Department of Microbiology & Immunology, Virginia Commonwealth University, Richmond, VA 23298, USA.
Department of Pediatrics, University of California San Francisco, Oakland, CA 94609, USA.
Viruses. 2023 Apr 21;15(4):1023. doi: 10.3390/v15041023.
Human cytomegalovirus (CMV) has evolved to replicate while causing minimal damage, maintain life-long latency, reactivate sub-clinically, and, in spite of robust host immunity, produce and shed infectious virus in order to transmit to new hosts. The CMV temperance factor RL13 may contribute to this strategy of coexistence with the host by actively restricting viral replication and spread. Viruses with an intact gene grow slowly in cell culture, release little extracellular virus, and form small foci. By contrast, viruses carrying disruptive mutations in the gene form larger foci and release higher amounts of cell-free infectious virions. Such mutations invariably arise during cell culture passage of clinical isolates and are consistently found in highly adapted strains. The potential existence in such strains of other mutations with roles in mitigating RL13's restrictive effects, however, has not been explored. To this end, a mutation that frame shifts the gene in the highly cell culture-adapted laboratory strain Towne was repaired, and a C-terminal FLAG epitope was added. Compared to the frame-shifted parental virus, viruses encoding wild-type or FLAG-tagged wild-type RL13 produced small foci and replicated poorly. Within six to ten cell culture passages, mutations emerged in that restored replication and focus size to those of the -frame-shifted parental virus, implying that none of the numerous adaptive mutations acquired by strain Towne during more than 125 cell culture passages mitigate the temperance activity of RL13. Whilst RL13-FLAG expressed by passage zero stocks was localized exclusively within the virion assembly compartment, RL13-FLAG with a E208K substitution that emerged in one lineage was mostly dispersed into the cytoplasm, suggesting that localization to the virion assembly compartment is likely required for RL13 to exert its growth-restricting activities. Changes in localization also provided a convenient way to assess the emergence of mutations during serial passage, highlighting the usefulness of RL13-FLAG Towne variants for elucidating the mechanisms underlying RL13's temperance functions.
人类巨细胞病毒（CMV）已经进化到在造成最小损害的情况下进行复制，维持终身潜伏，亚临床激活，并在强大的宿主免疫下产生和释放感染性病毒，以便传播给新宿主。CMV 的 RL13 温和因子可能通过积极限制病毒复制和传播，为与宿主共存做出贡献。基因完整的病毒在细胞培养中生长缓慢，释放的细胞外病毒很少，形成的焦点也很小。相比之下，携带 基因破坏性突变的病毒形成的焦点更大，释放的无细胞感染性病毒颗粒更多。这种突变在临床分离株的细胞培养传代过程中总是会出现，并且在高度适应的菌株中始终存在。然而，在这些菌株中，是否存在其他突变来减轻 RL13 的限制作用，目前尚未得到探索。为此，修复了高度适应细胞培养的实验室株 Towne 中的 基因框移突变，并添加了 C 端 FLAG 表位。与框移亲本病毒相比，编码野生型或 FLAG 标记野生型 RL13 的病毒形成的焦点较小，复制能力较差。在六到十个细胞培养传代过程中，出现了突变，使 恢复了复制和焦点大小，与框移亲本病毒相当，这表明 Towne 株在超过 125 个细胞培养传代过程中获得的无数适应性突变没有一个能减轻 RL13 的温和活性。尽管零传代储备中表达的 RL13-FLAG 仅局限于病毒组装隔室，但在一个谱系中出现的 E208K 取代 RL13-FLAG 主要分散到细胞质中，这表明定位到病毒组装隔室可能是 RL13 发挥其生长限制活性所必需的。定位的变化也为评估 在连续传代过程中的出现提供了一种方便的方法，突出了 RL13-FLAG Towne 变体在阐明 RL13 温和功能机制方面的有用性。
