Baines J D, Poon A P, Rovnak J, Roizman B
Department Microbiology, Immunology and Parasitology, Cornell University, Ithaca, New York 14853.
J Virol. 1994 Dec;68(12):8118-24. doi: 10.1128/JVI.68.12.8118-8124.1994.
Previous studies have shown that a ts mutant [herpes simplex virus 1 (mP)ts66.4] in the UL15 gene fails to package viral DNA into capsids (A. P. W. Poon and B. Roizman, J. Virol. 67:4497-4503, 1993) and that although the intron separating the first and second exons of the UL15 gene contains UL16 and UL17 open reading frames, replacement of the first exon with a cDNA copy of the entire gene does not affect viral replication (J.D. Baines, and B. Roizman, J. Virol. 66:5621-5626, 1992). We report that (i) a polyclonal rabbit antiserum generated against a chimeric protein consisting of the bacterial maltose-binding protein fused in frame to the majority of sequences contained in the second exon of the UL15 gene reacted with two proteins with M(r) of 35,000 and 75,000, respectively, in cells infected with a virus containing the authentic gene yielding a spliced mRNA or with a virus in which the authentic UL15 gene was replaced with a cDNA copy. (ii) Insertion of 20 additional codons into the C terminus of UL15 exon II caused a reduction in the electrophoretic mobility of both the apparently 35,000- and 75,000-M(r) proteins, unambiguously demonstrating that both share the carboxyl terminus of the UL15 exon II. (iii) Accumulation of the 35,000-M(r) protein was reduced in cells infected and maintained in the presence of phosphonoacetate, an inhibitor of viral DNA synthesis. (iv) The UL15 proteins were localized in the perinuclear space at 6 h after infection and largely in the nucleus at 12 h after infection. (v) Viral DNA accumulating in cells infected with herpes simplex virus 1(mP)ts66.4 and maintained at the nonpermissive temperature was in an endless (concatemeric) form, and therefore UL15 is required for the cleavage of mature, unit-length molecules for packaging into capsids.
先前的研究表明，UL15基因中的一个温度敏感突变体[单纯疱疹病毒1（mP）ts66.4]无法将病毒DNA包装到衣壳中（A.P.W.潘和B.罗伊兹曼，《病毒学杂志》67:4497 - 4503，1993），并且尽管分隔UL15基因第一和第二外显子的内含子包含UL16和UL17开放阅读框，但用整个基因的cDNA拷贝替换第一外显子并不影响病毒复制（J.D.贝恩斯和B.罗伊兹曼，《病毒学杂志》66:5621 - 5626，1992）。我们报告如下：（i）针对一种嵌合蛋白产生的多克隆兔抗血清，该嵌合蛋白由与UL15基因第二外显子中包含的大部分序列框内融合的细菌麦芽糖结合蛋白组成，在感染含有产生剪接mRNA的真实基因的病毒或真实UL15基因被cDNA拷贝替换的病毒的细胞中，分别与分子量为35,000和75,000的两种蛋白发生反应。（ii）在UL15外显子II的C末端插入另外20个密码子导致明显分子量为35,000和75,000的两种蛋白的电泳迁移率降低，明确表明两者共享UL15外显子II的羧基末端。（iii）在感染并在病毒DNA合成抑制剂膦甲酸存在下维持的细胞中，分子量为35,000的蛋白积累减少。（iv）UL15蛋白在感染后6小时定位于核周间隙，在感染后12小时大部分定位于细胞核。（v）在感染单纯疱疹病毒1（mP）ts66.4并在非允许温度下维持的细胞中积累的病毒DNA呈无端（串联体）形式，因此UL15是切割成熟的单位长度分子以包装到衣壳中所必需的。
