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
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
复制标题
DOI：
10.1128/jvi.02697-07
复制
发表时间：
2008-06-01
影响因子：
5.4
通讯作者：
Gibson, Wade
中科院分区：
文献类型：
--
作者：
Nguyen, Nang L.;Loveland, Amy N.;Gibson, Wade
球状原核和真核病毒的支架蛋白在衣壳组装中起着关键作用。巨细胞病毒的主要骨架成分称为组装蛋白前体(Pap，pUL80.5)和成熟蛋白前体(PPR，pUL80a)，它们包含两个核定位序列(NLS1和NLS2)，在共表达实验中，至少需要一个序列才能将主要衣壳蛋白(MCP，pUL85)转移到细胞核中。在本文报道的工作中，我们在人类巨细胞病毒(HCMV，AD169株)巴克米德衍生病毒中分别或联合突变了NLS1和NLS2，以测试它们对病毒复制的影响。与早期的转染/共表达实验结果一致，两个单突变菌株都能产生感染性病毒，但双突变菌株没有。与野生型病毒相比，两个突变体都表现出较慢的细胞间传播速度；感染性病毒的产量降低(NLS1(-)降低3倍，NLS2(-)降低140倍)；Pap、PPR和MCP核转位的效率降低(NLS1(-)降低6倍，NLS2(-)降低8倍)；120 kDa的MCP片段数量增加；核内衣壳数量减少。NLS2(-)突变体的所有影响都比NLS1(-)突变体更严重，感染NLS2(-)突变体的细胞的一个显著特征是细胞核内积累了大的含UL80蛋白的结构。我们的结论是，这些NLS有助于在HCMV复制过程中MCP的核转位，而NLS2可能在复制过程中有额外的参与，NLS2是β疱疹病毒UL80同源物所特有的。
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.