The Virion Host Shutoff RNase Plays a Key Role in Blocking the Activation of Protein Kinase R in Cells Infected with Herpes Simplex Virus 1

Earlier studies have shown that active MEK blocks the activation of protein kinase R (PKR), a component of antiviral innate immune responses. In this report we show that the herpes simplex virus 1 virion host shutoff (VHS) RNase protein and MEK (mitogen-activated protein kinase kinase) act cooperatively in blocking the activation of PKR. This conclusion is based on the following. (i) In contrast to viral gene expression in the parental cell line or a cell line expressing a constitutively active MEK, the replication of a VHS mutant is particularly impaired in cells expressing dominant negative MEK. In this cell line PKR is activated by phosphorylation, and the accumulation of several viral proteins is delayed. (ii) In transfected cells, wild-type VHS blocked the activation of PKR, whereas PKR was activated in cells transfected with a mutant VHS or with plasmids encoding the VHS RNase and VP16 and VP22, the two viral proteins that neutralize the RNase activity of VHS. The results suggest that early in infection the VHS RNase degrades RNAs that activate PKR. Coupled with published data, the results suggest that inhibition of activation of PKR or its effect on viral replication is staged early in infection by VHS, postsynthesis of VP16 and VP22 by the γ₁34.5 protein, and very late in infection by the U_S11 protein.     

伪狂犬病病毒宿主关闭蛋白的基因克隆及结构分析

为研究伪狂犬病病毒 (pseudorabiesvires ,PRV)UL4 1基因编码的病毒宿主关闭蛋白 (VHS)的结构与功能 ,通过PCR扩增得到含UL4 1基因完整编码区的 1174bp片段 ,将该片段克隆到表达载体pGEX KG中GST下游 ,在大肠杆菌BL2 1(DE3)中实现了GST VHS融合蛋白的高效表达 .序列分析发现VHS蛋白具有 4个保守区 ,并且第 3个保守区与核酸内切酶结构域FEN 1(1A76 )高度同源 .PROSPECT软件预测的伪狂犬病病毒VHS蛋白三维结构中含有 10个α螺旋和 2 2个β折叠 ,与单纯疱疹病毒Ⅰ型的VHS蛋白三维结构十分相似 .     

Trichosanthin affects HSV-1 replication in Hep-2 cells

Trichosanthin (TCS) is a type I ribosome-inactivating protein that inhibits the replication of both human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 1 (HSV-1). The mechanism of inhibition is not clear. This investigation explored the effects of TCS on the stages of HSV-1 infection in Hep-2 cells, from attachment to release. We demonstrated that TCS reduced HSV-1 antigen and DNA content and interfered with viral replication as early as 3-15 h after infection. TCS had no effect on HSV-1 attachment, penetration or immediate-early gene expression. However, the expression of early and late genes and virion release were diminished. In summary, this study demonstrates that TCS primarily affects HSV-1 replication in Hep-2 cells during the early to late infection period.     

Expression of recombinant genes containing herpes simplex virus delayed-early and immediate-early regulatory regions and trans activation by herpesvirus infection.

The promoter-regulatory regions from the herpes simplex virus type 1 (HSV-1) gene for the immediate-early, 175,000-molecular-weight (175K) protein and the HSV-2 delayed-early gene for a 38K protein were linked to the readily assayable bacterial gene for the enzyme chloramphenicol acetyltransferase (CAT). Unexpectedly, in measurements of the constitutive expression of the recombinant genes 40 to 50 h after transfection of Vero cells, enzyme levels expressed from the delayed-early 38K-promoter-CAT construct (p38KCAT) were at least as high as those from the immediate-early 175K-promoter-CAT construct (p175KCAT). In contrast, enzyme levels expressed after transfection of a similar recombinant gene containing a second delayed-early promoter region, that of the HSV-1 thymidine kinase gene, were ca. 20-fold lower. The amounts of enzyme expressed from both p38KCAT and p175KCAT could be increased by up to 20- to 40-fold after infection of the transfected cells with HSV. In comparison, virus infection had no significant effect on enzyme levels expressed from recombinant CAT genes containing the simian virus 40 early promoter region, with or without the 72-base-pair enhancer element. Experiments with the temperature-sensitive mutants HSV-1 tsB7 and HSV-1 tsK indicate that induction of expression from p175KCAT was mediated by components of the infecting virus particle, whereas that from p38KCAT required de novo expression of virus immediate-early proteins. In addition, we show that functions required to induce expression from both p175KCAT and p38KCAT could also be provided by infection with pseudorabies virus and cytomegalovirus.     

Control of expression of the vaccinia virus thymidine kinase gene.

mRNA extracted from vaccinia virus-infected cells early after infection directs cell-free synthesis of enzymatically active viral thymidine kinase (Hruby and Ball, Virology, in press). We used this assay for a specific vaccinia virus mRNA to study the induction and repression of the viral thymidine kinase gene during infection of thymidine kinase-deficient L-cells. As observed previously by other workers, the synthesis of thymidine kinase occurred immediately after infection but was switched off after 4 h later. We observed similar kinetics of accumulation and shutoff under conditions where viral DNA synthesis and late gene expression were inhibited. Cell-free translation of mRNA from infected cells showed that the concentration of functional message for viral thymidine kinase reached a peak 3 to 4 h after infection and then decreased with a half-life of about 1 h. These kinetics indicated that significant levels of thymidine kinase mRNA persisted in cells which had stopped synthesizing the enzyme. Under conditions where late gene expression was inhibited, high concentrations of functional mRNA could be isolated from cells at late times after infection. On the basis of these results, we conclude that the repression of thymidine kinase expression is mediated at the translational level by one or more early or delayed early viral genes. Repression is accompanied by, but does not depend on, the inactivation or degradation of thymidine kinase mRNA, which is a late gene function.