Members of the family Flaviviridae are important human pathogens and fall within the NIAID class A, B and C priorities, depending on the virus species. Like many other viruses, flaviviruses have evolved complex mechanisms to interact with the host immune response. The earliest immune responses to infection involve components of the innate immune system, starting with the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), and intracellular RNA helicases, such as RIG-I and Mda-5. PAMP recognition results in activation of specific signal transduction pathways that result in production of proinflammatory cytokines, such as type I interferon (IFN), TNFa and others. Type I interferon is one of the most important mediators of the early immune response. Mice deficient in IFN signaling are more susceptible to flaviviruses and pretreatment of mice with type I IFN can prevent flavivirus disease. One of the major pathways of IFN synthesis in viral infection is the stimulation of plasmacytoid dendritic cells (pDCs) via TLRs 7, 8 or 9. While several studies have indicated that flaviviruses are able to stimulate pDCs to produce IFNa, the mechanisms of this stimulation are not understood. Our preliminary data indicates that mosquito-derived flaviviruses are unable to stimulate TLR7 while viruses grown in mammalian cells strongly stimulate TLR7. We hypothesize that differential glycosylation of viruses grown in either mammalian or insect cells accounts for theses differences. In addition, our previous work has shown that WNV, and more specifically its NS1 protein, is able to interfere with TLRS and TLR7 signal transduction which could prevent further activation of these pathways after initial nfection has been established. TLR7 is a PRR that recognizes ssRNA and activates the transcription factors NFkB and interferon-regulatory factor 7 (IRF7), resulting in transcription of IFNa and other cytokines. NS1 is glycoprotein secreted to high levels during flavivirus infection that can be endocytosed by several cell types. We hypothesize that secreted NS1 is able to inhibit TLR7 function and thus contributes to viral pathogenesis. We have developed three specific Aims to address our hypotheses.
In Aim 1 we will identify mechanisms of flavivirus recognition by TLR7 with a specific focus on differential glycosylation of viruses grown in either mammalian or insect cells.
In Aim 2 we will determine mechanisms of TLR7 inhibition by the lavivirus NS1 proteins, focussing on a potential role for secreted NS in this inhibition.
In Aim 3 we will nvestigate the contribution of TLR7 signaling to WNV pathogenesis.

Public Health Relevance

IMPACT: We have identified a novel role for a flavivirus nonstructural protein, the inhibition of TLR signaling. This proposal aims to delineate the mechanism(s) of this interference and the roles that TLRs play in the response to flavivirus infection. We expect to gain important information about viral immune evasion mechanisms and a novel role for a replication-associated protein in this process.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Specialized Center--Cooperative Agreements (U54)
Project #
Application #
Study Section
Special Emphasis Panel (ZAI1-DDS-M)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of North Carolina Chapel Hill
Chapel Hill
United States
Zip Code
Guo, Haitao; Gao, Jianmei; Taxman, Debra J et al. (2014) HIV-1 infection induces interleukin-1? production via TLR8 protein-dependent and NLRP3 inflammasome mechanisms in human monocytes. J Biol Chem 289:21716-26
Emery, Felicia D; Stabenow, Jennifer M; Miller, Mark A (2014) Efficient inactivation of Burkholderia pseudomallei or Francisella tularensis in infected cells for safe removal from biosafety level 3 containment laboratories. Pathog Dis 71:276-81
Pop, Laurentiu M; Barman, Stephen; Shao, Chunli et al. (2014) A reevaluation of CD22 expression in human lung cancer. Cancer Res 74:263-71
Rice, Amanda D; Adams, Mathew M; Lindsey, Scott F et al. (2014) Protective properties of vaccinia virus-based vaccines: skin scarification promotes a nonspecific immune response that protects against orthopoxvirus disease. J Virol 88:7753-63
Zellweger, Raphaƫl M; Eddy, William E; Tang, William W et al. (2014) CD8+ T cells prevent antigen-induced antibody-dependent enhancement of dengue disease in mice. J Immunol 193:4117-24
Agnihothram, Sudhakar; Yount Jr, Boyd L; Donaldson, Eric F et al. (2014) A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant. MBio 5:e00047-14
Krumm, Stefanie A; Yan, Dan; Hovingh, Elise S et al. (2014) An orally available, small-molecule polymerase inhibitor shows efficacy against a lethal morbillivirus infection in a large animal model. Sci Transl Med 6:232ra52
Zhao, Jincun; Li, Kun; Wohlford-Lenane, Christine et al. (2014) Rapid generation of a mouse model for Middle East respiratory syndrome. Proc Natl Acad Sci U S A 111:4970-5
de Alwis, Ruklanthi; de Silva, Aravinda M (2014) Measuring antibody neutralization of dengue virus (DENV) using a flow cytometry-based technique. Methods Mol Biol 1138:27-39
Blake, Lauren E; Garcia-Blanco, Mariano A (2014) Human genetic variation and yellow fever mortality during 19th century U.S. epidemics. MBio 5:e01253-14

Showing the most recent 10 out of 288 publications