In the work reported here, we demonstrate that when the ACD is deleted (deltaACD) or disrupted by a point mutation (L47A), the bacterially expressed mutant protein sediments as a monomer during rate-velocity centrifugation, whereas the wild-type protein sediments mainly as oligomers. We also show that the L47A mutation reduces the production of infectious virus by at least 90%, results in the formation of irregular nuclear capsids, gives rise to tube-like structures in the nucleus that resemble the capsid core in cross-section and contain UL80 proteins, slows nuclear translocation of the major capsid protein, and may slow cleavage by the maturational protease. We provide physical corroboration that mutating the ACD disrupts self-interaction of the UL80 proteins and biological support for the proposal that the ACD has a critical role in capsid assembly and production of infectious virus.
Loveland
Amy N
AN
Virology Laboratories, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205, USA.
Nguyen
Nang L
NL
Brignole
Edward J
EJ
Gibson
Wade
W
D002213:Capsid / Q000378:metabolism*; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000821:virology; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008856:Microscopy, Fluorescence; D017354:Point Mutation; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly
D014764:Viral Proteins; D010450:Endopeptidases; C087225:human cytomegalovirus protease UL80