The vaccinia virus G1L putative metalloproteinase is essential for viral replication in vivo
The vaccinia virus G1L putative metalloproteinase is essential for viral replication in vivo
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DOI：
10.1128/jvi.78.18.9947-9953.2004
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发表时间：
2004-09-01
影响因子：
5.4
通讯作者：
Hruby, DE
中科院分区：
文献类型：
--
作者：
Hedengren-Olcott, M;Byrd, CM;Hruby, DE
由牛痘病毒G1 L基因编码的推定的金属蛋白酶的功能是未知的。为了解决这个问题，我们已经产生了一种牛痘病毒株，其中当在表达四环素阻遏物的细胞系中进行感染时，G1 L基因的表达依赖于四环素（泰特）的添加。当存在泰特时，vvtetOG 1 L病毒在这些细胞中复制类似于野生型西储（WR）病毒，但在不存在泰特时，在病毒成熟的晚期阶段被阻止。这种停滞导致98.5%的圆形未成熟病毒颗粒的积累，相比之下，当存在泰特时，在相似的时间点为6.9%。同样，当vvtetOG 1 L病毒在不存在泰特的情况下繁殖时，感染性病毒子代的滴度降低98.9%+/-0.97%。突变体病毒复制部分拯救质粒编码的G1 L，但不是由G1 L含有HXXEH基序突变为RXXQR。G1 L的建模揭示了与酿酒酵母线粒体加工肽酶（α-MPP）的α-亚基的预测结构相似性。在该模型中，G1 L的HXXEH基序与α-MPP的HXXDR基序完全重叠。这些结果表明，G1 L是病毒成熟所必需的，并表明G1 L是一种与α-MPP具有结构同源性的金属蛋白酶。然而，与泰特和野生型WR对照的结果相比，在不存在泰特的情况下，在G1 L条件突变体中未观察到对牛痘病毒主要核心蛋白的表达和加工的明显影响，表明在病毒形态发生的这一步骤之后需要G1 L活性。
The function of the putative metalloproteinase encoded by the vaccinia virus G1L gene is unknown. To address this question, we have generated a vaccinia virus strain in which expression of the G1L gene is dependent on the addition of tetracycline (TET) when infection proceeds in a cell line expressing the tetracycline repressor. The vvtetOG1L virus replicated similarly to wild-type Western Reserve (WR) virus in these cells when TET was present but was arrested at a late stage in viral maturation in the absence of TET. This arrest resulted in the accumulation of 98.5% round immature virus particles compared to 6.9% at a similar time point when TET was present. Likewise, the titer of infectious virus progeny decreased by 98.9%+/-0.97% when the vvtetOG1L virus was propagated in the absence of TET. Mutant virus replication was partially rescued by plasmid-encoded G1L, but not by G1L containing an HXXEH motif mutated to RXXQR. Modeling of G1L revealed a predicted structural similarity to the alpha-subunit of Saccharomyces cerevisiae mitochondrial processing peptidase (alpha-MPP). The HXXEH motif of G1L perfectly overlaps the HXXDR motif of alpha-MPP in this model. These results demonstrate that G1L is essential for virus maturation and suggest that G1L is a metalloproteinase with structural homology to alpha-MPP. However, no obvious effects on the expression and processing of the vaccinia virus major core proteins were observed in the G1L conditional mutant in the absence of TET compared to results for the TET and wild-type WR controls, suggesting that G1L activity is required after this step in viral morphogenesis.