The type I interferon (IFN-1/2) system, if activated in the absence of virus antagonism, is very effective at blocking the replication of multiple viruses, either preventing or substantially reducing disease in animal models. Furthermore, some components of this system have evolved such that they can effectively distinguish between host and viral structural and metabolic processes, suppressing the viral processes with minimal non- target effects upon the host. These attributes provide an excellent conceptual model for development of new antiviral drugs: a virus suppression system that distinguishes "self" (host) from "nonself" (virus) and effectively suppresses nonself, in some cases with broad spectrum activity versus multiple viruses. While this system is generally effective, we have found that the human-virulent alphavirus, Venezuelan equine encephalitis virus (VEEV), is much more resistant to the activity of the antiviral state than the human-avirulent Sindbis virus (SINV) and that the virulence of the viruses in animal models is largely reflective of these differences. Using global transcription profiling of dendritic cells, a cell-type highly relevant to alphavirus dissemination, amplification and disease in vivo, we have identified two IFN-1/2-inducible proteins with potent antiviral activity versus SINV, the ISG20 exoribonuclease and the Mx2 GTPase. To discover the mechanisms of antiviral activity employed by these proteins and the means by which VEEV resists these effectors of the antiviral state, we propose to characterize the relative efficacy of inhibition and mechanisms of antiviral activity exerted by each protein versus SINV and VEEV. To achieve this, we will use over-expression and interfering-RNA knockdown techniques to identify the point in the virus replication cycle at which each effector acts followed by mutagenesis to identify the responsible functional domains in the individual effectors and subcellular localization and protein-protein interaction studies to identify the viral structures and/or processes targeted for inhibition. The results of these studies will: i) determine the mechanism(s) of action versus alphaviruses of Mx2 and ISG20, ii) identify points of vulnerability of each virus as a first step in design of antiviral therapeutics, and iii) provide insights into molecular mechanisms underlying the resistance of VEEV to the activities of the antiviral state that be utilized in design of vaccines.

Public Health Relevance

The results of these studies will identify the mechanisms of action of two highly active IFN-induced antiviral effector proteins that are involved in interferon-mediated suppression of alphavirus replication and the relative sensitivity of benign and highly virulent viruses to the effectors. This information can be used in the design of antiviral drugs that artificially mimic the activity or artificially stimulate the induction of these effectors. Furthermore, these studies will provide information regarding the contribution of resistance to particular effectors to the virulence of alphaviruses in mice and humans that can be used in design of alphavirus vaccines.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI083383-04
Application #
8520159
Study Section
Special Emphasis Panel (ZRG1-IDM-Q (02))
Program Officer
Repik, Patricia M
Project Start
2010-09-01
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
4
Fiscal Year
2013
Total Cost
$317,218
Indirect Cost
$107,833
Name
University of Pittsburgh
Department
Genetics
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Bayer, Avraham; Delorme-Axford, Elizabeth; Sleigher, Christie et al. (2015) Human trophoblasts confer resistance to viruses implicated in perinatal infection. Am J Obstet Gynecol 212:71.e1-8
Sun, Chengqun; Gardner, Christina L; Watson, Alan M et al. (2014) Stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease. J Virol 88:2035-46
Hyde, Jennifer L; Gardner, Christina L; Kimura, Taishi et al. (2014) A viral RNA structural element alters host recognition of nonself RNA. Science 343:783-7
Trobaugh, Derek W; Gardner, Christina L; Sun, Chengqun et al. (2014) RNA viruses can hijack vertebrate microRNAs to suppress innate immunity. Nature 506:245-8
Gardner, Christina L; Hritz, Jozef; Sun, Chengqun et al. (2014) Deliberate attenuation of chikungunya virus by adaptation to heparan sulfate-dependent infectivity: a model for rational arboviral vaccine design. PLoS Negl Trop Dis 8:e2719
Gardner, Christina L; Choi-Nurvitadhi, Jo; Sun, Chengqun et al. (2013) Natural variation in the heparan sulfate binding domain of the eastern equine encephalitis virus E2 glycoprotein alters interactions with cell surfaces and virulence in mice. J Virol 87:8582-90
Gardner, Christina L; Burke, Crystal W; Higgs, Stephen T et al. (2012) Interferon-alpha/beta deficiency greatly exacerbates arthritogenic disease in mice infected with wild-type chikungunya virus but not with the cell culture-adapted live-attenuated 181/25 vaccine candidate. Virology 425:103-12