Mester J C, Pitha P M, Glorioso J C
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, PA 15261, USA.
Gene Ther. 1995 May;2(3):187-96.
Mutant herpes simplex virus type I (HSV-1) vectors were engineered to express murine alpha 1 interferon (IFN) and assessed for their ability to inhibit the replication of challenge viruses in infection of monolayer cell cultures. The alpha 1 IFN gene was placed under control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) region in the thymidine kinase (tk) locus of both the wild-type HSV-1 strain KOS and the replication-defective KOS mutant dl20, in which both copies of the ICP4 immediate-early (IE) gene are deleted. To evaluate IFN expression, vector-infected cell culture media from epithelial, fibroblast and neuronal cells were assayed for alpha IFN release. These cells did not secrete detectable levels of alpha IFN when infected with control KOS or d120-based viruses. Cells infected at a multiplicity of infection (MOI) of 10 with either RSV-LTR-IFN vector produced from approximately 12 to 100 U of alpha 1-IFN per millilitre of culture fluid in 24 h, as determined by a standard vesicular stomatitis virus (VSV) replication inhibition assay. Vector-derived alpha 1-IFN expression did not inhibit the growth of the KOS-RSV-IFN virus in IFN sensitive cell lines. However, pretreatment of murine L cells with IFN produced from the RSV-IFN vectors blocked the replication of HSV-1. Additionally, L cells infected with d120-RSV-IFN (at an MOI of 0.5 to 0.06) were protected from superinfection with VSV. Thus, pre-infection of a small percentage of cells with a nonreplicating HSV-IFN expression vector provided complete protection of tissue culture cell monolayers from the cytopathic effect of a challenge virus infection. These vectors should be useful for in vivo analysis of the antiviral potential of IFN expression from within the nervous system.
突变的I型单纯疱疹病毒（HSV-1）载体经改造后可表达小鼠α1干扰素（IFN），并对其在单层细胞培养感染中抑制攻击病毒复制的能力进行了评估。α1干扰素基因被置于野生型HSV-1毒株KOS和复制缺陷型KOS突变体dl20的胸苷激酶（tk）基因座中的劳斯肉瘤病毒（RSV）长末端重复序列（LTR）区域的控制之下，在dl20中，ICP4立即早期（IE）基因的两个拷贝均被删除。为了评估IFN的表达，对来自上皮细胞、成纤维细胞和神经元细胞的载体感染细胞培养基进行了α干扰素释放检测。当用对照KOS或基于d120的病毒感染时，这些细胞不会分泌可检测水平的α干扰素。用标准水泡性口炎病毒（VSV）复制抑制试验测定，以感染复数（MOI）为10感染任何一种RSV-LTR-IFN载体的细胞，在24小时内每毫升培养液可产生约12至100单位的α1-IFN。载体衍生的α1-IFN表达不会抑制KOS-RSV-IFN病毒在IFN敏感细胞系中的生长。然而，用RSV-IFN载体产生的IFN对小鼠L细胞进行预处理可阻断HSV-1的复制。此外，用d120-RSV-IFN（MOI为0.5至0.06）感染的L细胞可免受VSV的超感染。因此，用非复制性HSV-IFN表达载体对一小部分细胞进行预感染，可使组织培养细胞单层完全免受攻击病毒感染的细胞病变效应。这些载体应有助于对神经系统内IFN表达的抗病毒潜力进行体内分析。
