232: Inhibition of IFN-mediated STAT1 activation in intestinal epithelial cells by NSP1 – The rotavirus-encoded inhibitor of IFN induction

Abstract

Rotavirus (RV) replication in intestinal epithelial cells (IECs) in vivo is robust despite the vigorous activation of local early host interferon (IFN) responses. Using single cell microfluidics qRT-PCR coupled with flow sorting, we recently demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo and, using genetic approaches, associated this function with the RV NSP1 protein. Paradoxically, RV also induces robust type I IFNs in the intestinal hemaotopoietic compartment in a relatively replication-independent manner raising the possibility that RV replication and spread in IECs occurs despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. However, viral factor(s) responsible for the possible inhibition of STAT1 function are unknown. Here we report the ability of RV to mediate direct and remote inhibition of IFN-mediated STAT1 activation in IECs and identify the RV NSP1 protein as a direct inhibitor of the pathway. Infection of human IECs with RV strains that inhibit IFN induction by two distinct strategies targeting either NF-kB or IRF3 both resulted in effective regulation of IFN secretion. At early times during infection, neither type of RV strain could effectively inhibit the activation of STAT1 in response to exogenously added IFN. However, at later times in infection, both types of RV could inhibit IFN-mediated STAT1 activation, and by single cell analysis this blockage occurred in both infected and bystander cell populations. We found that the rotavirus NSP1 protein, when expressed by itself, is an efficient inhibitor of STAT1 activation. Analysis of NSP1 determinants in the inhibition of IFN induction and signaling pathways revealed that these properties are encoded on discrete domains. These findings explain previously observed remote regulation of the host IFN response in the intestine and uncover a novel function of NSP1 as a STAT1 antagonist.