Mapping of Sequences in Pseudorabies Virus pUL34 That Are Required for Formation and Function of the Nuclear Egress Complex
Mapping of Sequences in Pseudorabies Virus pUL34 That Are Required for Formation and Function of the Nuclear Egress Complex
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DOI：
10.1128/jvi.00021-13
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发表时间：
2013-04-01
影响因子：
5.4
通讯作者：
Mettenleiter, Thomas C.
中科院分区：
文献类型：
--
作者：
Passvogel, Lars;Truebe, Patricia;Mettenleiter, Thomas C.
新合成的疱疹病毒核衣壳从细胞核到细胞质的有效易位需要核出口复合物（NEC）。它由 II 型膜蛋白 pUL34 组成，该蛋白与内核膜 (INM) 处的 pUL31 相互作用。为了绘制 pUL34 内核膜靶向和 pUL31 相互作用所需的区域，我们构建了缺失/取代突变。此前，我们发现伪狂犬病病毒（PrV）pUL34的50个C端氨基酸（aa），包括跨膜结构域，可以在功能上被细胞层相关多肽2β（Lap2β）序列取代。相反，C 端 100 个氨基酸的替换消除了互补作用，但没有消除 pUL31 相互作用。为了进一步描绘该区域内的必需序列，生成了与 Lap2 beta 序列融合的 60、70、80、85 和 90 个氨基酸的 C 端 pUL34 截短。虽然截短至 85 个氨基酸是有功能的，但 C 端 90 个氨基酸的删除会消除功能，这表明重要区域位于氨基酸 171 和 176 之间。氨基酸 173 至 175 代表 RQR，这是一个介导 INM 靶向的基序。 RQG 诱变表明，突变蛋白表现出明显的高尔基体定位，但仍有一小部分到达 INM。 pUL34 N 端结构域的缺失突变表明，前 4 个氨基酸的缺失是可以耐受的，而删除 9 个或更多残基会产生无功能的蛋白质。此外，三个保守半胱氨酸的突变并没有消除pUL34的功能，而保守谷氨酰胺/酪氨酸序列的改变产生了无功能的蛋白质。
The nuclear egress complex (NEC) is required for efficient translocation of newly synthesized herpesvirus nucleocapsids from the nucleus to the cytosol. It consists of the type II membrane protein pUL34 which interacts with pUL31 at the inner nuclear membrane (INM). To map regions within pUL34 required for nuclear membrane targeting and pUL31 interaction, we constructed deletion/substitution mutations. Previously, we showed that 50 C-terminal amino acids (aa) of pseudorabies virus (PrV) pUL34, including the transmembrane domain, could be functionally replaced by cellular lamina-associated polypeptide 2 beta (Lap2 beta) sequences. In contrast, replacement of the C-terminal 100 aa abrogated complementation but not pUL31 interaction. To further delineate essential sequences within this region, C-terminal pUL34 truncations of 60, 70, 80, 85, and 90 aa fused to Lap2 beta sequences were generated. While truncations up to 85 aa were functional, deletion of the C- terminal 90 aa abrogated function, which indicates that the important region is located between aa 171 and 176. Amino acids 173 to 175 represent RQR, a motif suggested to mediate INM targeting. Mutagenesis to RQG revealed that the mutant protein exhibited pronounced Golgi localization, but a fraction still reached the INM. Deletion mutations in the N-terminal domain of pUL34 demonstrated that absence of the first 4 aa was tolerated, while removal of 9 or more residues resulted in a nonfunctional protein. In addition, mutation of three conserved cysteines did not abrogate pUL34 function, whereas alteration of a conserved glutamine/tyrosine sequence yielded a nonfunctional protein.