Martinez R, Shao L, Bronstein J C, Weber P C, Weller S K
Department of Microbiology, University of Connecticut Health Center, Farmington 06030, USA.
Virology. 1996 Jan 15;215(2):152-64. doi: 10.1006/viro.1996.0018.
Alkaline nuclease, a relatively abundant viral phosphoprotein in herpes simplex virus type 1 (HSV-1)- or HSV-2-infected cells, is encoded by a 2.3-kb mRNA (R. H. Costa, K. G. Draper, L. Banks, K. L. Powell, G. Cohen, R. Eisenberg, and E. K. Wagner, 1983. J. Virol. 48, 591-603). This mRNA is a member of a family of five unspliced 3'-coterminal messages. Costa et al. proposed that another member of this family of mRNAs (1.9-kb) may encode an N-terminally truncated protein which shares its carboxy-terminus with the alkaline nuclease protein. We previously described the isolation of AN-1, a deletion/insertion mutant of the alkaline nuclease gene (S. K. Weller, R. M. Seghatoleslami, L. Shao, D. Rowse, and E. P. Carmichael, 1990. J. Gen. Virol. 71, 2941-2952). The deletion in AN-1 would be predicted to abolish gene products of both the 2.3- and the 1.9-kb mRNAs. To investigate whether the putative truncated version of alkaline nuclease encoded by the 1.9-kb mRNA has enzymatic activity and plays a role in the viral life cycle, a viral mutant (AN-F1) was constructed which is predicted to abolish the gene product of the 2.3-kb mRNA (full-length alkaline nuclease) but leave intact the putative product of the 1.9-kb mRNA. Using a highly sensitive polyclonal antiserum raised against a bacterially expressed full-length alkaline nuclease, we observed a 60-kDa protein in KOS- and AN-F1-infected cells but not in AN-1-infected cells. This suggests that the 60-kDa protein is likely to be expressed from the 1.9-kb mRNA; the open reading frame is now designated UL12.5. Despite the presence of the 60-kDa band, AN-F1 failed to exhibit any alkaline exonuclease activity. This result suggests that the truncated polypeptide (UL12.5) is not enzymatically active, has low levels of activity, or possesses enzymatic activity which is not detected because of the low abundance of the polypeptide. AN-1 and AN-F1 are both severely restricted with respect to growth in Vero cells, as viral yields are 100- to 1000-fold lower than those of wild-type virus. We previously reported that the major defect in AN-1 is in the ability of DNA-containing capsids which form in the nucleus to mature into the cytoplasm (L. Shao, L. M. Rapp, and S. K. Weller, 1993. Virology 196, 146-162); AN-F1 exhibits the same defect. These results indicate that although the 1.9-kb mRNA encodes a 60-kDa protein presumably from the UL12.5 open reading frame, this polypeptide cannot substitute for the full-length UL12 product.
碱性核酸酶是单纯疱疹病毒1型（HSV-1）或HSV-2感染细胞中一种相对丰富的病毒磷蛋白，由一个2.3 kb的mRNA编码（R. H. 科斯塔、K. G. 德雷珀、L. 班克斯、K. L. 鲍威尔、G. 科恩、R. 艾森伯格和E. K. 瓦格纳，1983年。《病毒学杂志》48卷，591 - 603页）。该mRNA是一个由五条未剪接的3' 共末端信使RNA组成的家族成员。科斯塔等人提出，这个mRNA家族的另一个成员（1.9 kb）可能编码一种N端截短的蛋白质，其羧基末端与碱性核酸酶蛋白相同。我们之前描述了碱性核酸酶基因的一个缺失/插入突变体AN-1的分离（S. K. 韦勒、R. M. 塞加托莱斯拉米、L. 邵、D. 劳斯和E. P. 卡迈克尔，1990年。《普通病毒学杂志》71卷，2941 - 2952页）。预计AN-1中的缺失会消除2.3 kb和1.9 kb mRNA的基因产物。为了研究由1.9 kb mRNA编码的假定截短型碱性核酸酶是否具有酶活性并在病毒生命周期中发挥作用，构建了一个病毒突变体（AN-F1），预计该突变体将消除2.3 kb mRNA（全长碱性核酸酶）的基因产物，但保留1.9 kb mRNA的假定产物。使用针对细菌表达的全长碱性核酸酶产生的高灵敏度多克隆抗血清，我们在感染KOS和AN-F1的细胞中观察到一种60 kDa的蛋白质，但在感染AN-1的细胞中未观察到。这表明该60 kDa的蛋白质可能由1.9 kb的mRNA表达；现在将开放阅读框命名为UL12.5。尽管存在60 kDa的条带，但AN-F1未能表现出任何碱性外切核酸酶活性。该结果表明截短的多肽（UL12.5）没有酶活性、活性水平低，或者具有由于多肽丰度低而未检测到的酶活性。AN-1和AN-F1在Vero细胞中的生长都受到严重限制，因为病毒产量比野生型病毒低100至1000倍。我们之前报道过，AN-1的主要缺陷在于在细胞核中形成的含DNA衣壳成熟进入细胞质的能力（L. 邵、L. M. 拉普和S. K. 韦勒，1993年。《病毒学》196卷，146 - 162页）；AN-F1表现出相同的缺陷。这些结果表明，尽管1.9 kb的mRNA可能从UL12.5开放阅读框编码一种60 kDa的蛋白质，但该多肽不能替代全长的UL12产物。
