SUMMARY RIG-I is a cytosolic sensor of viral RNA that plays crucial roles in the induction of type I interferons. The C-terminal domain (CTD) of RIG-I is responsible for the recognition of viral RNA with 5′ triphosphate (5′ ppp). However, the mechanism of viral RNA recognition by RIG-I is still not fully understood. Here we show that RIG-I CTD binds 5′ ppp dsRNA or ssRNA, as well as blunt-ended dsRNA, and exhibits the highest affinity for 5′ ppp dsRNA. Crystal structures of RIG-I CTD bound to 5′ ppp dsRNA with GC- and AU- rich sequences revealed that RIG-I recognizes the termini of the dsRNA and interacts with the 5′ triphosphate through extensive electrostatic interactions. Mutagenesis and RNA binding studies demonstrated that similar binding surfaces are involved in the recognition of different forms of RNA. Mutations of key residues at the RNA binding surface affected RIG-I signaling in cells.

The RIG-I-like receptors (RLRs), RIG-I and MDA5, recognize single-stranded RNA with 5 triphosphates and doublestranded RNA (dsRNA) to initiate innate antiviral immune responses.LGP2, a homolog of RIG-I and MDA5 that lacks signaling capability, regulates the signaling of the RLRs. To establish the structural basis of dsRNA recognition by the RLRs, we have determined the 2.0-Å resolution crystal structure of human LGP2 C-terminal domain bound to an 8-bp dsRNA. Two LGP2 C-terminal domain molecules bind to the termini of dsRNA with minimal contacts between the protein molecules. Gel filtration chromatography and analytical ultracentrifugation demonstrated that LGP2 binds blunt-ended dsRNA of different lengths, forming complexes with 2:1 stoichiometry. dsRNA with protruding termini bind LGP2 and RIG-I weakly and do not stimulate the activation of RIG-I efficiently in cells. Surprisingly, full-length LGP2 containing mutations that abolish dsRNA binding retained the ability to inhibit RIG-I signaling.The innate immune response is the first line of defense against invading pathogens; it is the ubiquitous system of defense against microbial infections (1). Toll-like receptors (TLRs) 3 and RIG-I (retinoic acid-inducible gene 1)-like receptors (RLRs) play key roles in innate immune response toward viral infection (2-5). Toll-like receptors TLR3, TLR7, and TLR8 sense viral RNA released in the endosome following phagocytosis of the pathogens (6). RIG-I-like receptors RIG-I and MDA5 detect viral RNA from replicating viruses in infected cells (3,7,8). Stimulation of these receptors leads to the induction of type I interferons (IFNs) and other proinflammatory cytokines, conferring antiviral activity to the host cells and activating the acquired immune responses (4, 9).RIG-I discriminates between viral and host RNA through specific recognition of the uncapped 5Ј-triphosphate of singlestranded RNA (5Ј ppp ssRNA) generated by viral RNA polymerases (10, 11). In addition, RIG-I also recognizes doublestranded RNA generated during RNA virus replication (7,12). Transfection of cells with synthetic double-stranded RNA stimulates the activation of RIG-I (13, 14). Synthetic dsRNA mimics, such as polyinosinic-polycytidylic acid (poly(I⅐C)), can activate MDA5 when introduced into the cytoplasm of cells. Digestion of poly(I⅐C) with RNase III transforms poly(I⅐C) from a ligand for MDA5 into a ligand for RIG-I, suggesting that MDA5 recognizes long dsRNA, whereas RIG-I recognizes short dsRNA (15). Studies of RIG-I and MDA5 knock-out mice confirmed the essential roles of these receptors in antiviral immune responses and demonstrated that they sense different sets of RNA viruses (12, 16).RIG-I and MDA5 contain two caspase recruiting domains (CARDs) at their N termini, a DEX(D/H) box RNA helicase domain, and a C-terminal regulatory or repressor domain (CTD). The helicase domain and the CTD are responsible for viral RNA binding, whereas the CARDs are required for signaling (3, 8). The current model of RIG-I activation suggests that under resting ...

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

Recognition of double-stranded RNA by Toll-like receptor 3 (TLR3) will increase the production of cytokines and chemokines through transcriptional activation by the NF-B protein.Over 136 single-nucleotide polymorphisms (SNPs) in TLR3 have been identified in the human population. Of these, four alter the sequence of the TLR3 protein. Molecular modeling suggests that two of the SNPs, N284I and L412F, could affect the packing of the leucine-rich repeating units in TLR3. Notably, L412F is reported to be present in 20% of the population and is higher in the asthmatic population. To examine whether the four SNPs affect TLR3 function, each were cloned and tested for their ability to activate the expression of TLR3-dependent reporter constructs. SNP N284I was nearly completely defective for activating reporter activity, and L412F was reduced in activity. These two SNPs did not obviously affect the level of TLR3 expression or their intracellular location in vesicles. However, N284I and L412F were underrepresented on the cell surface, as determined by flow cytometry analysis, and were not efficiently secreted into the culture medium when expressed as the soluble ectodomain. They were also reduced in their ability to act in a dominant negative fashion on the wild type TLR3 allele. These observations suggest that N284I and L412F affect the activities of TLR3 needed for proper signaling.

Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV-infected patients. Since zinc supplementation therapy is known to be safe in healthy individuals and since high-dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV-associated health problems following a similar treatment regimen.