Hayashi M L, Blankenship C, Shenk T
Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014, USA.
Proc Natl Acad Sci U S A. 2000 Mar 14;97(6):2692-6. doi: 10.1073/pnas.050587597.
Human cytomegalovirus blocks cell-cycle progression in the G(1) compartment upon infection of primary human fibroblasts. The virus-coded UL69 protein can institute a G(1) block when expressed in cells in the absence of virus infection. We have constructed a cytomegalovirus mutant, TNsubUL69, that lacks the UL69 coding region. This virus grows slowly in fibroblasts, but produces a wild-type yield after an extended delay. It grows with normal kinetics in cells coinfected with a recombinant retrovirus, retroUL69, which expresses UL69 protein, demonstrating that its growth defect results from the mutation in the UL69 gene. UL69 protein is packaged within virus particles, and it was possible for us to produce two types of virus stocks. TNsubUL69(+pUL69) lacks the UL69 gene but contains UL69 protein in virus particles. It is produced by growth in fibroblasts that are coinfected with retroUL69. TNsubUL69(-pUL69) lacks the UL69 gene and protein. It is produced by growth in fibroblasts that do not contain UL69 protein. The mutant virions lacking both the UL69 gene and protein fail to induce a cell-cycle block with normal efficiency, whereas the mutant particles lacking the gene but containing the protein can institute the block. These results are consistent with the view that the UL69 protein contributes to the cytomegalovirus-induced cell-cycle block, and they suggest that UL69 protein delivered to cells within virions can induce the block without the synthesis of additional UL69 protein encoded by the infecting viral genome.
人巨细胞病毒感染原代人成纤维细胞后会在G(1)期阻断细胞周期进程。病毒编码的UL69蛋白在无病毒感染的细胞中表达时可引发G(1)期阻断。我们构建了一种巨细胞病毒突变体TNsubUL69，它缺失UL69编码区。这种病毒在成纤维细胞中生长缓慢，但经过较长延迟后可产生野生型产量。它在与表达UL69蛋白的重组逆转录病毒retroUL69共感染的细胞中以正常动力学生长，表明其生长缺陷是由UL69基因突变所致。UL69蛋白被包装在病毒颗粒内，我们能够制备两种类型的病毒株。TNsubUL69(+pUL69)缺失UL69基因，但病毒颗粒中含有UL69蛋白。它是通过在与retroUL69共感染的成纤维细胞中生长产生的。TNsubUL69(-pUL69)既缺失UL69基因也缺失该蛋白。它是通过在不含UL69蛋白的成纤维细胞中生长产生的。既缺失UL69基因又缺失该蛋白的突变病毒粒子不能以正常效率诱导细胞周期阻断，而缺失基因但含有该蛋白的突变颗粒则可引发阻断。这些结果与以下观点一致，即UL69蛋白促成了巨细胞病毒诱导的细胞周期阻断，并且表明病毒粒子内传递到细胞的UL69蛋白可诱导阻断，而无需合成由感染性病毒基因组编码的额外UL69蛋白。
