Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication.
Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication.
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DOI：
10.1128/jvi.00217-17
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发表时间：
2017-05-01
影响因子：
5.4
通讯作者：
Solari R
中科院分区：
文献类型：
--
作者：
Guedán A;Swieboda D;Charles M;Toussaint M;Johnston SL;Asfor A;Panjwani A;Tuthill TJ;Danahay H;Raynham T;Mousnier A;Solari R
已知小核糖核酸病毒复制引起高尔基体和内质网膜的广泛重塑，并且已经鉴定了参与病毒复制复合物的许多宿主蛋白，包括氧固醇结合蛋白（OSBP）和磷脂酰肌醇4-激酶III β（PI 4KB）。由于OSBP和PI 4KB都是蛋白激酶D（PKD）的底物，并且已知PKD参与高尔基体膜囊泡和脂质转运的控制，因此我们假设PKD在病毒复制中起作用。我们提出了多条证据线来支持这一假设。首先，用人鼻病毒（HRV）感染HeLa细胞诱导PKD的磷酸化。其次，PKD抑制剂以浓度依赖性方式减少HRV基因组复制、蛋白表达和滴度，并且还阻断脊髓灰质炎病毒（PV）和口蹄疫病毒（FMDV）在多种细胞中的复制。第三，HRV复制显着减少HeLa细胞过度表达野生型和突变形式的PKD 1。第四，HRV基因组复制在HAP 1细胞中减少，其中PKD 1基因被成簇规则间隔短回文重复序列（CRISPR）-Cas9敲除。虽然我们还没有确定PKD调节病毒复制的分子机制，但我们的数据表明，这不是由于增强的干扰素信号传导或抑制网格蛋白介导的内吞作用，并且PKD抑制剂不需要存在于病毒摄取过程中。我们的数据首次表明，用小分子靶向PKD可以抑制HRV，PV和FMDV的复制，因此，PKD可能代表药物发现的新的抗病毒靶点。小核糖核酸病毒是人类和动物病原体的一个重要家族，我们的抗病毒剂非常有限。HRV是普通感冒的病原体，其本身是一种相对轻微的感染;然而，在哮喘和慢性阻塞性肺病（COPD）患者中，这种病毒是导致药物使用增加，症状恶化以及经常住院的加重的主要原因。因此，HRV代表了大量的医疗保健和经济负担，对此没有批准的疗法。我们试图确定一种新的宿主靶点作为潜在的抗HRV治疗。HRV感染诱导PKD的磷酸化，该激酶的抑制剂在病毒生命周期的早期阶段有效地阻断HRV复制。此外，PKD抑制剂还阻断PV和FMDV复制。这是第一次描述PKD可能代表抗病毒药物发现的靶点。
Picornavirus replication is known to cause extensive remodeling of Golgi and endoplasmic reticulum membranes, and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi membrane vesicular and lipid transport, we hypothesized that PKD played a role in viral replication. We present multiple lines of evidence in support of this hypothesis. First, infection of HeLa cells with human rhinovirus (HRV) induced the phosphorylation of PKD. Second, PKD inhibitors reduced HRV genome replication, protein expression, and titers in a concentration-dependent fashion and also blocked the replication of poliovirus (PV) and foot-and-mouth disease virus (FMDV) in a variety of cells. Third, HRV replication was significantly reduced in HeLa cells overexpressing wild-type and mutant forms of PKD1. Fourth, HRV genome replication was reduced in HAP1 cells in which the PKD1 gene was knocked out by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Although we have not identified the molecular mechanism through which PKD regulates viral replication, our data suggest that this is not due to enhanced interferon signaling or an inhibition of clathrin-mediated endocytosis, and PKD inhibitors do not need to be present during viral uptake. Our data show for the first time that targeting PKD with small molecules can inhibit the replication of HRV, PV, and FMDV, and therefore, PKD may represent a novel antiviral target for drug discovery. IMPORTANCE Picornaviruses remain an important family of human and animal pathogens for which we have a very limited arsenal of antiviral agents. HRV is the causative agent of the common cold, which in itself is a relatively trivial infection; however, in asthma and chronic obstructive pulmonary disease (COPD) patients, this virus is a major cause of exacerbations resulting in an increased use of medication, worsening symptoms, and, frequently, hospital admission. Thus, HRV represents a substantial health care and economic burden for which there are no approved therapies. We sought to identify a novel host target as a potential anti-HRV therapy. HRV infection induces the phosphorylation of PKD, and inhibitors of this kinase effectively block HRV replication at an early stage of the viral life cycle. Moreover, PKD inhibitors also block PV and FMDV replication. This is the first description that PKD may represent a target for antiviral drug discovery.