CHARACTERIZATION OF A UBIQUITINATED PROTEIN WHICH IS EXTERNALLY LOCATED IN AFRICAN SWINE FEVER VIRIONS
CHARACTERIZATION OF A UBIQUITINATED PROTEIN WHICH IS EXTERNALLY LOCATED IN AFRICAN SWINE FEVER VIRIONS
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DOI：
10.1128/jvi.69.3.1785-1793.1995
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发表时间：
1995-03-01
影响因子：
5.4
通讯作者：
DIXON, LK
中科院分区：
文献类型：
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作者：
HINGAMP, PM;LEYLAND, ML;DIXON, LK
针对非洲猪瘟病毒（ASFV）编码的泛素偶联酶（UBCv1）培养了一种抗血清，并通过免疫印迹法（免疫印迹法）和免疫荧光法证明，该酶存在于纯化的细胞外病毒粒子中，在感染ASFV细胞后的早期和晚期均有表达，并且位于细胞质中。抗泛素血清用于鉴定ASFV感染期间出现的新型泛素偶联物。这种抗血清染色的病毒在感染后很晚才开始制造，提示病毒粒子蛋白可能被泛素化。Western blotting证实了这种可能性，在纯化的细胞外病毒粒子中鉴定出三种主要的抗泛素免疫反应蛋白，分子量分别为5、18和58 kDa；在相对较低浓度的洗涤剂n-辛基- β - d -glucopyranoside下，该18kda蛋白被病毒粒子溶解，这表明它位于外部，可能位于病毒衣壳内。纯化了18kda蛋白，n端氨基酸测序证实该蛋白泛素化，是ASFV编码蛋白。对编码该蛋白的ASFV基因（PIG1）进行测序，并在大肠杆菌表达载体中表达该蛋白。重组PIG1在off的存在下泛素化。大肠杆菌在体外表达UBCv1。这些结果表明PIG1可能是UBCv1的底物。预测的PIG1蛋白和重组泛素化蛋白的分子质量大于病毒粒子中泛素化蛋白的18 kda分子质量。因此，在病毒复制过程中，前体蛋白可能进行有限的蛋白水解以生成泛素化的18kda蛋白
An antiserum was raised against the African swine fever virus (ASFV)-encoded ubiquitin-conjugating enzyme (UBCv1) and used to demonstrate by Western blotting (immunoblotting) and immunofluorescence that the enzyme is present in purified extracellular virions, is expressed both early and late after infection of cells with ASFV, and is cytoplasmically located. Antiubiquitin serum was used to identify novel ubiquitin conjugates present during ASFV infections. This antiserum stained virus factories late after infection, suggesting that virion proteins may be ubiquitinated. This possibility was confirmed by Western blotting, which identified three major antiubiquitin-immunoreactive proteins with molecular masses of 5, 18, and 58 kDa in purified extracellular virions; The 18-kDa protein was solubilized from virions at relatively low concentrations of the detergent n-octyl-beta-D-glucopyranoside, indicating that it is externally located and is possibly in the virus capsid. The 18-kDa protein was purified, and N-terminal amino acid sequencing confirmed that the protein was ubiquitinated and was ASFV encoded. The ASFV gene encoding this protein (PIG1) was sequenced, and the encoded protein expressed in an Escherichia coli expression vector. Recombinant PIG1 was ubiquitinated in the presence off. coli expressed UBCv1 in vitro. These results suggest that PIG1 may be a substrate for UBCv1. The predicted molecular masses of the PIG1 protein and recombinant ubiquitinated protein were larger than the 18-kDa molecular mass of the ubiquinated protein present in virions. Therefore, during viral replication, a precursor protein may undergo limited proteolysis to generate the ubiquitinated 18-kDa protein