Rasty S, Poliani P L, Fink D J, Glorioso J C
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
J Neurovirol. 1997 Aug;3(4):247-64. doi: 10.3109/13550289709029466.
A distinctive feature of the genetic make-up of herpes simplex virus type 1 (HSV-1), a human neurotropic virus, is that approximately half of the 81 known viral genes are not absolutely required for productive infection in Vero cells, and most can be individually deleted without substantially impairing viral replication in cell culture. If large blocks of contiguous viral genes could be replaced with foreign DNA sequences, it would be possible to engineer highly attenuated recombinant HSV-1 gene transfer vectors capable of carrying large cellular genes or multiple genes having related functions. We report the isolation and characterization of an HSV-1 mutant, designated d311, containing a 12 kb deletion of viral DNA located between the L-S Junction a sequence and the U(S)6 gene, spanning the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5. Replication of d311 was totally inhibited in rat B103 and mouse Neuro-2A neuroblastoma cell lines, and was reduced by over three orders of magnitude in human SK-N-SH neuroblastoma cells compared to wild-type (wt) HSV-1 KOS. This suggested that the deleted genes, while nonessential for replication in Vero cells, play an important role in HSV replication in neuronal cells, particularly those of rodent origin. Unlike wt KOS which replicated locally and spread to other regions of brain following stereotactic inoculation into rat hippocampus, d311 was unable to replicate and spread within the brain, and did not cause any apparent local neuronal cell damage. These results demonstrate that d311 is highly attenuated for the rat central nervous system. d311 and other mutants of HSV containing major deletions of the nonessential genes within U(S) have the potential to serve as useful tools for gene transfer applications to brain.
人嗜神经病毒单纯疱疹病毒1型（HSV - 1）基因组成的一个显著特征是，在已知的81个病毒基因中，约有一半对于在非洲绿猴肾细胞（Vero细胞）中进行有效感染并非绝对必需，并且大多数基因可以单独删除而不会严重损害病毒在细胞培养中的复制。如果大片连续的病毒基因能够被外源DNA序列取代，那么就有可能构建出高度减毒的重组HSV - 1基因转移载体，该载体能够携带大的细胞基因或多个具有相关功能的基因。我们报告了一种HSV - 1突变体的分离和鉴定，命名为d311，其病毒DNA在L - S连接序列和U(S)6基因之间有一个12 kb的缺失，跨越S组分反向重复序列c'以及非必需基因U(S)1至U(S)5。与野生型（wt）HSV - 1 KOS相比，d311在大鼠B103和小鼠神经母细胞瘤Neuro - 2A细胞系中的复制完全被抑制，在人神经母细胞瘤SK - N - SH细胞中的复制减少了三个数量级以上。这表明，缺失的基因虽然对于在Vero细胞中复制并非必需，但在HSV在神经元细胞（特别是啮齿动物来源的神经元细胞）的复制中起重要作用。与立体定向接种到大鼠海马体后在局部复制并扩散到大脑其他区域的wt KOS不同，d311无法在脑内复制和扩散，也未引起任何明显的局部神经元细胞损伤。这些结果表明，d311对大鼠中枢神经系统具有高度减毒作用。d311和其他在U(S)内含有非必需基因主要缺失的HSV突变体有潜力作为向脑内进行基因转移应用的有用工具。
