The UL25 protein of pseudorabies virus associates with capsids and localizes to the nucleus and to microtubules
The UL25 protein of pseudorabies virus associates with capsids and localizes to the nucleus and to microtubules
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DOI：
10.1128/jvi.74.1.474-482.2000
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发表时间：
2000-01-01
影响因子：
5.4
通讯作者：
Flamand, A
中科院分区：
文献类型：
--
作者：
Kaelin, K;Dezélée, S;Flamand, A
伪狂犬病病毒（Pseudorabiesvirus，PrV）的UL 25基因编码一个分子量约为57 kDa的蛋白质，该蛋白质在疱疹病毒中高度保守。单纯疱疹病毒1型（Herpessimplexvirustype 1，HSV）的UL 25蛋白是一种衣壳蛋白，参与病毒的穿透和衣壳的成熟。为了鉴定和表征PrV的UL 25基因产物，针对细菌表达的融合蛋白产生多克隆小鼠抗UL 25抗体。在免疫印迹和免疫沉淀试验的PrV感染的细胞裂解物中，这些抗UL 25抗血清特异性地识别预期大小的蛋白质，具有后期表达动力学。这种57 kDa的产物也存在于纯化的病毒体中，并被发现与所有类型的衣壳相关。从真核表达质粒pCG-UL 25指导在相同大小点迁移的蛋白质的合成。为了确定UL 25的亚细胞定位，在用PrV感染或用pCG-UL 25转染的Nonidet P-40提取的COS-7细胞上用抗UL 25抗血清进行免疫荧光研究。在PRV感染的细胞中，新合成的UL 25主要针对不同的核隔室，而在不存在其他病毒蛋白的情况下表达的UL 25更均匀地分布在细胞核中，并且还与微管共定位。为了研究UL 25在感染早期阶段的命运，在存在或不存在特异性地去除细胞骨架组分的药物的情况下，对侵入的PrV颗粒进行免疫荧光实验。我们发现，传入的核衣壳共定位与微管在其运输到细胞核和UL 25仍然与核衣壳在此运输过程中。
The UL25 gene of pseudorabies virus (PrV) can encode a protein of about 57 kDa which is well conserved among herpesviruses, The UL25 protein of herpes simplex virus type 1 is a capsid constituent involved in virus penetration and capsid maturation. To identify and characterize the UL25 gene product of PrV, polyclonal mouse anti-UL25 antibodies were raised to a bacterially expressed fusion protein. In immunoblotting and immunoprecipitation assays of PrV-infected cell lysates, these anti-UL25 antisera specifically recognized a protein of the expected size with late expression kinetics. This 57-kDa product was also present in purified virions and was found to be associated with all types of capsids. Synthesis of a protein migrating at the same size point was directed from the eukaryotic expression plasmid pCG-UL25. To determine the subcellular localization of UL25, immunofluorescence studies with anti-UL25 antisera were performed on Nonidet P-40-extracted COS-7 cells infected with PrV or transfected with pCG-UL25. In PRV-infected cells, newly synthesized UL25 is directed mainly to distinct nuclear compartments, whereas UL25 expressed in the absence of other viral proteins is distributed more uniformly in the nucleus and colocalizes also with microtubules. To study the fate of UL25 at very early stages of infection, immunofluorescence experiments were performed on invading PrV particles in the presence or absence of drugs that specifically depolymerize components of the cytoskeleton. We found that the incoming nucleocapsids colocalize with microtubules during their transport to the nucleus and that UL25 remains associated with nucleocapsids during this transport.