Little is known about the mechanism by which IFNs inhibit human cytomegalovirus (HCMV) replication. Indeed, infection of fibroblasts with HCMV initiates the expression of a subset of type I IFN-inducible genes whose role in the infectious process is unclear. We describe here the identification of a cytoplasmic antiviral protein that is induced by IFNs, by HCMV infection, and by the HCMV envelope protein, glycoprotein B (gB). Stable expression of the protein in fibroblasts inhibits productive HCMV infection, down-regulating several HCMV structural proteins (gB, pp28, and pp65) known to be indispensable for viral assembly and maturation. We have named the protein viperin (for v irus i nhibitory p rotein, e ndoplasmic r eticulum-associated, in terferon-inducible). HCMV infection causes the redistribution of the induced viperin from its normal endoplasmic reticulum association, first to the Golgi apparatus and then to cytoplasmic vacuoles containing gB and pp28. Expression before HCMV infection reduces viperin redistribution from the endoplasmic reticulum to the Golgi apparatus and prevents vacuolar localization, perhaps reflecting the mechanism used by HCMV to evade the antiviral function.

Type I IFNs induce the expression of IFN-stimulated gene 15 (ISG15)and its conjugation to cellular targets. ISGylation is a multistep process involving IFN-inducible Ube1L, UbcH8, and a yet-to-be identified E3 ligase. Here we report the identification of an IFNinduced HECT-type E3 protein ligase, HERC5͞Ceb1, which mediates ISGylation. We also defined a number of proteins modified by ISG15 after IFN triggering or HERC5 overexpression. A reduction in endogenous HERC5 by small interfering RNA inhibition blocks the IFN-induced ISG15 conjugation. Conversely, HERC5 coexpression with Ube1L and UbcH8 induces the ISG15 conjugation in vivo independent of IFN stimulation. A targeted substitution of Cys-994 to Ala in the HECT domain of HERC5 completely abrogates its E3 protein ligase activity. Therefore, this study demonstrates that HERC5͞Ceb1 is involved in the conjugation of ISG15 to cellular proteins.ISG15 ͉ Ceb1 ͉ innate immunity ͉ antiviral proteins T ype I IFNs (IFN-␣͞␤) play an essential role in both innate antiviral and adaptive immune responses and are rapidly produced in response to microbial infection (1-3). They exert signals through the activation of the Janus kinase-signal transducer and activator of transcription pathway that mediates rapid induction of IFN-stimulated genes (ISGs) (4, 5). ISG15 is one of the most strongly induced genes after IFN treatment (6, 7) and is also significantly induced by viral infection (8, 9) and LPS treatment (10, 11). The ISG15 protein starts with two ubiquitin-related domains that have Ϸ27% sequence identity to ubiquitin and terminate in a conserved 152 LRLRGG 157 ubiquitin C-terminal motif. This study suggests that ISG15 could act in a similar way to ubiquitin and other ubiquitin-like proteins such as SUMO by forming an isopeptide bond with cellular proteins (6, 12, 13). The crystal structure of ISG15 revealed that ISG15 consists of two domains with ubiquitinlike folds joined by a linker sequence (14).Conjugation of ISG15 to cellular proteins occurs in a parallel but distinct mechanism to that of ubiquitin (15-17). The E1 enzyme for ISG15, Ube1L, is a single-subunit enzyme and is identified in vitro by its ability to catalyze the formation of a thioester bond between ISG15 and Ube1L (17,18). The Ube1L protein is highly similar to the E1 enzyme for ubiquitin at the protein level. However, this protein does not form a conjugate with ubiquitin, indicating that Ube1L is an E1 enzyme for the ISG15 conjugation system (ISGylation). Influenza B virus blocks protein ISGylation by inhibiting the activation step through the interaction of the NS1B viral protein with ISG15 (18). This finding was the first suggestion that ISGylation might be important for protecting cells from viral infection.Two groups recently found that a member of the ubiquitin E2-conjugating enzyme family, UbcH8, is involved in the ISGylation (19,20). Like ISG15 and Ube1L, the expression of UbcH8 is also induced by IFN (21). The suppression of UbcH8 protein expression by RNA interference is shown to dramatically inhib...

Chikungunya virus (CHIKV) is a mosquito-borne arthralgia arbovirus that is reemergent in sub-Saharan Africa and Southeast Asia. CHIKV infection has been shown to be self-limiting, but the molecular mechanisms of the innate immune response that control CHIKV replication remain undefined. Here, longitudinal transcriptional analyses of PBMCs from a cohort of CHIKV-infected patients revealed that type I IFNs controlled CHIKV infection via RSAD2 (which encodes viperin), an enigmatic multifunctional IFN-stimulated gene (ISG). Viperin was highly induced in monocytes, the major target cell of CHIKV in blood. Anti-CHIKV functions of viperin were dependent on its localization in the ER, and the N-terminal amphipathic α-helical domain was crucial for its antiviral activity in controlling CHIKV replication. Furthermore, mice lacking Rsad2 had higher viremia and severe joint inflammation compared with wild-type mice. Our data demonstrate that viperin is a critical antiviral host protein that controls CHIKV infection and provide a preclinical basis for the design of effective control strategies against CHIKV and other reemerging arthrogenic alphaviruses.

Background: BRD4 interacts with P-TEFb, which regulates Pol II elongation. Results: Disruption of BRD4 binding by JQ1 resulted in reduced Pol II Ser-2 in CD4ϩ T cells. Conclusion: BRD4 affects Pol II Ser-2 phosphorylation at a subset of lineage-specific active genes in primary human CD4ϩ T cells. Significance: BRD4 binding may represent a means of identifying active promoters and lineage-specific enhancer elements.