Isolation and characterization of noncytopathic pestivirus mutants reveals a role for nonstructural protein NS4B in viral cytopathogenicity
Isolation and characterization of noncytopathic pestivirus mutants reveals a role for nonstructural protein NS4B in viral cytopathogenicity
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DOI：
10.1128/jvi.75.22.10651-10662.2001
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发表时间：
2001-11-01
影响因子：
5.4
通讯作者：
Rice, CM
中科院分区：
文献类型：
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作者：
Qu, L;McMullan, LK;Rice, CM
牛病毒性腹泻病毒（BVDV）是鼠疫病毒的原型，根据其对培养细胞的影响可分为细胞病变型（cp）和非细胞病变型（ncp）。cp病毒与ncp病毒的不同之处在于产生病毒非结构蛋白NS3。然而，cp病毒在细胞培养中诱导细胞病变的机制尚不清楚。在这里，我们使用遗传方法分离出由一种低频率的cp病毒产生的ncp变异。建立了以嘌呤霉素乙酰转移酶为显性选择标记的双链BVDV （cp菌株NADL）。这种双链病毒表现出比NADL稍慢的生长动力学和更小的斑块，但仍然是cp。通过嘌呤霉素选择分离出许多独立的ncp变体。值得注意的是，这些ncp变异产生NS3和病毒RNA的水平与cp亲本相当。序列分析发现NS3没有变化，但在所有ncp变异中，NS4B残基15处发现了Y2441C的替代。将Y2441C替换引入NADL或双电子cp病毒中，重建了ncp表型。Y2441在鼠疫病毒中高度保守，位于内质网膜细胞质侧的NS4B区域。Y2441的其他工程替代也影响了病毒的细胞致病性和活力，其中Y2441V为cp， Y2441A为ncp，而Y2441D使病毒无法复制。对Y2441进行ncp替换后，NS2-3的表达水平相对于NS3略有升高。我们还发现，在nadl感染的细胞中，NS3、NS4B和NS5A可以化学交联，表明它们作为多蛋白复合物的组成部分相关。虽然机制仍有待阐明，但这些结果表明，尽管产生NS3，但NS413的突变可以减弱BVDV的细胞致病性。
Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viruses by the production of viral nonstructural protein NS3. However, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics and smaller plaques than NADL but remained cp. A number of independent ncp variants were isolated by puromycin selection. Remarkably, these ncp variants produced NS3 and viral RNA at levels comparable to those of the cp parent. Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasmic reticulum membrane. Other engineered substitutions for Y2441 also affected viral cytopathogenicity and viability, with Y2441V being cp, Y2441A being ncp, and Y2441D rendering the virus unable to replicate. The ncp substitutions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-linked in NADL-infected cells, indicating that they are associated as components of a multiprotein complex. Although the mechanism remains to be elucidated, these results demonstrate that mutations in NS413 can attenuate BVDV cytopathogenicity despite NS3 production.