Scaffolding proteins of spherical prokaryotic and eukaryotic viruses have critical roles in capsid assembly. The primary scaffolding components of cytomegalovirus, called the assembly protein precursor (pAP, pUL80.5) and the maturational protease precursor (pPR, pUL80a), contain two nuclear localization sequences (NLS1 and NLS2), at least one of which is required in coexpression experiments to translocate the major capsid protein (MCP, pUL85) into the nucleus. In the work reported here, we have mutated NLS1 and NLS2, individually or together, in human cytomegalovirus (HCMV, strain AD169) bacmid-derived viruses to test their effects on virus replication. Consistent with results from earlier transfection/coexpression experiments, both single-mutant bacmids gave rise to infectious virus but the double mutant did not. In comparisons with the wild-type virus, both mutants showed slower cell-to-cell spread; decreased yields of infectious virus (3-fold lower for NLS1- and 140-fold lower for NLS2-); reduced efficiency of pAP, pPR, and MCP nuclear translocation (sixfold lower for NLS1- and eightfold lower for NLS2-); increased amounts of a 120-kDa MCP fragment; and reduced numbers of intranuclear capsids. All effects were more severe for the NLS2 - mutant than the NLS1- mutant, and a distinguishing feature of cells infected with the NLS2- mutant was the accumulation of large, UL80 protein-containing structures within the nucleus. We conclude that these NLS assist in the nuclear translocation of MCP during HCMV replication and that NLS2, which is unique to the betaherpesvirus UL80 homologs, may have additional involvements during replication.
Nuclear localization sequences in cytomegalovirus capsid assembly proteins (UL80 proteins) are required for virus production : Inactivating NLS1, NLS2, or both affects replication to strikingly different extents. / Nguyen, Nang L.; Loveland, Amy N.; Gibson, D Wade.In: Journal of Virology, Vol. 82, No. 11, 06.2008, p. 5381-5389.
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