CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses
CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses
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DOI：
10.1128/jvi.01714-18
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发表时间：
2019-03-01
影响因子：
5.4
通讯作者：
Tautz, N.
中科院分区：
文献类型：
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作者：
Isken, O.;Postel, A.;Tautz, N.
像牛病毒性腹泻病毒(BVDV)这样的有害病毒是对牲畜的威胁。对于鼠疫病毒，在细胞培养中区分细胞致病株(Cp)和非细胞致病株(Nncp)。BVDV的非cp生物型能够建立持续性感染，这是疾病控制中的一个主要问题。非CP生物型依赖于病毒RNA复制的时间控制，由非结构蛋白2-3(NS2-3)的调节裂解介导。这种切割是由NS2中的自体蛋白酶催化的，其活性取决于其细胞辅因子DNAJC14。由于这种伴侣蛋白只有少量可用，并与NS2紧密结合，因此在感染后翻译的NS2-3不再被切割。由于NS3是病毒复制酶的重要组成部分，这种多蛋白加工的转变与RNA复制的下调有关。相反，主要通过RNA重组产生的cp BVDV毒株表现出高度可变的基因组结构和不受限制的NS3释放。到目前为止，DNAJC14对非cp鼠疫病毒的功能重要性仅对BVDV-1被证实。因此，其他非猪瘟病毒的复制是否也依赖于DNAJC14仍是一个谜。通过建立牛和猪的DNAJC14基因敲除细胞，我们可以证明：(1)6个不同的非cp瘟疫病毒物种(A到D，F和G)的复制依赖于DNAJC14，(Ii)在没有DNAJC14的情况下，鼠疫病毒复制酶NS3-5B可以组装成功能复合体，以及(Iii)所有cp瘟疫病毒复制它们的RNA并产生不依赖宿主DNAJC14的感染性后代。综上所述，这些发现证实了DNAJC14是复制和维持非cp生物型鼠疫病毒的关键细胞辅助因子。重要的是，只有非cp鼠疫病毒株能够建立终身持续感染，从而在田间产生病毒库。这种生物型的分子基础只有部分了解，也只对BVDV-1毒株进行了深入研究。病毒RNA复制的时间控制与非CP生物型相关，并通过限制细胞DNAJC14的数量来调节，DNAJC14激活病毒NS2蛋白酶以催化必要复制酶组分NS3的释放。在这里，我们证明了几种非冠状病毒依赖于DNAJC14进行RNA复制。此外，与非猪瘟病毒形成鲜明对比的是，所有的猪瘟病毒都独立于DNAJC14进行复制。在持续感染的动物体内产生cp BVDV是引起粘膜疾病的原因。因此，在DNAJC14依赖中观察到的严格的生物型特异性差异应该进一步研究它在细胞类型/组织趋向性和这种致命疾病的发病机制中的作用。
Pestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.IMPORTANCE Only noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.