Genetic retrieval of viral genome sequences from herpes simplex virus transformed cells

Oncogenic transformation of cultured cells by inactivated herpes simplex virus (HSV) types 1 and 2 has been demonstrated. Expression of HSV information in these transformed cells has been shown by immunofluorescence studies, detection of HSV neutralizing antibody in sera from tumour-bearing animals and by hybridization of HSV-specific RNA. Molecular hybridization studies of DNA from HSV-2 transformed hamster cells have detected up to 40% of the HSV genome present in several copies. Complementation of three HSV-2 temperature-sensitive mutants when superinfecting the RE1 rat embryo cell line (transformed by the HSV-2 temperature-sensitive mutant ts1) suggests that resident viral genes can be expressed. Brown et al. used a similar approach to detect HSV information latent in human ganglia. We report here retrieval of intertypic HSV recombinants from HSV transformed cells after superinfection with ts mutants of the alternative serotype of HSV. Restriction enzyme analysis which clearly differentiates between HSV-1 and HSV-2 DNA has demonstrated the isolation of recombinants spanning the genome and of virus indistinguishable from the original transforming virus.     

An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection

Herpes simplex virus 2 (HSV-2) infection causes significant morbidity and is an important cofactor for the transmission of HIV infection. A microbicide to prevent sexual transmission of HSV-2 would contribute substantially to controlling the spread of HIV and other infections. Because RNA interference (RNAi) provides effective antiviral defence in plants and other organisms, several studies have focused on harnessing RNAi to inhibit viral infection. Here we show that vaginal instillation of small interfering RNAs (siRNAs) targeting HSV-2 protects mice from lethal infection. siRNAs mixed with lipid are efficiently taken up by epithelial and lamina propria cells and silence gene expression in the mouse vagina and ectocervix for at least nine days. Intravaginal application of siRNAs targeting the HSV-2 UL27 and UL29 genes (which encode an envelope glycoprotein and a DNA binding protein, respectively) was well tolerated, did not induce interferon-responsive genes or cause inflammation, and protected mice when administered before and/or after lethal HSV-2 challenge. These results suggest that siRNAs are attractive candidates for the active component of a microbicide designed to prevent viral infection or transmission.     

Ⅰ型单纯疱疹病毒包膜糖蛋白L基因片段的克隆及高效表达

 采用PCR方法扩增HSV-1病毒型特异性包膜糖蛋白L(gL)基因片段并克隆至原核表达载体pGEX-5X-1获得重组质粒pGEX-5X-1-gL,将重组质粒转化E.coli BL21表达菌后经IPTG诱导表达目的蛋白.SDS-PAGE蛋白检测表明,在分子质量56 ku处有HSV-1 GST-gL融合蛋白的高效表达,通过IPTG浓度筛选和诱导前表达菌扩增培养时间的比较分析对诱导条件进行了优化,GST-gL融合蛋白表达量可达到菌体蛋白总量的48.65%.Western blot中利用HSV-1灭活病毒获得的多克隆抗体确证所表达蛋白为HSV-1病毒组分.这一表达系统的建立和优化对进一步探讨HSV-1 gL蛋白功能及其免疫原性提供了有利条件.     

An immunologically active chimaeric protein containing herpes simplex virus type 1 glycoprotein D

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) cause both persistent and latent infections, including recurrent cutaneous disease, lethal neonatal disease, central nervous system disease and other clinical syndromes. Modified live vaccines or conventionally prepared subunit vaccines have generally been unsuccessful in the treatment of HSV-1 and HSV-2 infections from the standpoints of safety and efficacy. It has been established that HSV-1 and HSV-2 infectivity may be neutralized in vitro with antisera directed specifically against each of the four major glycoproteins of the virus (gA/gB, gC, gD and gE) and antisera against glycoprotein gD, of either HSV-1 or HSV-2, are capable of neutralizing both HSV-1 and HSV-2 infectivity in vitro and in vivo. We have previously reported on the identification, DNA sequence and expression at low level in Escherichia coli of the gD gene of HSV-1 strain Patton. Here we describe construction of a hybrid gene encoding a chimaeric protein containing HSV-1 gD, bacteriophage lambda Cro and E. coli beta-galactosidase (gD-beta-gal) protein, which is expressed at high level in E. coli. Moreover, the chimaeric protein elicits antibodies in rabbits that not only immunoprecipitate gD from cells infected with HSV-1 and HSV-2 but also neutralize HSV-1 and HSV-2 infectivity in vitro.