Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide 2′,5′ cGAMP that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and IFN-β reporter assays of cGAS mutants demonstrated that interactions at both DNA binding sites are essential for cGAS activation. Mutagenesis and DNA binding studies showed that the two sites bind dsDNA cooperatively and site B plays a critical role in DNA binding. The structure of mouse cGAS bound to dsDNA and 2′,5′ cGAMP provided insight into the catalytic mechanism of cGAS. These results demonstrated that cGAS is activated by dsDNA-induced oligomerization.

The STING (stimulator of interferon genes) protein can bind cyclic dinucleotides to activate the production of type I interferons and inflammatory cytokines. The cyclic dinucleotides can be bacterial second messengers c-di-GMP and c-di-AMP, 3’5’-3’5’ cyclic GMP-AMP (3’3’ cGAMP) produced by Vibrio cholerae and metazoan second messenger 2’5’-3’5’ Cyclic GMP-AMP (2’3’ cGAMP). Analysis of single nucleotide polymorphism (SNP) data from the 1000 Genome Project revealed that R71H-G230A-R293Q (HAQ) occurs in 20.4%, R232H in 13.7%, G230A-R293Q (AQ) in 5.2%, and R293Q in 1.5% of human population. In the absence of exogenous ligands, the R232H, R293Q and AQ SNPs had only modest effect on the stimulation of IFN-β and NF-κB promoter activities in HEK293T cells, while HAQ had significantly lower intrinsic activity. The decrease was primarily due to the R71H substitution. The SNPs also affected the response to the cyclic dinucleotides. In the presence of c-di-GMP, the R232H variant partially decreased the ability to activate IFN-βsignaling, while it was defective for the response to c-di-AMP and 3’3’ cGAMP. The R293Q dramatically decreased the stimulatory response to all bacterial ligands. Surprisingly, the AQ and HAQ variants maintained partial abilities to activate the IFN-β signaling in the presence of ligands due primarily to the G230A substitution. Biochemical analysis revealed that the recombinant G230A protein could affect the conformation of the C-terminal domain of STING and the binding to c-di-GMP. Comparison of G230A structure with that of WT revealed that the conformation of the lid region that clamps onto the c-di-GMP was significantly altered. These results suggest that hSTING variation can affect innate immune signaling and that the common HAQ haplotype expresses a STING protein with reduced intrinsic signaling activity but retained the ability to response to bacterial cyclic dinucleotides.

Although oxidative tissue injury often accompanies viral infection, there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase 2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals, suggesting critical regulation of the conformation of the NS3/4A protease and NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to trans-membrane and membrane-proximal residues within these proteins, and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a novel mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

RNA viruses use several initiation strategies to ensure that their RNAs are synthesized in appropriate amounts, have correct termini, and can be translated efficiently. Many viruses with genomes of single-stranded positive-, negative-, and double-stranded RNA initiate RNA synthesis by a de novo (primer-independent) mechanism. This review summarizes biochemical features and variations of de novo initiation in viral RNA replication.

The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3 of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible crosslinking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.The Nidoviruses contain three families of viruses, including the Coronaviridae that cause numerous diseases in humans (1). Severe acute respiratory syndrome coronavirus (SARS-CoV) 3 is a member of the Coronavirus genus (2, 3). It originated from animals but spread to humans, causing severe respiratory distress with a fatality rate of ϳ10% (as shown by the World Health Organization, www.who.int/csr/ sars/country/en/country2003_08_15.pdf). In addition to their medical importance, coronaviruses are of interest for their large ϳ30-kb positive-strand genome and novel mechanisms that have evolved to replicate and transcribe this large RNA (5, 6). In keeping with the novel strategies used, coronaviruses encode several unusual RNA-processing enzymes, including an RNA endoribonuclease, an RNA methyltransferase, a second RNA-dependent RNA polymerase that generates primers for coronavirus replication (7), and a mechanism to decrease replication errors (8).Coronavirus subgenomic RNAs are particularly interesting in that they all have the same 5Ј leader sequence derived from the 5Ј end of the genomic RNA. This organization requires recombination as part of transcription. Various mechanisms have been proposed for subgenomic RNA production, but a discontinuous transcription mechanism is increasingly favored (5). This model proposes that transcription regulatory sequences in the minus-strand RNA direct translocation of the ternary complex to the 5Ј leader sequence, where transcription resumes. The minus-strand RNAs serves as the template for subgenomic RNA transcription. Ribonucleases that process the RNA intermediates for transcription have been proposed (9 -11), but the mechanism for the process is still not completely understood.Nsp15 (nonstructural protein 15) was predicted to be an RNA endoribonuclease as part of a bioinformatics analysis of the SARSCoV genome (7). S...

RNA templates of 33 nucleotides containing the brome mosaic virus (BMV) core subgenomic promoter were used to determine the promoter elements recognized by the BMV RNA-dependent RNA polymerase (RdRp) to initiate RNA synthesis. Nucleotides at positions ؊17, ؊14, ؊13, and ؊11 relative to the subgenomic initiation site must be maintained for interaction with the RdRp. Changes to every other nucleotide at these four positions allow predictions for the base-specific functional groups required for RdRp recognition. RdRp contact of the nucleotide at position ؊17 was suggested with a template competition assay. Comparison of the BMV subgenomic promoter to those from other plant and animal alphaviruses shows a remarkable degree of conservation of the nucleotides required for BMV subgenomic RNA synthesis. We show that the RdRp of the plant-infecting BMV is capable of accurately, albeit inefficiently, initiating RNA synthesis from the subgenomic promoter of the animalinfecting Semliki Forest virus. The sequence-specific recognition of RNA by the BMV RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.Viral RNA replication, a process fundamental to pathogenicity, requires specific recognition of RNA features by proteins. RNA-dependent RNA polymerase (RdRp) is a complex composed of viral and cellular proteins that directs viral RNA synthesis from infecting RNA templates (1). Many viral RdRp proteins have been sequenced and analyzed (2); however, a comprehensive mechanism describing RNA synthesis is lacking. Consequently, general knowledge of RdRps is significantly less than that of other RNA and DNA polymerases.To investigate the mechanism of RNA-directed RNA synthesis, we study brome mosaic virus (BMV), type member of the bromovirus group of plant viruses in the alphavirus-like superfamily of (ϩ)-strand RNA viruses (3). The BMV genome is comprised of three RNAs designated 1, 2, and 3, and a subgenomic RNA4 which is initiated from (Ϫ)-strand RNA3. Enriched BMV RdRp preparations from infected barley can, in a highly specific manner, synthesize (Ϫ)-strand RNA from (ϩ)-strand templates and subgenomic (ϩ)-strand products from (Ϫ)-strand templates (4-6).We have developed a system to study BMV subgenomic RNA initiation from minimal templates, designated proscripts, because they contain the promoter and template for (ϩ)-strand RNA synthesis. We have shown that the 20 nt 3Ј of the subgenomic initiation nucleotide, recognized as the subgenomic core promoter (7,8), are sufficient to direct (ϩ)-strand RNA synthesis (6), permitting the design of proscripts focusing only on the core promoter.In this paper, we have used a functional assay to determine that nucleotides Ϫ17, Ϫ14, Ϫ13, and Ϫ11 relative to the subgenomic initiation site are required for RNA synthesis from the subgenomic core promoter. Moreover, at least one of these nucleotides, Ϫ17, was found likely to be contacted by the BMV RdRp. The resolution achieved in this study allows us to predict the base moieties contacted by RdRp and dem...

The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3 cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3 single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.Hepatitis C virus (HCV), a plus-strand RNA virus, is estimated to infect up to 3% of the world's population (44), causing liver cirrhosis and hepatocellular carcinoma (14). Following entry into the infected cell, the viral RNA directs the translation of a polyprotein that is proteolytically processed to produce 10 individual structural and nonstructural proteins (15, 32). Nonstructural protein 5B (NS5B) is at the C terminus of the polyprotein. NS5B is an RNA-dependent RNA polymerase (RdRp). Based on the paradigms of other RNA virus replication strategies (8), NS5B, along with viral and cellular proteins, forms a replicase that replicates the HCV genome. At present, functional HCV replicase has not been demonstrated in vitro. Therefore, studies of HCV RNA synthesis have focused on recombinant NS5B.Recombinant HCV NS5B can catalyze a number of reactions. In the presence of a primer-template duplex, NS5B catalyzes template-dependent but relatively nonspecific RNA synthesis (5, 23-25, 45, 46). In addition, NS5B has recently been reported to direct de novo (oligonucleotide primer-independent) synthesis (26, 30, 47), a mechanism used for the replication of many plus-strand RNA viruses (8). De novo initiation of RNA synthesis may be especially relevant for HCV since, to our knowledge, it does not contain a VPg-like protein that could mediate protein-primed RNA synthesis, and there is no evidence for a cap-snatching mechanism (32). De novo RNA synthesis directed by HCV NS5B prefers a cytidylate template and the substrate nucleotide GTP (26, 42), although ATP can also be used as an initiation nucleotide (29,42,47). In general, RNA polymerases have a higher K m for the initiation nucleotide than for the same nucleotide during elongating RNA synthesis (for examples, see references ...

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