Systematic identification of type I and type II interferon-induced antiviral factors Su-Yang Liua, David Jesse Sanchezb, Roghiyh Aliyaria, Sun Luc, and Genhong Chenga,1 aDepartment of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095; bDepartment of Pharmaceutical Sciences, Western University of Health Sciences, Pomona, CA 91766; and cGuangzhou FulenGen Co. Ltd., Guangzhou City 510663, China Edited by Owen N. Witte, University of California, Los Angeles, CA, and approved January 24, 2012 (received for review September 21, 2011) Type I and type II interferons (IFNs) are cytokines that establish the family, which includes rabies virus. It infects most cell types and cellular antiviral state through the induction of IFN-stimulated replicates lytically in the cytoplasm. MHV-68 is a member of the gammaherpes virus family that includes Kaposi sarcoma-associ- genes (ISGs). We sought to understand the basis of the antiviral – activity induced by type I and II IFNs in relation to the functions of ated herpes virus and Epstein Barr virus. They can infect partic- their ISGs. Based on gene expression studies, we systematically ular cell types and are thought to predominantly establish latency fi in B cells (5). These viruses are models for virology and several identi ed antiviral ISGs by performing blinded, functional screens human-related diseases, yet a systematic identification of ISGs on 288 type I and type II ISGs. We assessed and validated the that inhibit these viruses has not been done. antiviral activity of these ISGs against an RNA virus, vesicular In this study, we sought to compare IFNα and IFNγ induction stomatitis virus (VSV), and a DNA virus, murine gammaherpes virus of ISGs under an equivalent signaling input and systematically (MHV-68). Overall, we identified 34 ISGs that elicited an antiviral screened their effects on VSV and MHV-68 growth. We iden- effect on the replication of either one or both viruses. Fourteen tified and validated known and unique ISGs that have broad and ISGs have uncharacterized antiviral functions. We further de- specific antiviral activity against these viruses. fined ISGs that affect critical life-cycle processes in expression of VSV protein and MHV-68 immediate-early genes. Two previously Results undescribed antiviral ISGs, TAP1 and BMP2, were further validated. Both type I and type II IFNs broadly inhibit many types of viruses fi fi and elicit antiviral responses in various cell types. We hypothesized TAP1-de cient broblasts were more susceptible to VSV infection α γ but less so to MHV-68 infection. On the other hand, exogenous that IFN and IFN can activate distinct and common ISGs that IMMUNOLOGY govern their antiviral effects. Because IFNα and IFNγ are struc- BMP2 inhibits MHV-68 lytic growth but did not affect VSV growth. turally distinct cytokines that activate different receptors, we These results delineate common and distinct sets of type I and type normalized IFNα and IFNγ concentrations based on phosphory- II IFN-induced genes as well as identify unique ISGs that have either lation of the shared and obligatory signaling factor STAT1 (6). broad or specific antiviral effects on these viruses. Titration of both IFNs showed IFNγ phosphorylates STAT1 more than IFNα at equivalent units; for example, 1 U/mL of IFNγ interferon stimulated genes | antiviral effectors | phosphorylates STAT1 to an equivalent level as 62 U/mL of IFNα murine gammaherpes virus 68 (Fig. S1 A and B). Based on this equivalent biological input, pri- mary bone marrow-derived macrophages (BMMs) were stimu- α γ he immune system inhibits viral growth through expression of lated with IFN or IFN for 2.5 h in biological triplicates and processed for gene expression profiling by microarray (Fig. 1). a diverse set of antiviral genes. Interferons (IFNs) are potent T Overall, IFNα and IFNγ regulated a common set of genes that had activators of these antiviral factors. Type I IFNs, which include a significant correlation coefficient of 0.59. There were more ISGs IFNα,-β, and -ω, are rapidly activated during viral infection and α γ “ ” α fi induced by IFN than IFN over untreated controls. Genes that considered antiviral partly because IFN receptor-de cient were induced by IFNα threefold over IFNγ were classified as mice are highly susceptible to viral infections (1). Type II IFN, “ α fi ” γ γ IFN -speci c, and genes that were induced by IFN threefold which is represented only by IFN , also inhibits growth of viral more than IFNα were classified as “IFNγ-specific.” Based on this and other pathogenic infections. Both types of IFN induce ex- categorization, 165 ISGs were IFNα-specific, 17 were IFNγ-spe- pression of IFN-stimulated genes (ISGs) that have a variety of cific, and 203 ISGs were commonly induced by both IFNs (Fig. 1, functions ranging from direct inhibition of viral components to Inset and Table S1). These results suggest that under an equivalent activation of other immune cell types. signaling input, IFNα is a more efficient activator of gene ex- Type I and type II IFNs have distinct physiological roles, but pression than IFNγ. they both can activate the cellular antiviral response. Many cell To further understand the basis of type I and type II antiviral types, particularly macrophages and dendritic cells, secrete type I activity, we next sought to identify IFNα- and IFNγ-induced ISGs IFN through the activation of intracellular and extracellular that inhibit replication of VSV and MHV-68. The ISGs identi- sensors of viral components. Type I IFN acts in paracrine and fied in Fig. 1 and Table S1 were used to generate a list of human autocrine fashion to activate IFN receptors (IFNARs) present on ISGs based on homology for study in HEK293T cells. ISGs in the most cell types. Upon receptor ligation, IFNAR is phosphorylated same family were included, and some genes were omitted be- by the kinases JAK1 and TYK2, which recruit and phosphorylate cause they were not found in the human genome. We obtained STAT1 and STAT2 proteins. STAT1 can homodimerize or het- 288 genes in pCMV-driven expression constructs. First, we per- erodimerize with STAT2 and translocate into the nucleus to ac- tivate specific target promoters (2). Type II IFN is largely secreted by T and natural killer cells and is induced predominantly by IL-12 γ Author contributions: S.-Y.L., D.J.S., and G.C. designed research; S.-Y.L. and R.A. performed and IL-18 (3). IFN receptor (IFNGR) activation leads to phos- research; S.L. contributed new reagents/analytic tools; S.-Y.L. and G.C. analyzed data; and phorylation and homodimerization of STAT1 and target gene S.-Y.L. and D.J.S. wrote the paper. expression (3). The authors declare no conflict of interest. IFN-stimulated genes have diverse effects on different viruses and operate through distinct mechanisms. A comparison of ISGs This article is a PNAS Direct Submission. that inhibit various RNA viruses shows that there are some that Freely available online through the PNAS open access option. broadly inhibit RNA virus growth and others that are specific for Data deposition: The microarray data reported in this paper have been deposited in the Gene particular viruses (4). We sought to identify and compare ISGs Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE35825). that inhibit growth of vesicular stomatitis virus (VSV), a negative- 1To whom correspondence should be addressed. E-mail: [email protected]. strand RNA virus, and murine gammaherpes virus 68 (MHV-68), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a DNA virus. VSV is a neurotropic virus in the Rhabdoviridae 1073/pnas.1114981109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1114981109 PNAS | March 13, 2012 | vol. 109 | no. 11 | 4239–4244 Downloaded by guest on September 29, 2021 GPR146, MITD1, TAP1, RHOH, TRIM62, and FCGR1A (Table S2). Twenty-four candidate genes that demonstrated high antiviral activity were chosen for further verification with biological trip- licates. The amounts of VSV-GFP infection in total, DsRed- − positive (DsRed+), and DsRed-negative (DsRed ) populations were measured for these ISGs and normalized to the control transfected cells of the respective populations (Fig. 2F). The DsRed-positive population should have high expression of the particular ISG that is cotransfected with DsRed, whereas the DsRed-negative population should have very low or no expres- sion of the ISG. Comparison of both populations should indicate whether a particular ISG has an antiviral effect intrinsic to the cell expressing the gene or whether the ISG can confer pro- tection in trans for cells expressing the gene. Genes that had the most pronounced inhibitory effect were IRF1, IFIH1 (MDA5), CH25H, and DDX58 (RIG-I). These genes inhibited VSV-GFP expression in all populations, suggesting that they amplify anti- viral response to confer resistance on cells that do not express or express low levels of the ISG (DsRed-negative population), likely through induction of IFN (8, 9). On the other hand, ISGs such as ISG20, IFITM3, OAS1, MGAT1, GPR146, PARP12, LY6E, APOBEC proteins, TAP1, MX2, CD64, TRIM62, OASL, and others inhibited VSV-GFP expression in DsRed-positive cells but had little effect in DsRed-negative cells. These ISGs likely inhibit viral growth within the cell but not in surrounding cells. To validate this idea, we collected conditioned media Fig. 1. Gene expression profile of BMMs treated for 2.5 h with IFNα and from the supernatants of HEK293T cells transfected with IRF1, IFNγ at 62 U/mL and 1 U/mL, respectively. Axes represent fold change in IFIH1, ISG20, and TBK and transferred them onto freshly response to IFNα or IFNγ over untreated cells.