OPEN READING FRAMES UL44, IRS1 TRS1, AND UL36-38 ARE REQUIRED FOR TRANSIENT COMPLEMENTATION OF HUMAN CYTOMEGALOVIRUS ORILYT-DEPENDENT DNA-SYNTHESIS
OPEN READING FRAMES UL44, IRS1 TRS1, AND UL36-38 ARE REQUIRED FOR TRANSIENT COMPLEMENTATION OF HUMAN CYTOMEGALOVIRUS ORILYT-DEPENDENT DNA-SYNTHESIS
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DOI：
10.1128/jvi.67.5.2575-2582.1993
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发表时间：
1993-05-01
影响因子：
5.4
通讯作者：
ANDERS, DG
中科院分区：
文献类型：
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作者：
PARI, GS;KACICA, MA;ANDERS, DG
先前的结果表明，如果HCMV感染提供了必需的反式作用因子，则含有人巨细胞病毒（HCMV）oriLyt的质粒在转染到允许细胞中后复制（D. G.安德斯，M。A. Kacica湾S. Pari和S. M. Punturieri，J. Virol. 66：3373-3384，1992）。我们现在已经使用oriLyt作为HCMV DNA复制的瞬时互补试验中的报告，其中共转染的粘粒克隆，而不是HCMV感染，提供了必要的反式作用因子。互补复制是oriLyt依赖和膦甲酸敏感，并产生典型的HCMV裂解期DNA合成的串联阵列。因此，该检测提供了一种有效的遗传检测，以发现以前未鉴定的DNA合成所必需的基因，并证实核苷酸序列比较和生化分析所作的功能预测。五个cosestrin是必要的，足以产生来源依赖的DNA合成，所有这些所需的cosestrin，但其中一个含有至少一个候选人的同源性单纯疱疹病毒1型复制基因。我们进一步使用该测定法来确定两个所需的cosplay中的必需区域，pCM 1017和pCM 1052。结果表明，UL 44，提出的基础上的生化证据是HCMV DNA聚合酶辅助蛋白，所需的互补。此外，在该测定中还需要编码调节蛋白的三个基因组区域来产生来源依赖性DNA合成：（i）IRS 1/TRS 1，其与主要的立即早期蛋白协作以激活UL 44表达;（ii）UL 36 -38;和（iii）包括IE 1和IE 2的主要立即早期区域。结合起来，这些结果明确地建立了这种方法的实用性，HCMV复制基因的定位。因此，现在可以确定启动和进行裂解期DNA合成所必需和足够的HCMV基因组，以及鉴定在人成纤维细胞中表达所需的那些病毒基因。
Previous results showed that plasmids containing human cytomegalovirus (HCMV) oriLyt are replicated after transfection into permissive cells if essential trans-acting factors are supplied by HCMV infection (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). We have now used oriLyt as a reporter of HCMV DNA replication in a transient complementation assay in which cotransfected cosmid clones, instead of HCMV infection, provided essential trans-acting factors. Complemented replication was oriLyt dependent and phosphonoformic acid sensitive and produced tandem arrays typical of HCMV lytic-phase DNA synthesis. Thus, this assay provides a valid genetic test to find previously unidentified genes that are essential for DNA synthesis and to corroborate functional predictions made by nucleotide sequence comparisons and biochemical analyses. Five cosmids were necessary and sufficient to produce origin-dependent DNA synthesis; all but one of these required cosmids contain at least one candidate homolog of herpes simplex virus type 1 replication genes. We further used the assay to define essential regions in two of the required cosmids, pCM1017 and pCM1052. Results presented show that UL44, proposed on the basis of biochemical evidence to be the HCMV DNA polymerase accessory protein, was required for complementation. In addition, three genomic regions encoding regulatory proteins also were needed to produce origin-dependent DNA synthesis in this assay: (i) IRS1/TRS1, which cooperates with the major immediate-early proteins to activate UL44 expression; (ii) UL36-38; and (iii) the major immediate-early region comprising IE1 and IE2. Combined, these results unequivocally establish the utility of this approach for mapping HCMV replication genes. Thus, it will now be possible to define the set of HCMV genes necessary and sufficient for initiating and performing lytic-phase DNA synthesis as well as to identify those virus genes needed for their expression in human fibroblasts.