ORF73-Null murine gammaherpesvirus 68 reveals roles for mLANA and p53 in virus replication
ORF73-Null murine gammaherpesvirus 68 reveals roles for mLANA and p53 in virus replication
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DOI：
10.1128/jvi.00111-07
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发表时间：
2007-11-01
影响因子：
5.4
通讯作者：
Speck, Samuel H.
中科院分区：
文献类型：
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作者：
Forrest, J. Craig;Paden, Clinton R.;Speck, Samuel H.
伽马疱疹病毒在宿主淋巴细胞中建立终生的潜伏感染，在此期间，有限的病毒基因产物亚集有助于病毒Episome的维持。在伽马-2疱疹病毒亚家族中，这包括保守的ORF73编码的LANA蛋白的表达。我们先前通过功能丧失突变证明，小鼠伽马疱疹病毒68(MHV68)ORF73基因产物mLANA是建立小鼠鼻内接种后潜伏期所必需的(N.J.Moorman，D.O.Willer，和S.H.Spek，J.Virol)。77：10295-10303,2003年)。MLANA缺陷病毒在感染小鼠的肺部也表现出急性病毒复制的缺陷。后一项观察使我们研究了mLANA在病毒复制中的作用。我们评估了mLANA缺陷病毒(73)在低感染复数(MOI)条件下在细胞培养中的复制能力，发现73.停止生长在小鼠成纤维细胞中受到损害，但在Vero细胞中没有。表达mLANA-绿色荧光蛋白(GFP)融合的重组病毒显示，mLANA在病毒复制周期中都有表达。此外，在高MOIS条件下停止感染小鼠成纤维细胞比感染基因修复的标记拯救病毒(73 MR)具有更强的细胞毒性，这种表型与病毒基因表达的动力学增强和P53的激活有关。值得注意的是，细胞死亡增加、病毒基因表达和P53诱导与病毒DNA复制无关。成纤维细胞中mLANA-GFP融合蛋白的表达与p53诱导激动剂治疗后P53稳定性降低和细胞死亡减少有关。在P53基因缺陷的小鼠胚胎成纤维细胞中，停止感染减少了。更值得注意的是，P53的缺失导致了73.Stop和73.MR病毒复制的整体延迟，这与病毒基因表达延迟有关，表明P53在MRV68复制中起作用。与这些发现一致的是，促进复制的病毒基因的表达受到p53过度表达或p53激动剂依托泊苷治疗的积极影响。总体而言，这些数据证明了mLANA在MHV68复制中的重要性，并表明LANA蛋白限制了细胞应激反应的诱导，以调节病毒基因表达级联反应，并限制宿主细胞损伤。
Gammaherpesviruses establish lifelong, latent infections in host lymphocytes, during which a limited subset of viral gene products facilitates maintenance of the viral episome. Among the gamma-2-herpesvirus (rhadinovirus) subfamily, this includes expression of the conserved ORF73-encoded LANA proteins. We previously demonstrated by loss-of-function mutagenesis that the murine gammaherpesvirus 68 (MHV68) ORF73 gene product, mLANA, is required for the establishment of latency following intranasal inoculation of mice (N. J. Moorman, D. O. Willer, and S. H. Speck, J. Virol. 77:10295-10303, 2003). mLANA-deficient viruses also exhibited a defect in acute virus replication in the lungs of infected mice. The latter observation led us to examine the role of mLANA in productive viral replication. We assessed the capacity of mLANA-deficient virus (73.Stop) to replicate in cell culture at low multiplicities of infection (MOIs) and found that 73.Stop growth was impaired in murine fibroblasts but not in Vero cells. A recombinant virus expressing an mLANA-green fluorescent protein (GFP) fusion revealed that mLANA is expressed throughout the virus replication cycle. In addition, 73.Stop infection of murine fibroblasts at high MOIs was substantially more cytotoxic than infection with a genetically repaired marker rescue virus (73.MR), a phenotype that correlated with enhanced kinetics of viral gene expression and increased activation of p53. Notably, augmented cell death, viral gene expression, and p53 induction were independent of viral DNA replication. Expression of a mLANA-GFP fusion protein in fibroblasts correlated with both reduced p53 stabilization and reduced cell death following treatment with p53-inducing agonists. In agreement, accentuated cell death associated with 73.Stop infection was reduced in p53-deficient murine embryonic fibroblasts. Additionally, replication of 73.Stop in p53-deficient cells was restored to levels comparable to those of 73.MR. More remarkably, the absence of p53 led to an overall delay in replication for both 73.Stop and 73.MR viruses, which correlated with delayed viral gene expression, indicating a role for p53 in MRV68 replication. Consistent with these findings, the expression of replication-promoting viral genes was positively influenced by p53 overexpression or treatment with the p53 agonist etoposide. Overall, these data demonstrate the importance of mLANA in MHV68 replication and suggest that LANA proteins limit the induction of cellular stress responses to regulate the viral gene expression cascade and limit host cell injury.