Replicative forms of human cytomegalovirus DNA with joined termini are found in permissively infected human cells but not in non-permissive Balb/c-3T3 mouse cells.
Replicative forms of human cytomegalovirus DNA with joined termini are found in permissively infected human cells but not in non-permissive Balb/c-3T3 mouse cells.
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DOI：
10.1099/0022-1317-64-2-373
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发表时间：
1983-02
期刊：
影响因子：
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通讯作者：
R. Lafemina;G. Hayward
中科院分区：
文献类型：
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作者：
R. Lafemina;G. Hayward
发现Balb/c-3 T3小鼠细胞是人巨细胞病毒（HCMV）Towne株的高度限制性非允许宿主。这些细胞不产生感染性子代病毒粒子，不允许病毒DNA复制，并且仅允许表达单一的、主要的、病毒特异性的立即早期多肽。通过三种不同的实验方法检查病毒DNA合成。在感染的Balb/c-3 T3细胞中，在CsCl梯度中的病毒密度下没有发现32 P标记的新合成的DNA，并且在用限制性内切酶切割后没有检测到病毒特异性片段。类似地，杂交实验显示，总病毒DNA没有超过输入病毒特异性DNA序列的量的净增加。与此相反，受感染的容许人成纤维细胞合成32 P标记的病毒特异性DNA片段，并积累了大量的总杂交病毒DNA。用克隆的BamHI L-S接头片段探针进行的实验提供了形成环状或多联体复制形式的HCMV DNA的证据，其中所有半摩尔末端片段缺失，四分之一摩尔接头片段的比例增加。这些形式是丰富的，在第一个48小时后，感染的人细胞和成熟的线性单体形式积累之后。在非允许Balb/c-3 T3细胞或人成纤维细胞中，在膦酰乙酸存在下，未检测到输入病毒DNA末端的连接。在感染的Balb/c-3 T3细胞中，放线菌酮逆转后2小时内可检测到对应于68 K立即早期多肽的单一主要蛋白。很少，如果有的话，其他病毒蛋白质在以后的时间或在没有抑制剂的情况下合成。68 K蛋白在Balb/c-3 T3细胞中过量产生，以至于它成为核组分的主要成分，并且可以通过聚丙烯酰胺凝胶中的直接染色程序容易地检测到。
Summary Balb/c-3T3 mouse cells were found to be highly restricted non-permissive hosts for human cytomegalovirus (HCMV) strain Towne. These cells did not produce infectious progeny virions, did not permit virus DNA replication, and allowed expression of only a single, major, virus-specific, immediate-early polypeptide. Virus DNA synthesis was examined by three different experimental approaches. In infected Balb/c-3T3 cells, no 32P-labelled newly synthesized DNA was found at the virus density in CsCl gradients and no virus-specific fragments were detected after cleavage with restriction enzymes. Similarly, hybridization experiments revealed no net increase in total virus DNA over the amount of input virus-specific DNA sequences. In contrast, infected permissive human fibroblast cells synthesized 32P-labelled virus-specific DNA fragments and accumulated greatly increased amounts of total hybridizing virus DNA. Experiments with a cloned BamHI L-S joint fragment probe provided evidence for the formation of either circular or concatemeric replicative forms of HCMV DNA in which all half-molar terminal fragments were missing and the proportion of quarter-molar joint fragments increased. These forms were abundant in the first 48 h after infection of permissive human cells and mature linear monomeric forms accumulated thereafter. No detectable joining of the termini of input virus DNA occurred in either non-permissive Balb/c-3T3 cells or in human fibroblast cells in the presence of phosphonoacetic acid. In the infected Balb/c-3T3 cells a single major protein corresponding to the 68K immediate-early polypeptide could be detected within 2 h after cycloheximide reversal. Few, if any, other virus proteins were synthesized at later times or in the absence of inhibitors. The 68K protein was overproduced in Balb/c-3T3 cells to such an extent that it became a major component of the nuclear fraction and could be readily detected by direct staining procedures in polyacrylamide gels.