Melancon Jeffrey M, Fulmer Preston A, Kousoulas Konstantin G
Division of Biotechnology and Molecular Medicine, School of Veterinary Medicine, Louisiana State University, Baton Rouge, USA.
Virol J. 2007 Nov 8;4:120. doi: 10.1186/1743-422X-4-120.
The HSV-1 UL20 protein (UL20p) and glycoprotein K (gK) are both important determinants of cytoplasmic virion morphogenesis and virus-induced cell fusion. In this manuscript, we examined the effect of UL20 mutations on the coordinate transport and Trans Golgi Network (TGN) localization of UL20p and gK, virus-induced cell fusion and infectious virus production. Deletion of 18 amino acids from the UL20p carboxyl terminus (UL20 mutant 204t) inhibited intracellular transport and cell-surface expression of both gK and UL20, resulting in accumulation of UL20p and gK in the endoplasmic reticulum (ER) in agreement with the inability of 204t to complement UL20-null virus replication and virus-induced cell fusion. In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids (UL20 mutants 211t and 216t, respectively) allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization. However, while both 211t and 216t failed to complement for infectious virus production, 216t complemented for virus-induced cell fusion, but 211t did not. These results indicated that the carboxyl terminal six amino acids of UL20p were crucial for infectious virus production, but not involved in intracellular localization of UL20p/gK and concomitant virus-induced cell fusion. In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites. UL20p tyrosine-modified mutants with both tyrosine residues changed enabled efficient intracellular transport and TGN localization of UL20p and gK, but failed to complement for either infectious virus production, or virus-induced cell fusion. These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion.
单纯疱疹病毒1型UL20蛋白（UL20p）和糖蛋白K（gK）都是细胞质病毒体形态发生和病毒诱导细胞融合的重要决定因素。在本论文中，我们研究了UL20突变对UL20p和gK的协同转运及反式高尔基体网络（TGN）定位、病毒诱导细胞融合和感染性病毒产生的影响。从UL20p羧基末端缺失18个氨基酸（UL20突变体204t）抑制了gK和UL20的细胞内转运及细胞表面表达，导致UL20p和gK在内质网（ER）中积累，这与204t无法互补UL20缺失病毒的复制及病毒诱导的细胞融合相一致。相比之下，分别缺失11个或6个氨基酸的不太严重的羧基末端缺失（分别为UL20突变体211t和216t）允许UL20p和gK进行高效的细胞内转运、细胞表面表达及TGN共定位。然而，虽然211t和216t都不能互补感染性病毒的产生，但216t能互补病毒诱导的细胞融合，而211t不能。这些结果表明，UL20p的羧基末端6个氨基酸对于感染性病毒的产生至关重要，但不参与UL20p/gK的细胞内定位及伴随的病毒诱导细胞融合。在UL20的氨基末端，产生了UL20p突变体，改变了假定磷酸化位点内发现的Y38和Y49这一个或两个残基。两个酪氨酸残基都发生改变的UL20p酪氨酸修饰突变体能够实现UL20p和gK的高效细胞内转运及TGN定位，但不能互补感染性病毒的产生或病毒诱导的细胞融合。这些结果表明，UL20p在细胞质包膜中的功能与UL20在UL20p细胞内转运、细胞表面表达和病毒诱导细胞融合中的功能是可分离的。
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