Chowdhury S I, Batterson W
Department of Pathology and Microbiology, College of Veterinary Medicine, Kansas State University, Manhattan 66506.
Virus Res. 1994 Jul;33(1):67-87. doi: 10.1016/0168-1702(94)90018-3.
This study demonstrates that cells expressing a dysfunctional analog of a herpes simplex virus (HSV) capsid protein inhibits HSV replication. Vero cell lines expressing HSV-1 capsid protein VP19c/beta-galactosidase fusion proteins were constructed and tested for their kinetics of expression, intracellular location, and ability to interfere with HSV replication. Two chimeric genes were constructed for these studies. The larger chimeric gene encodes the amino terminal 327 amino acids (aa) of VP19c fused to the carboxy terminal 1026 aa of beta-galactosidase, and the shorter chimeric gene encodes VP19c aa 1-30 and 302-327 fused to the carboxy-terminal 1026 aa of beta-galactosidase. Cell lines V32G-1 and V32G-2 containing the larger and the shorter chimeric genes, respectively, were isolated after cotransfection with plasmid pSV2-neo DNA, cell selection, and limiting-dilution cloning. The chimeric VP19c/beta-galactosidase genes resident in V32G-1 and V32G-2 cell lines were induced by early gene products of superinfecting wild-type HSV-1 and HSV-2, but were not constitutively expressed. The hybrid proteins expressed in infected V32G-1 and V32G-2 cells both colocalized with infected cell protein 8 (ICP8) into virus-replicative compartments in the cell nuclei. HSV-1 and HSV-2 growth in V32G-1 cells (which express the larger chimeric gene) was significantly reduced compared to growth in V32G-2 and control Vero cells. The data suggest that the larger VP19c/beta-galactosidase hybrid protein interferes with virus capsid assembly or morphogenesis in a competitive manner. Results also demonstrate that a small portion of VP19c containing the predicted endoplasmic reticulum signal sequence for this capsid protein (aa 1-30) promotes incorporation of the VP19c/beta-galactosidase fusion proteins into nuclear viral replication compartments.
本研究表明，表达单纯疱疹病毒（HSV）衣壳蛋白功能失调类似物的细胞可抑制HSV复制。构建了表达HSV-1衣壳蛋白VP19c/β-半乳糖苷酶融合蛋白的Vero细胞系，并对其表达动力学、细胞内定位以及干扰HSV复制的能力进行了检测。为这些研究构建了两个嵌合基因。较大的嵌合基因编码与β-半乳糖苷酶羧基末端1026个氨基酸（aa）融合的VP19c氨基末端327个氨基酸，较短的嵌合基因编码与β-半乳糖苷酶羧基末端1026个氨基酸融合的VP19c第1-30和302-327位氨基酸。分别含有较大和较短嵌合基因的细胞系V32G-1和V32G-2在与质粒pSV2-neo DNA共转染、细胞筛选和有限稀释克隆后分离得到。V32G-1和V32G-2细胞系中存在的嵌合VP19c/β-半乳糖苷酶基因由超感染的野生型HSV-1和HSV-2的早期基因产物诱导，但不是组成性表达。在感染的V32G-1和V32G-2细胞中表达的杂交蛋白均与感染细胞蛋白8（ICP8）共定位到细胞核中的病毒复制区室。与在V32G-2和对照Vero细胞中的生长相比，HSV-1和HSV-2在V32G-1细胞（表达较大嵌合基因）中的生长显著降低。数据表明，较大的VP19c/β-半乳糖苷酶杂交蛋白以竞争方式干扰病毒衣壳组装或形态发生。结果还表明，含有该衣壳蛋白预测内质网信号序列（第1-30位氨基酸）的一小部分VP19c促进VP19c/β-半乳糖苷酶融合蛋白掺入核病毒复制区室。
