title: Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication.
abstract: 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.IMPORTANCEPicornaviruses 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.
authors.affiliation: National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, United Kingdom.
authors.forename: Anabel
authors.initials: A
authors.lastname: Guedán
authors.affiliation: National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, United Kingdom.
authors.forename: Dawid
authors.initials: D
authors.lastname: Swieboda
authors.affiliation: CRT Discovery Laboratories, Cambridge, United Kingdom.
authors.forename: Mark
authors.initials: M
authors.lastname: Charles
authors.affiliation: National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, United Kingdom.
authors.forename: Marie
authors.initials: M
authors.lastname: Toussaint
authors.affiliation: National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, United Kingdom.
authors.forename: Sebastian L
authors.initials: SL
authors.lastname: Johnston
authors.affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: Amin
authors.initials: A
authors.lastname: Asfor
authors.affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: Anusha
authors.initials: A
authors.lastname: Panjwani
authors.affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: Tobias J
authors.initials: TJ
authors.lastname: Tuthill
authors.affiliation: Enterprise Therapeutics, Sussex Innovation Centre, University of Sussex, Brighton, United Kingdom.
authors.forename: Henry
authors.initials: H
authors.lastname: Danahay
authors.affiliation: CRT Discovery Laboratories, Cambridge, United Kingdom.
authors.forename: Tony
authors.initials: T
authors.lastname: Raynham
authors.affiliation: National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, United Kingdom.|Centre for Experimental Medicine, Queen's University Belfast, Belfast, United Kingdom.
authors.forename: Aurelie
authors.initials: A
authors.lastname: Mousnier
authors.affiliation: National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, United Kingdom r.solari@imperial.ac.uk.
authors.forename: Roberto
authors.initials: R
authors.lastname: Solari
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D006224:Cricetinae; D004261:DNA Replication / Q000235:genetics*; D004279:DNA, Viral / Q000096:biosynthesis; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000254:growth & development*; D055786:Gene Knockout Techniques; D006367:HeLa Cells; D006801:Humans; D007370:Interferon Type I / Q000378:metabolism; D010766:Phosphorylation; D017955:Poliovirus / Q000235:genetics / Q000254:growth & development*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D012229:Rhinovirus / Q000235:genetics* / Q000254:growth & development*; D014779:Virus Replication / Q000235:genetics*
chemical_list: C551536:CRT 0066101; D004279:DNA, Viral; D007370:Interferon Type I; D011743:Pyrimidines; C089212:protein kinase D; D011493:Protein Kinase C
keywords: Golgi membrane; antiviral; picornavirus; protein kinase D; rhinovirus; viral replication