Mechanism of Cell Culture Adaptation of an Enteric Calicivirus, the Porcine Sapovirus Cowden Strain
Mechanism of Cell Culture Adaptation of an Enteric Calicivirus, the Porcine Sapovirus Cowden Strain
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DOI：
10.1128/jvi.02197-15
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发表时间：
2016-02-01
影响因子：
5.4
通讯作者：
Saif, Linda J.
中科院分区：
文献类型：
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作者：
Lu, Zhongyan;Yokoyama, Masaru;Saif, Linda J.
猪痘病毒（SaV）（PoSaV）Cowden株是仅有的几种可培养的肠杯状病毒之一。与野生型（WT）PoSaV Cowden株相比，组织培养适应（TC）PoSaV在RNA依赖性RNA聚合酶（RdRp）中具有两个保守的氨基酸取代，在衣壳蛋白（VP 1）中具有六个保守的氨基酸取代。通过使用反向遗传学系统，我们确定了4个氨基酸的取代在VP 1（残基178，289，324和328），但不是在RdRp区域的取代，是关键的PoSaV Cowden株的细胞培养适应。VP 1中的另外两个取代（残基291和295）在体外减少病毒复制。VP 1的三维结构分析表明，第178位残基位于二聚体-二聚体界面附近，可能影响VP 1的组装和寡聚化，第289、291、324和328位残基位于VP 1的突出亚结构域2（P2），可能影响病毒与细胞受体的结合;第295位残基位于两个单体蛋白的界面，可能影响了VP 1的二聚化。虽然在291或295位残基的突变从TC菌株的WT的逆转减少病毒在体外的复制，它增强了病毒在体内的复制，和回复突变体诱导更高水平的血清和粘膜抗体反应比TC PoSaV Cowden菌株诱导的。我们的研究结果揭示了PoSaV适应细胞培养的分子基础。这些发现可能为其他PoSaV毒株和人SaV或诺如病毒的细胞培养适应性提供新的关键信息。
The porcine sapovirus (SaV) (PoSaV) Cowden strain is one of only a few culturable enteric caliciviruses. Compared to the wildtype (WT) PoSaV Cowden strain, tissue culture-adapted (TC) PoSaV has two conserved amino acid substitutions in the RNA-dependent RNA polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse-genetics system, we identified that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328), but not the substitutions in the RdRp region, were critical for the cell culture adaptation of the PoSaV Cowden strain. The other two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro. Three-dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at protruding subdomain 2 (P2) of VP1, which may influence virus binding to cellular receptors; and residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutation at residue 291 or 295 from that of the TC strain to that of the WT reduced virus replication in vitro, it enhanced virus replication in vivo, and the revertants induced higher-level serum and mucosal antibody responses than those induced by the TC PoSaV Cowden strain. Our findings reveal the molecular basis for PoSaV adaptation to cell culture. These findings may provide new, critical information for the cell culture adaptation of other PoSaV strains and human SaVs or noroviruses.