Engel J P, Boyer E P, Goodman J L
Department of Medicine, East Carolina University School of Medicine, Greenville, North Carolina 27858.
Virology. 1993 Jan;192(1):112-20. doi: 10.1006/viro.1993.1013.
We previously reported on a variant of the herpes simplex type 1 (HSV-1) strain 17 syn+, named 17 hep syn, capable of forming giant polykaryocytes (syncytia) in tissue culture and which induced a striking alteration in the pathogenesis of infection in vivo. Following footpad inoculation of mice, 17 hep syn infection resulted in a marked clinicopathologic acute inflammatory response of the inoculated limb and mice died without antecedant limb paralysis typical of the wild-type 17 syn+ infection. The syncytial and pathogenic phenotypes were mapped to a cloned 670-base pair Kpnl-Pstl (0.345-0.351 map units) DNA fragment encoding the carboxy terminal portion of the glycoprotein B (gB). In this report, we focus on the genetics of the region of the 17 hep syn gB gene that conferred both the syncytial and pathogenic phenotypes to 17 syn+. Five 17 syn+ x 17 hep syn syncytial recombinant viruses, R1-R5, generated in marker transfer experiments with cloned 17 hep syn fragments containing gB sequences, produced 17 hep syn-like disease in mice. Sequence analysis of the Kpnl-Pstl fragment of 17 hep syn revealed a single base pair change when compared to the 17 syn+ sequence, predicting an alanine (GCC codon) to valine (GTC codon) amino acid substitution at residue 825 of the mature gB protein, plus loss of an Ncol restriction endonuclease site. Southern blot analysis of Ncol digests of viral DNAs showed that all of the recombinants except R4 contained the same mutation as 17 hep syn. The syncytial phenotype of R4 was, however, mapped to the same region as 17 hep syn and the other recombinants, and the DNA sequence of the 670-base pair Kpnl-Pstl clone of R4 revealed another single base pair change predicting a leucine (CTC codon) to histadine (CAC codon) amino acid substitution at residue 787 of gB. The mutant gBs did not effect viral growth as all of the recombinant viruses had similar in vitro replication kinetics to wild-type HSV-1. These data provide direct evidence that at least two mutations can exist in the carboxy terminus of gB of HSV-1 that promote syncytial formation in vitro and effect pathogenesis in vivo.
我们之前报道过单纯疱疹病毒1型（HSV-1）株17 syn+的一个变种，名为17 hep syn，它能够在组织培养中形成巨大多核细胞（多核体），并且在体内感染的发病机制中引起显著改变。在小鼠足垫接种后，17 hep syn感染导致接种肢体出现明显的临床病理急性炎症反应，小鼠死亡，且无前驱肢体麻痹，而野生型17 syn+感染则有典型的肢体麻痹。多核体和致病表型被定位到一个克隆的670碱基对的Kpnl-Pstl（0.345 - 0.351图谱单位）DNA片段上，该片段编码糖蛋白B（gB）的羧基末端部分。在本报告中，我们聚焦于17 hep syn gB基因区域的遗传学，该区域赋予了17 syn+多核体和致病表型。在使用含有gB序列的克隆17 hep syn片段进行的标记转移实验中产生的5种17 syn+×17 hep syn多核体重组病毒R1 - R5，在小鼠中引发了类似17 hep syn的疾病。与17 syn+序列相比，17 hep syn的Kpnl-Pstl片段的序列分析显示有一个单碱基对变化，预测成熟gB蛋白第825位残基处的丙氨酸（GCC密码子）被缬氨酸（GTC密码子）取代，同时丢失了一个Ncol限制性内切酶位点。对病毒DNA的Ncol酶切产物进行Southern印迹分析表明，除R4外的所有重组体都含有与17 hep syn相同的突变。然而，R4的多核体表型被定位到与17 hep syn和其他重组体相同的区域，R4的670碱基对Kpnl-Pstl克隆的DNA序列显示另一个单碱基对变化，预测gB第787位残基处的亮氨酸（CTC密码子）被组氨酸（CAC密码子）取代。突变的gB不影响病毒生长，因为所有重组病毒在体外的复制动力学与野生型HSV-1相似。这些数据提供了直接证据，表明HSV-1的gB羧基末端至少可以存在两个突变，这些突变在体外促进多核体形成，并在体内影响发病机制。
