Xu Y, Zhong J. studies exhibited that NEURL3 inhibited HCV assembly by directly binding HCV envelope glycoprotein E1 to interfere with the E1/E2 heterodimerization, an important prerequisite for virion morphogenesis. Finally, we showed that knockout of NEURL3 significantly enhanced HCV contamination. In summary, we identified NEURL3 as a novel inducible antiviral host factor that suppresses HCV assembly. Our results not only shed new insight into how host innate immunity acts against HCV but also revealed a new important biological function for NEURL3. IMPORTANCE The exact biological function of NEURL3, a putative E3 ligase, remains largely unknown. In this study, we found that NEURL3 could be upregulated upon HCV contamination in a manner dependent on pattern recognition receptor-mediated innate immune response. NEURL3 inhibits HCV assembly by directly binding viral E1 envelope VGX-1027 glycoprotein to disrupt its interaction with E2, an action that requires its Neuralized homology repeat (NHR) domain but not the RING domain. Furthermore, we found that NEURL3 has a pangenotypic anti-HCV activity and interacts with E1 of genotypes 2a, 1b, 3a, and 6a but does not inhibit other closely related RNA viruses, such as ZIKV, DENV, and VSV. To our knowledge, our study is the first report to demonstrate that NEURL3 functions as an antiviral host factor. Our results not only shed new insight into how host innate immunity acts against HCV, but also revealed a new important biological function for NEURL3. family. Its 9.6-kb single-stranded RNA genome consists of a single open reading frame (ORF) flanked by highly conserved 5- and 3-untranslated regions (UTRs). Translation of the ORF produces a 3,000-amino-acid polyprotein, which is processed by cellular and viral proteases into the structural proteins VGX-1027 (core, E1, and E2) and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The nonstructural proteins, including NS3, NS4A, NS4B, NS5A, and NS5B, comprise the membrane-bound replication complex that contributes to HCV replication. Two envelope glycoproteins, E1 and E2, form a heterodimer that can be incorporated into the virion envelope when nascent HCV capsids bud on the endoplasmic reticulum membrane. Numerous studies have demonstrated that the E1/E2 heterodimerization ensures the functional conformation of both of the viral envelope proteins and plays crucial roles in HCV entry and morphogenesis (1). The innate immune response functions as the first line of host defense against viral infection. Virus can Rabbit Polyclonal to 53BP1 (phospho-Ser25) be sensed as pathogen-associated molecular patterns (PAMPS) VGX-1027 by pattern recognition receptors (PRRs) in the host cell, leading to activation of the innate immune response. There are several types of PRRs, including membrane-associated Toll-like receptors (TLRs), C-type lectin receptors (CLRs), cytosolic NOD-like receptors (NLRs), AIM2-like receptors (ALRs), and RIG-I-like receptors (RLRs) (2). TLRs and RLRs have been reported to recognize HCV (3, 4). We recently found that HCV VGX-1027 PAMP can be detected by MDA5 with the help of LGP2 in the context of HCV infection (5, 6). Upon activation, the N-terminal caspase recruitment domain (CARD) of MDA5 interacts with MAVS, which subsequently activates the transcription factors NF-B and IRF3. The activated transcription factors translocate into the nucleus to induce the transcription of type I and III interferons (IFNs), which subsequently activate the expression of IFN-stimulated genes (ISGs). Dozens of ISGs have been reported to restrict HCV infection (4, 7,C9). IFITM1 has been demonstrated to inhibit HCV entry and replication (10, 11); 2,5-oligoadenylate synthetases (OASs), ADAR1, MxA, viperin, TRIM22, and IFIT1 have been reported to suppress HCV replication (12,C17); tetherin/BST-2 has been reported to restrict HCV production and release (18,C20). Some ISGs, such as DExD/H box RNA helicase family (RIG-I, MDA5, LGP2, and DDX60) and IRF families (IRF1 and IRF7), VGX-1027 can serve as PRRs or their signaling partners to reinforce the innate immune response by further inducing IFN or ISGs (7). In addition.