Global Human-Kinase Screening Identifies Therapeutic Host Targets against Influenza.
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.
Southern Research Institute, Birmingham, AL, USA University of Alabama at Birmingham, Birmingham, AL, USA.
Colm
C
Atkins
Southern Research Institute, Birmingham, AL, USA.
Carrie W
CW
Evans
ActivX Biosciences, La Jolla, CA, USA.
Brian
B
Nordin
Wellspring Biosciences, LLC, San Diego, CA, USA.
Matthew P
MP
Patricelli
University of Alabama at Birmingham, Birmingham, AL, USA.
Robert
R
Reynolds
The Institute for Molecular Medicine Finland, Helsinki, Finland.
Krister
K
Wennerberg
Southern Research Institute, Birmingham, AL, USA University of Alabama at Birmingham, Birmingham, AL, USA j.noah@southernresearch.org.
James W
JW
Noah
D000072283:A549 Cells; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D017209:Apoptosis; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D002470:Cell Survival; D002853:Chromatography, Liquid; D055808:Drug Discovery; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000502:physiology*; D007251:Influenza, Human / Q000201:enzymology*; D013058:Mass Spectrometry; D000072380:NIMA-Related Kinase 1 / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000090742:Serine-Threonine Kinase 3; D019562:Viral Load; D014779:Virus Replication; D000099317:Hippo Kinases
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001710:Biotin; D000072380:NIMA-Related Kinase 1; D010455:Peptides; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D000090742:Serine-Threonine Kinase 3; C004749:desthiobiotin; C109163:STK24 protein, human; D000099317:Hippo Kinases; C000606914:NEK1 protein, human; C097835:STK3 protein, human; C491614:STK38 protein, human
influenza; kinase; profiling; siRNA