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Cell-specific regulation of nucleic acid sensor cascades: a controlling interest in the antiviral response.

TitleCell-specific regulation of nucleic acid sensor cascades: a controlling interest in the antiviral response.
Publication TypeJournal Article
Year of Publication2012
AuthorsStein SC, Lam E, Falck-Pedersen E
JournalJ Virol
Volume86
Issue24
Pagination13303-12
Date Published2012 Dec
ISSN1098-5514
KeywordsAdenoviridae Infections, Animals, Cell Line, Interferon Regulatory Factor-3, Mice, Nucleic Acids, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction
Abstract

In this study, we examined the capacities of non-antigen-presenting cell types to propagate antiviral signals following infection with recombinant adenovirus or by direct nucleic acid transfection. Three murine cell lines (RAW264.7 macrophages as a positive control, FL83B hepatocytes, and MS1 endothelial cells) were assessed following exposure to adenovirus, DNA, or RNA ligands. Based on primary (interferon response factor 3 [IRF3] phosphorylation) and secondary (STAT1/2 phosphorylation) response markers, we found each cell line presented a unique response profile: RAW cells were highly responsive, MS1 cells were modified in their response, and FL83B cells were essentially nonresponsive. Comparative reverse transcription-quantitative PCR (RT-qPCR) of nucleic acid sensing components revealed major differences between the three cell types. A prominent difference was at the level of adaptor molecules; TRIF, MyD88, MAVS, and STING. TRIF was absent in MS1 and FL83B cells, whereas MyD88 levels were diminished in FL83B hepatocytes. These differences resulted in compromised TLR-mediated activation. While the cytosolic adaptor MAVS was well represented in all cell lines, the DNA adaptor STING was deficient in FL83B hepatocytes (down by nearly 3 log units). The absence of STING provides an explanation for the lack of DNA responsiveness in these cells. This hypothesis was confirmed by acquisition of IRF3 activation in Flag-STING FL83B cells following DNA transfection. To consolidate the central role of adaptors in MS1 endothelial cells, short hairpin RNA (shRNA) knockdown of STING and MAVS resulted in a ligand-specific loss of IRF3 responsiveness. In contrast to the requirement for specific adaptor proteins, a requirement for a specific DNA sensor (AIM2, DDx41, or p204) in the IRF3 activation response was not detected by shRNA knockdown in MS1 cells. The data reveal that cell-specific regulation of nucleic acid sensing cascade components influences antiviral recognition responses, that controlling levels of adaptor molecules is a recurring strategy in regulating antiviral recognition response functions, and that comparative RT-qPCR has predictive value for antiviral/innate response functions in these cells.

DOI10.1128/JVI.02296-12
Alternate JournalJ Virol
PubMed ID23015711
PubMed Central IDPMC3503059
Grant ListR01 AI094050 / AI / NIAID NIH HHS / United States

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