The Latent Herpes Simplex Virus Type 1 Genome Copy Number in Individual Neurons Is Virus Strain Specific and Correlates with Reactivation

The viral genetic elements that determine the in vivo reactivation efficiencies of fully replication competent wild-type herpes simplex virus (HSV) strains have not been identified. Among the common laboratory strains, KOS reactivates in vivo at a lower efficiency than either strain 17syn+ or strain McKrae. An important first step in understanding the molecular basis for this observation is to distinguish between viral genetic factors that regulate the establishment of latency from those that directly regulate reactivation. Reported here are experiments performed to determine whether the reduced reactivation of KOS was associated with a reduced ability to establish or maintain latent infections. For comparative purposes, latent infections were quantified by (i) quantitative PCR on DNA extracted from whole ganglia, (ii) the number of latency-associated transcript (LAT) promoter-positive neurons, using KOS and 17syn+ LAT promoter–β-galactosidase reporter mutants, and (iii) contextual analysis of DNA. Mice latently infected with 17syn+-based strains contained more HSV type 1 (HSV-1) DNA in their ganglia than those infected with KOS strains, but this difference was not statistically significant. The number of latently infected neurons also did not differ significantly between ganglia latently infected with either the low- or high-reactivator strains. In addition to the number of latent sites, the number of viral genome copies within the individual latently infected neurons has recently been demonstrated to be variable. Interestingly, neurons latently infected with KOS contained significantly fewer viral genome copies than those infected with either 17syn+ or McKrae. Thus, the HSV-1 genome copy number profile is viral strain specific and positively correlates with the ability to reactivate in vivo. This is the first demonstration that the number of HSV genome copies within individual latently infected neurons is regulated by viral genetic factors. These findings suggest that the latent genome copy number may be an important parameter for subsequent induced reactivation in vivo.

目前尚未明确鉴定出可决定完全复制型野生型单纯疱疹病毒（herpes simplex virus, HSV）毒株在体内再激活效率的病毒遗传元件。在常见实验室毒株中，KOS株的体内再激活效率低于17syn+株与McKrae株。解析这一现象的分子基础，首要关键步骤是区分调控潜伏感染建立的病毒遗传因子与直接调控再激活的病毒遗传因子。本文报道的实验旨在探究KOS株的再激活能力减弱是否与其建立或维持潜伏感染的能力下降相关。
为开展比较分析，我们通过以下三种方式对潜伏感染进行定量：(i) 对从全神经节中提取的DNA进行定量聚合酶链反应（quantitative PCR）；(ii) 利用携带KOS与17syn+株潜伏相关转录本（latency-associated transcript, LAT）启动子-β-半乳糖苷酶（β-galactosidase）报告基因的突变株，统计LAT启动子阳性神经元的数量；(iii) 对DNA进行背景分析。
以17syn+株为骨架构建的毒株感染的小鼠，其神经节内的1型单纯疱疹病毒（herpes simplex virus type 1, HSV-1）DNA拷贝数高于KOS株感染的小鼠，但该差异未达到统计学显著性水平。无论是低再激活株还是高再激活株感染的神经节，其潜伏感染神经元的数量同样未呈现显著差异。除潜伏位点的数量外，近期研究已证实，单个潜伏感染神经元内的病毒基因组拷贝数存在差异。值得注意的是，KOS株感染的神经元内的病毒基因组拷贝数显著低于17syn+株或McKrae株感染的神经元。
由此可见，HSV-1基因组拷贝数谱具有病毒毒株特异性，且与体内再激活能力呈正相关。本研究首次证实，单个潜伏感染神经元内的HSV基因组拷贝数受病毒遗传因子调控。上述研究结果表明，潜伏状态下的基因组拷贝数可能是影响体内诱导再激活的重要参数。