Identification of a role for TRIM29 in the control of innate immunity in the respiratory tract.

The respiratory tract is heavily populated with innate immune cells, but the mechanisms that control such cells are poorly defined. Here we found that the E3 ubiquitin ligase TRIM29 was a selective regulator of the activation of alveolar macrophages, the expression of type I interferons and the production of proinflammatory cytokines in the lungs. We found that deletion of TRIM29 enhanced macrophage production of type I interferons and protected mice from infection with influenza virus, while challenge of Trim29-/- mice with Haemophilus influenzae resulted in lethal lung inflammation due to massive production of proinflammatory cytokines by macrophages. Mechanistically, we demonstrated that TRIM29 inhibited interferon-regulatory factors and signaling via the transcription factor NF-κB by degrading the adaptor NEMO and that TRIM29 directly bound NEMO and subsequently induced its ubiquitination and proteolytic degradation. These data identify TRIM29 as a key negative regulator of alveolar macrophages and might have important clinical implications for local immunity and immunopathology.

The E3 ubiquitin ligase TRIM21 negatively regulates the innate immune response to intracellular double-stranded DNA.

DDX41 is a sensor of intracellular double-stranded DNA (dsDNA) in myeloid dendritic cells (mDCs) that triggers a type I interferon response via the signaling adaptor STING. We identified the E3 ligase TRIM21 as a DDX41-interacting protein and found that knockdown of or deficiency in TRIM21 resulted in enhanced type I interferon responses to intracellular dsDNA and DNA viruses. Overexpression of TRIM21 resulted in more degradation of DDX41 and less production of interferon-ß (IFN-ß) in response to intracellular dsDNA. The SPRY-PRY domain of TRIM21 interacted with the DEADc domain of DDX41. Lys9 and Lys115 of DDX41 were the targets of TRIM21-mediated ubiquitination. TRIM21 is therefore an interferon-inducible E3 ligase that induces the Lys48 (K48)-linked ubiquitination and degradation of DDX41 and negatively regulates the innate immune response to intracellular dsDNA.

Human NLRP3 inflammasome senses multiple types of bacterial RNAs.

Inflammasomes are multiprotein platforms that activate caspase-1, which leads to the processing and secretion of the proinflammatory cytokines IL-1ß and IL-18. Previous studies demonstrated that bacterial RNAs activate the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome in both human and murine macrophages. Interestingly, only mRNA, but neither tRNA nor rRNAs, derived from bacteria could activate the murine Nlrp3 inflammasome. Here, we report that all three types of bacterially derived RNA (mRNA, tRNA, and rRNAs) were capable of activating the NLRP3 inflammasome in human macrophages. Bacterial RNA's 5'-end triphosphate moieties, secondary structure, and double-stranded structure were dispensable; small fragments of bacterial RNA were sufficient to activate the inflammasome. In addition, we also found that 20-guanosine ssRNA can activate the NLRP3 inflammasome in human macrophages but not in murine macrophages. Therefore, human and murine macrophages may have evolved to recognize bacterial cytosolic RNA differently during bacterial infections.

DHX15 senses double-stranded RNA in myeloid dendritic cells.

Many members of the DEXD/H box helicase family play important roles in the innate immune system against viral infection. Therefore, we isolated dsRNA complex in myeloid dendritic cells. We found that DHx15, a DEXDc helicase family member, is one of the components of this complex. Knockdown of DHX15 expression by short hairpin RNA efficiently reduced the ability of myeloid dendritic cells to produce IFN-ß, IL-6, and TNF-α in response to dsRNA and RNA virus. DHX15 specifically bound polyinosine-polycytidylic acid via its helicase C-terminal domain. DHX15 interacted with MAVS and formed a complex following stimulation with polyinosine-polycytidylic acid. The N-terminal domain containing a DEXDc motif in DHX15 bound the C terminus of MAVS. DHX15 is required to activate IRF3 phosphorylation as well as NF-κB and MAPK signaling during RNA virus infection. We, therefore, identified DHX15 as a new RNA virus sensor mediated by MAVS to activate the immune responses to RNA.

The E3 ubiquitin ligase tripartite motif 33 is essential for cytosolic RNA-induced NLRP3 inflammasome activation.

NLRP3 is a key component of caspase-activating macromolecular protein complexes called inflammasomes. It has been found that DHX33 is a cytosolic dsRNA sensor for the NLRP3 inflammasome, which induces caspase-1-dependent production of IL-1ß and IL-18 upon activation. However, how the cytosolic dsRNAs induce the interaction between DHX33 and the NLRP3 inflammasome remains unknown. In this study, we report that TRIM33, a member of the tripartite motif (TRIM) family, can bind DHX33 directly and induce DHX33 ubiquitination via the lysine 218 upon dsRNA stimulation. Knocking down of TRIM33 abolished the dsRNA-induced NLRP3 inflammasome activation in both THP-1-derived macrophages and human monocyte-derived macrophages. The ubiquitination of DHX33 by TRIM33 is lysine 63 specific and is required for the formation of the DHX33-NLRP3 inflammasome complex.

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Different mechanisms of hepatitis C virus RNA polymerase activation by cyclophilin A and B in vitro.

BACKGROUND: Cyclophilins (CyPs) are cellular proteins that are essential to hepatitis C virus (HCV) replication. Since cyclosporine A was discovered to inhibit HCV infection, the CyP pathway contributing to HCV replication is a potential attractive stratagem for controlling HCV infection. Among them, CyPA is accepted to interact with HCV nonstructural protein (NS) 5A, although interaction of CyPB and NS5B, an RNA-dependent RNA polymerase (RdRp), was proposed first. METHODS: CyPA, CyPB, and HCV RdRp were expressed in bacteria and purified using combination column chromatography. HCV RdRp activity was analyzed in vitro with purified CyPA and CyPB. RESULTS: CyPA at a high concentration (50× higher than that of RdRp) but not at low concentration activated HCV RdRp. CyPB had an allosteric effect on genotype 1b RdRp activation. CyPB showed genotype specificity and activated genotype 1b and J6CF (2a) RdRps but not genotype 1a or JFH1 (2a) RdRps. CyPA activated RdRps of genotypes 1a, 1b, and 2a. CyPB may also support HCV genotype 1b replication within the infected cells, although its knockdown effect on HCV 1b replicon activity was controversial in earlier reports. CONCLUSIONS: CyPA activated HCV RdRp at the early stages of transcription, including template RNA binding. CyPB also activated genotype 1b RdRp. However, their activation mechanisms are different. GENERAL SIGNIFICANCE: These data suggest that both CyPA and CyPB are excellent targets for the treatment of HCV 1b, which shows the greatest resistance to interferon and ribavirin combination therapy.

Effect of the methyltransferase domain of Japanese encephalitis virus NS5 on the polymerase activity.

Japanese encephalitis virus (JEV) NS5 consists of an N-terminal guanylyltransferase/methyltransferase (MTase) domain and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. We purified JEV NS5 from bacteria and examined its RdRp activity in vitro. It showed exclusive specificity for Mn(2+) and alkaline conditions (pH 8-10) for RdRp activity. It showed strong RdRp activity with dinucleotide primers, and the order of template strength was poly(U)>(I)>(A)>(C). It showed weak transcription activity without primers, but could not transcribe poly(I) without primers. It bound homopolymeric RNA templates, but weakly bound poly(C). The Km (µM) values were 22.13±1.11 (ATP), 21.94±3.88 (CTP), 21.27±1.23 (GTP), and 9.91±0.30 (UTP), indicating low substrate affinity. Vmax (/min) values were 0.216±0.017 (ATP), 0.781±0.020 (CTP), 0.597±0.049 (GTP), and 0.347±0.022 (UTP), indicating high polymerization activity. The RdRp domain alone did not show RdRp activity; a structural and functional interaction between the MTase and RdRp domains via 299-EHPYRTWTYH-308 (MTase domain) and 739-LIGRARISPG-748 (RdRp domain) was predicted, because mutations in the MTase domain affected RdRp activity.

Fluorescent primer-based in vitro transcription system of viral RNA-dependent RNA polymerases.

Viral infection is a leading cause of disease and death. Although vaccines are the most effective method of controlling viral infections, antiviral drugs are also important. Here, we established an in vitro transcription system by using fluorescein isothiocyanate-conjugated primers for RNA polymerases of viruses that are important disease-causing human pathogens (influenza, hepatitis C, Japanese encephalitis viruses, and enterovirus 71). This technology will allow us to analyze RNA polymerase activity without using radioisotopes.

Biochemical characterization of enterovirus 71 3D RNA polymerase.

An unusual enterovirus 71 (EV71) epidemic has begun in China since 2008. EV71 RNA polymerases (3D(pol)) showed polymerase activity with an Mn(2+). Little activity was detected with Co(2+), and no activity was detected with Mg(2+), Ca(2+), Cu(2+), Ni(2+), Cd(2+), or Zn(2+). It is a primer-dependent polymerase, and the enzyme functioned with both di- and 10-nucleotide RNA primers. DNA primer, dT15, increased primer activity, similar to other enterovirus 3D(pol). However, EV71 3D(pol) initiated de novo transcription with a poly(C) template and genome RNA. Its RNA binding activity was weak. Terminal nucleotidyl transferase and reverse transcriptase activity were not detected. The Km and Vmax for EV71 3D(pol) were calculated from classic Lineweaver-Burk plots. The Km values were 2.35±0.05 (ATP), 5.40±0.93 (CTP), 1.12±0.10 (GTP) and 2.81±0.31 (UTP), and the Vmax values were 0.00078±0.00005/min (ATP), 0.011±0.0017/min (CTP), 0.050±0.0043/min (GTP) and 0.0027±0.0005/min (UTP). The Km of EV71 3D(pol) was similar to that of foot and mouth disease virus and rhinovirus. Polymerase activity of BrCr-TR strain and a strain from a clinical isolate in Beijing, 2008 were similar, indicating the potential for 3D(pol) as an antiviral drug target.

RNA polymerase activity and specific RNA structure are required for efficient HCV replication in cultured cells.

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3'X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3' untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3'X region. We next analyzed the 3' structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3'UTR were required for J6CF replication in cultured cells.

PA from an H5N1 highly pathogenic avian influenza virus activates viral transcription and replication and induces apoptosis and interferon expression at an early stage of infection.

BACKGROUND: Although gene exchange is not likely to occur freely, reassortment between the H5N1 highly pathogenic avian influenza virus (HPAIV) and currently circulating human viruses is a serious concern. The PA polymerase subunit of H5N1 HPAIV was recently reported to activate the influenza replicon activity. METHODS: The replicon activities of PR8 and WSN strains (H1N1) of influenza containing PA from HPAIV A/Cambodia/P0322095/2005 (H5N1) and the activity of the chimeric RNA polymerase were analyzed. A reassortant WSN virus containing the H5N1 Cambodia PA (C-PA) was then reconstituted and its growth in cells and pathogenicity in mice examined. The interferon promoter, TUNEL, and caspase 3, 8, and 9 activities of C-PA-infected cells were compared with those of WSN-infected cells. RESULTS: The activity of the chimeric RNA polymerase was slightly higher than that of WSN, and C-PA replicated better than WSN in cells. However, the multi-step growth of C-PA and its pathogenicity in mice were lower than those of WSN. The interferon promoter, TUNEL, and caspase 3, 8, and 9 activities were strongly induced in early infection in C-PA-infected cells but not in WSN-infected cells. CONCLUSIONS: Apoptosis and interferon were strongly induced early in C-PA infection, which protected the uninfected cells from expansion of viral infection. In this case, these classical host-virus interactions contributed to the attenuation of this strongly replicating virus.

Sphingomyelin activates hepatitis C virus RNA polymerase in a genotype-specific manner.

Hepatitis C virus (HCV) replication and infection depend on the lipid components of the cell, and replication is inhibited by inhibitors of sphingomyelin biosynthesis. We found that sphingomyelin bound to and activated genotype 1b RNA-dependent RNA polymerase (RdRp) by enhancing its template binding activity. Sphingomyelin also bound to 1a and JFH1 (genotype 2a) RdRps but did not activate them. Sphingomyelin did not bind to or activate J6CF (2a) RdRp. The sphingomyelin binding domain (SBD) of HCV RdRp was mapped to the helix-turn-helix structure (residues 231 to 260), which was essential for sphingomyelin binding and activation. Helix structures (residues 231 to 241 and 247 to 260) are important for RdRp activation, and 238S and 248E are important for maintaining the helix structures for template binding and RdRp activation by sphingomyelin. 241Q in helix 1 and the negatively charged 244D at the apex of the turn are important for sphingomyelin binding. Both amino acids are on the surface of the RdRp molecule. The polarity of the phosphocholine of sphingomyelin is important for HCV RdRp activation. However, phosphocholine did not activate RdRp. Twenty sphingomyelin molecules activated one RdRp molecule. The biochemical effect of sphingomyelin on HCV RdRp activity was virologically confirmed by the HCV replicon system. We also found that the SBD was the lipid raft membrane localization domain of HCV NS5B because JFH1 (2a) replicon cells harboring NS5B with the mutation A242C/S244D moved to the lipid raft while the wild type did not localize there. This agreed with the myriocin sensitivity of the mutant replicon. This sphingomyelin interaction is a target for HCV infection because most HCV RdRps have 241Q.

Biochemical and kinetic analysis of the influenza virus RNA polymerase purified from insect cells.

The influenza virus RNA polymerase (RdRp) was purified from insect cells (around 0.2mg/l). The RdRp catalyzed all the biochemical reactions of influenza virus transcription and replication in vitro; dinucleotide ApG and globin mRNA-primed transcription, de novo initiation (replication), and polyadenylation. The optimal Mg concentration, pH and temperature were 8mM, 8.0 and 25 degrees C, respectively, which were slightly different from those measured for RdRp of virions. This system is a single-round transcription system. K(m) (microM) were 10.74+/-0.26 (GTP), 33.22+/-3.37 (ATP), 28.93+/-0.48 (CTP) and 22.01+/-1.48 (UTP), and V(max) (fmol nucleotide/pmol RdRp/min) were 2.40+/-0.032 (GTP), 1.95+/-0.17 (ATP), 2.07+/-0.17 (CTP), and 1.52+/-0.38 (UTP), which agreed with high mutation of influenza viruses.

Modification of hepatitis C virus 1b RNA polymerase to make a highly active JFH1-type polymerase by mutation of the thumb domain.

Hepatitis C virus (HCV) JFH1 efficiently replicates and produces infectious virus particles in cultured cells. We compared polymerase activity between JFH1 and 1b strains in vitro. The RNA polymerase activity of 1b was 6.4% of that of JFH1. In order to study the mechanism and identify domains responsible for the high polymerase activity of JFH1, we converted the amino acids of 1b RdRp to those of JFH1, and compared their Km, Vmax and template binding activity. Four amino acid mutations in the thumb domain of 1b RdRp, S377R, A450S, E455N and Y561F increased 1b polymerase activity, and their activity was 23.1, 45.8, 28.9, and 36.1% of JFH1, respectively. Vmax and RNA binding activity of JFH1, 1bwt and 1bA450S was JFH1 > 1bA450S > 1b, which indicated both high processivity and slightly higher template binding activity contributed to the high polymerase activity of JFH1.

NFATC3 promotes IRF7 transcriptional activity in plasmacy--toid dendritic cells.

Plasmacytoid dendritic cells (pDCs) rapidly produce large amounts of type 1 interferon (IFN) after Toll-like receptor 7 and 9 engagements. This specialized function of type 1 IFN production is directly linked to the constitutive expression of IRF7, the master transcription factor for type 1 IFN production. However, the IRF7 regulatory network in pDCs remains largely unknown. In this study, we identify that the transcription factor NFATC3 specifically binds to IRF7 and enhances IRF7-mediated IFN production. Furthermore, knockout of NFATC3 greatly reduced the CpG DNA-induced nuclear translocation of IRF7, which resulted in impaired type 1 IFN production in vitro and in vivo. In addition, we found that NFATC3 and IRF7 both bound to type 1 IFN promoters and that the NFAT binding site in IFN promoters was required for IRF7-mediated IFN expression. Collectively, our study shows that the transcription factor NFATC3 binds to IRF7 and functions synergistically to enhance IRF7-mediated IFN expression in pDCs.

The interaction between the helicase DHX33 and IPS-1 as a novel pathway to sense double-stranded RNA and RNA viruses in myeloid dendritic cells.

In eukaryotes, there are at least 60 members of the DExD/H helicase family, many of which are able to sense viral nucleic acids. By screening all known family members, we identified the helicase DHX33 as a novel double-stranded RNA (dsRNA) sensor in myeloid dendritic cells (mDCs). The knockdown of DHX33 using small heteroduplex RNA (shRNA) blocked the ability of mDCs to produce type I interferon (IFN) in response to poly I:C and reovirus. The HELICc domain of DHX33 was shown to bind poly I:C. The interaction between DHX33 and IPS-1 is mediated by the HELICc region of DHX33 and the C-terminal domain of IPS-1 (also referred to MAVS and VISA). The inhibition of DHX33 expression by RNA interference blocked the poly I:C-induced activation of MAP kinases, NF-κB and IRF3. The interaction between the helicase DHX33 and IPS-1 was independent of RIG-I/MDA5 and may be a novel pathway for sensing poly I:C and RNA viruses in mDCs.

Detergent-induced activation of the hepatitis C virus genotype 1b RNA polymerase.

Recently, we found that sphingomyelin bound and activated hepatitis C virus (HCV) 1b RNA polymerase (RdRp), thereby recruiting the HCV replication complex into lipid raft structures. Detergents are commonly used for resolving lipids and purifying proteins, including HCV RdRp. Here, we tested the effect of detergents on HCV RdRp activity in vitro and found that non-ionic (Triton X-100, NP-40, Tween 20, Tween 80, and Brij 35) and twitterionic (CHAPS) detergents activated HCV 1b RdRps by 8-16.6 folds, but did not affect 1a or 2a RdRps. The maximum effect of these detergents was observed at around their critical micelle concentrations. On the other hand, ionic detergents (SDS and DOC) completely inactivated polymerase activity at 0.01%. In the presence of Triton X-100, HCV 1b RdRp did not form oligomers, but recruited more template RNA and increased the speed of polymerization. Comparison of polymerase and RNA-binding activity between JFH1 RdRp and Triton X-100-activated 1b RdRp indicated that monomer RdRp showed high activity because JFH1 RdRp was a monomer in physiological conditions of transcription. Besides, 502H plays a key role on oligomerization of 1b RdRp, while 2a RdRps which have the amino acid S at position 502 are monomers. This oligomer formed by 502H was disrupted both by high salt and Triton X-100. On the contrary, HCV 1b RdRp completely lost fidelity in the presence of 0.02% Triton X-100, which suggests that caution should be exercised while using Triton X-100 in anti-HCV RdRp drug screening tests.

The N-terminal helix α(0) of hepatitis C virus NS3 protein dictates the subcellular localization and stability of NS3/NS4A complex.

The N-terminal amphipathic helix α(0) of hepatitis C virus (HCV) NS3 protein is an essential structural determinant for the protein membrane association. Here, we performed functional analysis to probe the role of this helix α(0) in the HCV life cycle. A point mutation M21P in this region that destroyed the helix formation disrupted the membrane association of NS3 protein and completely abolished HCV replication. Mechanistically the mutation did not affect either protease or helicase/NTPase activities of NS3, but significantly reduced the stability of NS3 protein. Furthermore, the membrane association and stability of NS3 protein can be restored by replacing the helix α(0) with an amphipathic helix of the HCV NS5A protein. In summary, our data demonstrated that the amphipathic helix α(0) of NS3 protein determines the proper membrane association of NS3, and this subcellular localization dictates the functional role of NS3 in the HCV life cycle.