Respiratory syncytial virus influences NF-kappaB-dependent gene expression through a novel pathway involving MAP3K14/NIK expression and nuclear complex formation with NF-kappaB2.
A member of the Paramyxoviridae family of RNA viruses, respiratory syncytial virus (RSV), is a leading cause of epidemic respiratory tract infection in children. In children, RSV primarily replicates in the airway mucosa, a process that alters epithelial cell chemokine expression, thereby inducing airway inflammation. We investigated the role of the mitogen-activated protein kinase kinase kinase 14/NF-kappaB-inducing kinase (NIK) in the activation of NF-kappaB-dependent genes in alveolus-like A549 cells. RSV infection induces a time dependent increase of NIK mRNA and protein expression that peaks 12 to 24 h after viral exposure. Immunoprecipitation kinase assays indicate that NIK kinase activity is activated even more rapidly (within 6 h of RSV adsorption) associated with an endogenous approximately 50-kDa NF-kappaB2 substrate. Because NIK associates with IKKalpha to mediate processing of the 100-kDa NF-kappaB2 precursor into its 52-kDa DNA binding isoform ("p52"), the effects of RSV on NIK complex formation with IKKalpha and NF-kappaB2 were determined by coimmunoprecipitation assay. We find that NIK, IKKalpha, and both 100 kDa- and 52-kDa NF-kappaB2 isoforms strongly complex 15 h after exposure to RSV at times subsequent to NIK kinase activation. Western immunoblot and microaffinity DNA pull-down assays showed a parallel increase in nuclear translocation and DNA binding of the NF-kappaB2-Rel B complex. Interestingly, we make the novel observations that NIK also transiently translocates into the nucleus complexed with 52-kDa NF-kappaB2. Small interfering RNA-mediated NIK "knock-down" blocked RSV-inducible 52-kDa NF-kappaB2 processing and interfered with the early activation of a subset of NF-kappaB-dependent genes, indicating the importance of this activation pathway in the genomic NF-kappaB response to RSV. Together, these data indicate that RSV infection rapidly activates the noncanonical NF-kappaB activation pathway prior to the more potent canonical pathway activation. This appears to be through a novel mechanism involving induction of NIK kinase activity, expression, and nuclear translocation of a ternary complex with IKKalpha and processed NF-kappaB2.
Department of Medicine, The University of Texas Medical Branch, Galveston, TX 77555-1060, USA.
Sanjeev
S
Choudhary
Steve
S
Boldogh
Roberto
R
Garofalo
Mohammad
M
Jamaluddin
Allan R
AR
Brasier
D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism* / Q000502:physiology*; D052003:NF-kappa B p52 Subunit; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D012136:Respiratory Syncytial Viruses / Q000502:physiology*; D051841:Transcription Factor RelB; D014157:Transcription Factors / Q000378:metabolism; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000502:physiology; D000097782:NF-kappaB-Inducing Kinase
D004247:DNA; D016328:NF-kappa B; D052003:NF-kappa B p52 Subunit; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D051841:Transcription Factor RelB; D014157:Transcription Factors; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D000097782:NF-kappaB-Inducing Kinase; C497593:RELB protein, human