Autocatalytic Cleavage within Classical Swine Fever Virus NS3 Leads to a Functional Separation of Protease and Helicase
Autocatalytic Cleavage within Classical Swine Fever Virus NS3 Leads to a Functional Separation of Protease and Helicase
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DOI：
10.1128/jvi.00754-13
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发表时间：
2013-11-01
影响因子：
5.4
通讯作者：
Ruemenapf, Till
中科院分区：
文献类型：
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作者：
Lamp, Benjamin;Riedel, Christiane;Ruemenapf, Till
猪瘟病毒（classical swine fever virus，CSFV）是黄病毒科瘟病毒属（Pestivirus）的一种正链RNA病毒。加工大部分病毒多蛋白的关键是非结构蛋白3（NS 3）内的丝氨酸蛋白酶活性，其也具有RNA复制所必需的解旋酶和NTR活性。在CSFV感染的细胞中，NS 3以两种形式出现，80 kDa的完全加工的NS 3和120 kDa的前体分子NS 2 -3。在这里，我们报告的识别和映射的额外的自催化分子内裂解。Leu（1781）和Met（1782）之间存在一个可裂解的肽键，产生55 kDa的解旋酶亚基，并且根据底物，产生78 kDa的NS 2 -3片段（NS 2 - 3 p）或26 kDa的NS 3蛋白酶亚基（NS 3 p）。在使用NS 4 -5作为底物的转裂解测定中，NS 3 p充当能够加工多蛋白的全功能蛋白酶。NS 3 p包含最小必需蛋白酶，因为Leu 1781的缺失导致失活。第二个分子内切割定位于Leu（1748）/Lys（1749）肽键，产生蛋白水解无活性的NS 3片段。任何一个切割位点残基的缺失都会导致RNA感染性的丧失，这表明在相应位置上氨基酸同一性的功能重要性。我们的数据表明，NS 3部分内的内部裂解是一个共同的过程，进一步扩展了多功能NS 2 -3或NS 3的功能库，并代表了黄病毒科的复杂多蛋白加工的另一个水平。
Classical swine fever virus (CSFV) is a positive-strandedRNAvirus belonging to the genus Pestivirus within the Flaviviridae family. Pivotal for processing of a large portion of the viral polyprotein is a serine protease activity within nonstructural protein 3 (NS3) that also harbors helicase and NTPase activities essential forRNAreplication. In CSFV-infected cells, NS3 appears as two forms, a fully processed NS3 of 80 kDa and the precursor molecule NS2-3 of 120 kDa. Here we report the identification and mapping of additional autocatalytic intramolecular cleavages. One cleavable peptide bond occurs between Leu(1781) and Met(1782), giving rise to a helicase subunit of 55 kDa and, depending on the substrate, a NS2-3 fragment of 78 kDa (NS2-3p) or a NS3 protease subunit of 26 kDa (NS3p). In transcleavage assays using NS4-5 as a substrate, NS3p acts as a fully functional protease that is able to process the polyprotein. NS3p comprises the minimal essential protease, as deletion of Leu1781 results in inactivation. A second intramolecular cleavage was mapped to the Leu(1748)/Lys(1749) peptide bond that yields a proteolytically inactive NS3 fragment. Deletion of either of the cleavage site residues resulted in a loss ofRNAinfectivity, indicating the functional importance of amino acid identity at the respective positions. Our data suggest that internal cleavage within the NS3 moiety is a common process that further extends the functional repertoires of the multifunctional NS2-3 or NS3 and represents another level of the complex polyprotein processing of Flaviviridae.