Max von Pettenkofer Institute, Pettenkoferstrasse 9a, D-80336 Munich, Germany.
J Virol. 2011 Sep;85(18):9254-67. doi: 10.1128/JVI.00443-11. Epub 2011 Jun 29.
The gene M94 of murine cytomegalovirus (MCMV) as well as its homologues UL16 in alphaherpesviruses is involved in viral morphogenesis. For a better understanding of its role in the viral life cycle, a library of random M94 mutants was generated by modified transposon-based linker scanning mutagenesis. A comprehensive set of M94 mutants was reinserted into the MCMV genome and tested for their capacity to complement the M94 null mutant. Thereby, 34 loss-of-function mutants of M94 were identified, which were tested in a second screen for their capacity to inhibit virus replication. This analysis identified two N-terminal insertion mutants of M94 with a dominant negative effect. We compared phenotypes induced by the conditional expression of these dominant negative M94 alleles with the null phenotype of the M94 deletion. The viral gene expression cascade and the nuclear morphogenesis steps were not affected in either setting. In both cases, however, secondary envelopment did not proceed in the absence of functional M94, and capsids subsequently accumulated in the center of the cytoplasmic assembly complex. In addition, deletion of M94 resulted in a block of cell-to-cell spread. Moreover, the dominant negative mutant of M94 demonstrated a defect in interacting with M99, the UL11 homologue of MCMV.
鼠巨细胞病毒（MCMV）的 M94 基因及其α疱疹病毒中的同源物 UL16 参与病毒形态发生。为了更好地了解其在病毒生命周期中的作用，通过改良的转座子连接扫描诱变生成了随机 M94 突变体库。一组完整的 M94 突变体被重新插入 MCMV 基因组中，并测试它们补充 M94 缺失突变体的能力。因此，鉴定出 34 个 M94 的功能丧失突变体，在第二个筛选中测试它们抑制病毒复制的能力。该分析确定了具有显性负效应的 M94 的两个 N 端插入突变体。我们比较了这些显性负 M94 等位基因的条件表达诱导的表型与 M94 缺失的缺失表型。在这两种情况下，病毒基因表达级联和核形态发生步骤均不受影响。然而，在没有功能性 M94 的情况下，二次包膜不会进行，随后衣壳在细胞质组装复合物的中心积累。此外，M94 的缺失导致细胞间传播受阻。此外，M94 的显性负突变体在与 MCMV 的 UL11 同源物 M99 相互作用方面表现出缺陷。
