TY - JOUR

T1 - Global Human-Kinase Screening Identifies Therapeutic Host Targets against Influenza

AU - Atkins, Colm

AU - Evans, Carrie W

AU - Nordin, Brian

AU - Patricelli, Matthew P

AU - Reynolds, Robert

AU - Wennerberg, Krister

AU - Noah, James W

N1 - © 2014 Society for Laboratory Automation and Screening.

PY - 2014/7

Y1 - 2014/7

N2 - During viral infection of human cells, host kinases mediate signaling activities that are used by all viruses for replication; therefore, targeting of host kinases is of broad therapeutic interest. Here, host kinases were globally screened during human influenza virus (H1N1) infection to determine the time-dependent effects of virus infection and replication on kinase function. Desthiobiotin-labeled analogs of adenosine triphosphate and adenosine diphosphate were used to probe and covalently label host kinases in infected cell lysates, and probe affinity was determined. Using infected human A549 cells, we screened for time-dependent signal changes and identified host kinases whose probe affinities differed significantly when compared to uninfected cells. Our screen identified 10 novel host kinases that have not been previously shown to be involved with influenza virus replication, and we validated the functional importance of these novel kinases during infection using targeted small interfering RNAs (siRNAs). The effects of kinase-targeted siRNA knockdowns on replicating virus levels were measured by quantitative reverse-transcription PCR and cytoprotection assays. We identified several novel host kinases that, when knocked down, enhanced or reduced the viral load in cell culture. This preliminary work represents the first screen of the changing host kinome in influenza virus-infected human cells.

AB - During viral infection of human cells, host kinases mediate signaling activities that are used by all viruses for replication; therefore, targeting of host kinases is of broad therapeutic interest. Here, host kinases were globally screened during human influenza virus (H1N1) infection to determine the time-dependent effects of virus infection and replication on kinase function. Desthiobiotin-labeled analogs of adenosine triphosphate and adenosine diphosphate were used to probe and covalently label host kinases in infected cell lysates, and probe affinity was determined. Using infected human A549 cells, we screened for time-dependent signal changes and identified host kinases whose probe affinities differed significantly when compared to uninfected cells. Our screen identified 10 novel host kinases that have not been previously shown to be involved with influenza virus replication, and we validated the functional importance of these novel kinases during infection using targeted small interfering RNAs (siRNAs). The effects of kinase-targeted siRNA knockdowns on replicating virus levels were measured by quantitative reverse-transcription PCR and cytoprotection assays. We identified several novel host kinases that, when knocked down, enhanced or reduced the viral load in cell culture. This preliminary work represents the first screen of the changing host kinome in influenza virus-infected human cells.

KW - A549 Cells

KW - Adenosine Diphosphate/chemistry

KW - Adenosine Triphosphate/chemistry

KW - Apoptosis

KW - Biotin/analogs & derivatives

KW - Cell Survival

KW - Chromatography, Liquid

KW - Drug Discovery

KW - Humans

KW - Influenza A Virus, H1N1 Subtype/physiology

KW - Influenza, Human/enzymology

KW - Mass Spectrometry

KW - NIMA-Related Kinase 1/chemistry

KW - Peptides/chemistry

KW - Protein-Serine-Threonine Kinases/chemistry

KW - RNA, Small Interfering/genetics

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Viral Load

KW - Virus Replication

U2 - 10.1177/1087057113518068

DO - 10.1177/1087057113518068

M3 - Journal article

C2 - 24464431

VL - 19

SP - 936

EP - 946

JO - SLAS Discovery

JF - SLAS Discovery

SN - 2472-5552

IS - 6

ER -