FULL MANUSCRIPT Rapid Uptake and Inhibition of Viral Propagation by Extracellular OAS1 Karthiga Thavachelvam, 1 Hans Henrik Gad, 1 Mikkel Søes Ibsen, 1 Philippe Despre `s, 2 Marianne Hokland, 3 Rune Hartmann, 1 and Helle Kristiansen 4 The oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins that mediate antiviral activity through the synthesis of 2¢-5¢-linked oligoadenylates and subsequent activation of the endoribonuclease RNase L. However, we have recently demonstrated that exogenous recombinant OAS1 is taken up by cells and reduces viral replication both in cell culture and in vivo, independent of RNase L. These results demonstrate a novel paracrine antiviral activity of OAS working in parallel with the classical RNase L pathway. In this study, we investigate the uptake kinetics of recombinant porcine OAS1 and show that it is rapidly and efficiently internalized in a manner that can be blocked by heparin. Heparin, furthermore, abolishes the antiviral activity of OAS1, demonstrating the requirement of the intracellular localization of OAS1 to inhibit the virus. In addition, we demonstrate that exogenous OAS1 affects an early step of the viral replication cycle. Introduction I nterferon (IFN) is the collective name of a group of cytokines that can orchestrate an efficient antiviral de- fense. Three types of IFN exist: type I IFN (IFN-a/b), type II IFN (IFN-g), and type III IFN (IFN-l). Type II IFN is so named for historical reasons and although it does harbor an antiviral activity, it also has a pleiotropic effect on different immune cells. Type I and type III IFNs are what we consider classical IFNs, and though utilizing different receptor com- plexes, they signal through the same transcription factor, known as IFN-stimulated gene factor 3, comprising STAT1, STAT2, and IRF9 (ISGF3) and induce a similar set of genes known as IFN-stimulated genes (ISGs) (Zhou and others 2007; Kotenko 2011). One of these ISGs is oligoadenylate synthetase 1 (OAS1), which together with OAS2, OAS3, and OASL belongs to the OAS family of proteins. OAS1 to 3 are all active synthetases that synthesize 2¢-5¢-linked oligoadenylates (2-5As) in response to viral infections (Kristiansen and others 2011). The 2-5As then activate a latent endoribonuclease, RNase L, which cleaves both cellular and viral RNA present inside the cell. Thus, the classical OAS-RNase L pathway constitutes a regulated RNA decay pathway, which is activated in response to viral infections (Silverman 2007). However, we recently described a novel role for OAS1 in innate immunity. We demonstrated that recombinant OAS1 was taken up by cells and reduced viral replication in both cell cultures and mice treated with the OAS1 protein (Kristiansen and others 2010). This antiviral activity was independent of both the catalytic activity of OAS1 and of RNase L, but seems to act synergistically with the classical OAS-RNase L pathway (Kristiansen and others 2010). However, this work left several open questions regarding the kinetics of OAS1 uptake and antiviral activity, as well as which part of the viral replication cycle was affected. OAS activity is observed in the serum of patients chronically infected with hepatitis C virus (Shindo and others 2008; Mihm and others 2009), and the demonstration that OAS1 could act in a paracrine way opened a new way of looking at the antiviral activity of ISGs. Recently, it was shown that the cyclic dinucleotide cGAMP, which is a potent inducer of IFN through activation of stimulator of IFN genes (STING), could traverse the cell membrane through gap junctions and thereby initiate an IFN response in uninfected cells (Ablasser and others 2013). Thus, paracrine actions of ISGs or secondary messengers produced in response to viral infections constitute an emerging theme. In this study, we in- vestigate the uptake kinetics of recombinant porcine OAS1 (pOAS1), the potential role of heparin in pOAS1 internaliza- tion, and antiviral activity, as well as the specific step of the viral replication cycle, which is inhibited. Materials and Methods Cells and viruses Cell lines used in this study were HeLa (human cervical cancer cells) and Vero cells (African green monkey kidney 1 Department of Molecular Biology and Genetics, Centre for Structural Biology, Aarhus University, Aarhus, Denmark. 2 Unite ´ Interactions Mole ´culaires Flavivirus-Ho ˆtes, Institut Pasteur, Paris, France. Departments of 3 Biomedicine and 4 Animal Sciences, Aarhus University, Aarhus, Denmark. JOURNAL OF INTERFERON & CYTOKINE RESEARCH Volume 00, Number 00, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/jir.2014.0140 1