Barreca Cristina, O'Hare Peter
Marie Curie Research Institute, Oxted, The Chart, Surrey RH8 0TL, United Kingdom.
J Virol. 2004 Aug;78(16):8641-53. doi: 10.1128/JVI.78.16.8641-8653.2004.
Herpes simplex virus (HSV) normally undergoes productive infection in culture, causing cell destruction and plaque formation. Here we characterize an unusual pattern of HSV type 1 (HSV-1) infection in MDBK cells which surprisingly results in suppression of replication, cell recovery, and maintenance of virus. Compared to Vero cells, MDBK cells supported a normal productive infection at a high multiplicity with complete cell destruction. At low multiplicity, HSV also showed an identical initial specific infectivity in the two cell types. Thereafter, the progression of infection was radically different. In contrast to the rapid plaque expansion and eventual destruction in Vero monolayers, in MDBK cells, after initial plaque formation, plaque size actually decreased and, with time, monolayers recovered. Using a green fluorescent protein (GFP)-VP16-expressing virus, we monitored infection in live individual plaques. After early stages of intense GFP-VP16 expression, expression regressed to a thin boundary at the edge of the plaques and was completely suppressed by 10 days. Cells lacking expression then began to grow into the plaque boundaries. Furthermore, following media replacement, individual cells expressing GFP-VP16 could be observed reinitiating infection. The results indicated the production of a potent inhibitory component during infection in MDBK cells, and we show the continued and prolonged presence of interferon in the medium, at times when there was no longer evidence of ongoing productive infection. We exploited the ability of V protein of simian virus 5 to degrade Stat1 and prevent interferon signaling. We established MDBK cells constitutively expressing the V protein with the resultant loss of Stat1. In comparison to the parental cells, infection in these cells now progressed at a rapid rate with expanding plaque formation. We believe the conclusions have significant implications for the study of HSV-1 and interferon signaling both in culture and in animal models.
单纯疱疹病毒（HSV）通常在培养物中进行增殖性感染，导致细胞破坏和空斑形成。在此，我们描述了1型单纯疱疹病毒（HSV-1）在MDBK细胞中一种不寻常的感染模式，令人惊讶的是，这种感染模式导致病毒复制受到抑制、细胞恢复以及病毒维持。与Vero细胞相比，MDBK细胞在高感染复数时支持正常的增殖性感染，导致细胞完全破坏。在低感染复数时，HSV在这两种细胞类型中也表现出相同的初始特异性感染性。此后，感染进程截然不同。与Vero单层细胞中快速的空斑扩展和最终破坏相反，在MDBK细胞中，初始空斑形成后，空斑大小实际上减小了，并且随着时间推移，单层细胞恢复。使用表达绿色荧光蛋白（GFP）-VP16的病毒，我们监测了单个活空斑中的感染情况。在GFP-VP16强烈表达的早期阶段之后，表达退回到空斑边缘的一条细边界，并在10天时完全被抑制。缺乏表达的细胞随后开始生长到空斑边界。此外，更换培养基后，可以观察到表达GFP-VP16的单个细胞重新开始感染。结果表明在MDBK细胞感染过程中产生了一种有效的抑制成分，并且我们发现在培养基中干扰素持续且长时间存在，而此时不再有正在进行的增殖性感染的证据。我们利用猴病毒5的V蛋白降解Stat1并阻止干扰素信号传导的能力。我们构建了组成性表达V蛋白的MDBK细胞，导致Stat1缺失。与亲代细胞相比，这些细胞中的感染现在以快速的速度进行，空斑形成不断扩大。我们认为这些结论对于在培养物和动物模型中研究HSV-1和干扰素信号传导具有重要意义。
