Interferon epsilon promotes HIV restriction at multiple steps of viral replication Albert Garcia-Minambres1,2, Sahar G Eid1,2, Niamh E Mangan3,4, Corinna Pade1,2, San S Lim3,4, Antony Y Matthews3,4, Nicole A de Weerd3,4, Paul J Hertzog3,4, and Johnson Mak1,2 1School of Medicine, Deakin University, Geelong, Victoria, Australia; 2CSIRO Australian Animal Health Laboratory, Geelong, Victoria, Australia; 3Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia; 4Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia. Lorne Infectious and Immunity conference 2017 Contact: Albert Garcia agarciam@deakin.edu.au INTRODUCTION Interferon epsilon (IFNε) is a new gene in the type I IFN locus and it is expressed constitutively and most abundantly in the epithelial cells of the female reproductive tract. IFNε has previously shown protection of sexually transmitted infections (herpes simplex virus 2 and Chlamydia muridarum) in the mouse model by regulating mucosal immune responses. Conventional type I IFNs inhibit HIV infection by inducing the expression of many HIV restriction factors inhibiting the virus at various stages of its life cycle: (i) Non-human TRIM5a (ii) SAMHD1 (iii) MX2 (iv) HERC5 (v) Tetherin (BST2 gene) (vi) APOBEC family proteins and (vii) IFITM family proteins.   RESULTS IFNε inhibits HIV infection of epithelial cells, T-cells and primary activated PBLs cells Treatment of cells with 25 or 250 IU/ml of IFNε resulted in a significant dose-dependent inhibition of HIV infection in TZM-bl cells and Sup-T1 cells. HIV Infection of activated PBLs was significantly inhibited by IFNε at 25, 50, 100 or 200 IU/ml. These results confirm that IFNε is a potent inhibitor of HIV infection at a stage between virus entry and viral gene expression. IFNε inhibits HIV infection of T-cells post viral entry Sup-T1 cells infected with VSV-G pseudotyped EGFP reporter HIV showed a dose-dependent inhibition of infection by both type I IFNs. IFNε does not inhibit HIV release but suppresses infectivity of newly released progeny viruses No changes in the level of viral capsid proteins p24 were observed in the media of IFNε treated and untreated HIV producing cells (293T, HeLa, and activated PBLs). IFNε did not affect virion release when using a Vpu negative HIV which avoids neutralization by BST2 activity. These data imply that the level of BST2 expression induced by IFNε was not involved in, or sufficient to block HIV particle release.   RESULTS HIV particles produced from different cells (293T cells, HeLa and activated PBLs) treated with IFNε showed a 2 to 3-fold reduction in viral infectivity during infection of TZM-bl reporter cells. RT-qPCR results of virion producing 293T cells showed that both APOBEC3G and IFITM3 were upregulated by a factor of 5 to >20-fold by IFNε, consistent with their potential contributions to the suppressive activities against newly produced progeny HIV particles.   CONCLUSIONS IFNε has strong antiviral activities toward HIV at different steps of HIV replication cycle likely via the induction of HIV restriction factors expression Strong correlation of IFNε anti-HIV activity of epithelial cells, T-cells and PBL cells and gene expression upregulation of HIV restriction factor MX2 Upregulation of HIV restriction factors BST2 and HERC5 did not lead to a reduction of viral assembly and release IFITM3 gene expression upregulation of >20-fold could be the main contributor to the suppression of the HIV infectivity of the progeny viral particles produced in presence of IFNε RESULTS IFNε activates expression of HIV host-cell restriction factors IFNε induced the expression of HIV restriction factors TRIM5a, MX2, HERC5, BST2, IFITM3 and APOBEC3G in activated peripheral blood lymphocytes cells (PBLs) and Sup-T1. Based on induction of different classes of HIV restriction factors, IFNε has the potential to inhibit HIV infection at different stages of the viral life cycle.  Uncoating and nuclear import of the viral cDNA IMPORTANCE OF FINDINGS These results show the potential of IFNε as a new therapeutic that could be used to enhance female reproductive health to block a range of sexually transmitted infection. Viral assembly and release ACKNOWLEDGEMENTS This work was supported by: an Australian National Health and Medical Research Council (NHMRC) Senior Principal Research Fellowship (APP1027020) and Project Grant (APP1049686) awarded to PJH; an Australian Research Council Fellowship awarded to NEM (DP110103616); an Australian NHMRC Project Grant awarded to NAdW (APP1070782); grants awarded to PJH, NEM and JM by the Australian Centre for HIV and Hepatitis Virology Research (2013-34; 2015-12); a Monash University Faculty of Medicine, Nursing and Health Sciences Strategic Platform Grant (ECP003) awarded to NEM; and Deakin University scholarships to AGM and CP. Entry and reverse transcription of new progeny viruses PUBLICATION Albert Garcia-Minambres, Sahar G Eid, Niamh E Mangan, Corinna Pade, San S Lim, Antony Y Matthews, Nicole A de Weerd, Paul J Hertzog and Johnson Mak. Interferon epsilon promotes HIV restriction at multiple steps of viral replication. Immunology and Cell Biology. DOI: 10.1038/icb.2016.123