Expression of the hepatitis E virus ORF1.
Expression of the hepatitis E virus ORF1.
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DOI：
10.1007/s007050070093
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发表时间：
2000
影响因子：
2.7
通讯作者：
Frey TK
中科院分区：
文献类型：
--
作者：
Ropp SL;Tam AW;Beames B;Purdy M;Frey TK
戊型肝炎病毒（HEV）是一种未分类的正链RNA病毒，其基因组包含三个开放阅读框（ORF）。ORF1是HEV的5 ′端开放阅读框，编码与RNA复制有关的非结构蛋白，与正链RNA病毒的甲病毒样超家族成员的相应开放阅读框产物具有同源性。在该超家族的动物病毒成员（披膜病毒科的甲病毒属和风疹病毒属）中，该ORF的产物是一种非结构多聚蛋白（NSP），它被NSP内的木瓜蛋白酶样半胱氨酸蛋白酶（PCP）切割。为了确定HEV的NSP是否被类似地加工，将ORF 1引入质粒载体中，该质粒载体允许使用偶联的转录/翻译系统在体外表达和使用牛痘病毒驱动的瞬时表达系统在体内表达。构建了表达ORF 1的重组痘苗病毒。在标准条件下的体外和体内表达仅产生全长185 kDa的多蛋白。在体外添加辅因子，如二价阳离子和微粒体，已被证明激活其他病毒蛋白酶，未能改变这种表达模式。然而，在体内延长孵育（24 - 36小时）后，观察到107 kDa和78 kDa的两种潜在加工产物。使用N-和C-末端特异性免疫沉淀和缺失诱变来确定NSP内这些产物的顺序为NH $_2 $-78 kDa-107 kDa-COOH。然而，Cys483的位点特异性诱变，通过计算机比对预测是NSP内PCP的催化二联体的一个成员，未能消除这种切割。此外，HEV毒株之间的序列比对显示，该PCP的拟议催化二联体的另一个成员His590不保守。因此，在体内长时间表达后观察到的NSP的切割不是由该蛋白酶介导的，并且怀疑NSP内存在功能性PCP。在HEV感染的原代猴肝细胞中检测NSP表达和加工的尝试没有成功，因此这种蛋白水解切割不能被验证。总体而言，本研究的结果表明，要么HEV NSP不被加工，要么它在一个位点被α-样超家族病毒中新颖的病毒编码的蛋白酶或细胞蛋白酶切割。
Hepatitis E virus (HEV) is an unclassified, plus-strand RNA virus whose genome contains three open reading frames (ORFs). ORF1, the 5′ proximal ORF of HEV, encodes nonstructural proteins involved in RNA replication which share homology with the products of the corresponding ORF of members of the alphavirus-like superfamily of plus-strand RNA viruses. Among animal virus members of this superfamily (the alphavirus and rubivirus genera of the family Togaviridae), the product of this ORF is a nonstructural polyprotein (NSP) that is cleaved by a papain-like cysteine protease (PCP) within the NSP. To determine if the NSP of HEV is similarly processed, ORF1 was introduced into a plasmid vector which allowed for expression both in vitro using a coupled transcription/translation system and in vivo using a vaccinia virus-driven transient expression system. A recombinant vaccinia virus expressing ORF1 was also constructed. Both in vitro and in vivo expression under standard conditions yielded only the full-length 185 kDa polyprotein. Addition of co-factors in vitro, such as divalent cations and microsomes which have been shown to activate other viral proteases, failed to change this expression pattern. However, in vivo following extended incubations (24--36 hours), two potential processing products of 107 kDa and 78 kDa were observed. N- and C-terminus-specific immunoprecipitation and deletion mutagenesis were used to determine that the order of these products within the NSP is NH$_2$-78 kDa-107 kDa-COOH. However, site-specific mutagenesis of Cys483, predicted by computer alignment to be one member of the catalytic dyad of a PCP within the NSP, failed to abolish this cleavage. Additionally, sequence alignment across HEV strains revealed that the other member of the proposed catalytic dyad of this PCP, His590, was not conserved. Thus, the cleavage of the NSP observed following prolonged in vivo expression was not mediated by this protease and it is doubtful that a functional PCP exists within the NSP. Attempts to detect NSP expression and processing in HEV-infected primary monkey hepatocytes were not successful and therefore this proteolytic cleavage could not be authenticated. Overall, the results of this study indicate that either the HEV NSP is not processed or that it is cleaved at one site by a virally-encoded protease novel among alpha-like superfamily viruses or a cellular protease.
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影响因子：
14.9
作者：
DEVEREUX, J;HAEBERLI, P;SMITHIES, O
通讯作者：
SMITHIES, O
影响因子：
5.4
作者：
MACKETT, M;SMITH, GL;MOSS, B
通讯作者：
MOSS, B
影响因子：
5.4
作者：
KIRCHWEGER, R;ZIEGLER, E;SKERN, T
通讯作者：
SKERN, T
影响因子：
64.8
作者：
MOSS, B;ELROYSTEIN, O;FUERST, TR
通讯作者：
FUERST, TR
影响因子：
2.7
作者：
Pringle, C R
通讯作者：
Pringle, C R