Po Tien

The Adaptor Protein MITA Links Virus-Sensing Receptors to IRF3 Transcription Factor Activation

Viral infection triggers activation of transcription factors such as NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. Here, we identified MITA as a critical mediator of virus-triggered type I IFN signaling by expression cloning. Overexpression of MITA activated IRF3, whereas knockdown of MITA inhibited virus-triggered activation of IRF3, expression of type I IFNs, and cellular antiviral response. MITA was found to localize to the outer membrane of mitochondria and to be associated with VISA, a mitochondrial protein that acts as an adaptor in virus-triggered signaling. MITA also interacted with IRF3 and recruited the kinase TBK1 to the VISA-associated complex. MITA was phosphorylated by TBK1, which is required for MITA-mediated activation of IRF3. Our results suggest that MITA is a critical mediator of virus-triggered IRF3 activation and IFN expression and further demonstrate the importance of certain mitochondrial proteins in innate antiviral immunity.

Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors

Summary Background Studies on the fusion-inhibitory peptides derived from the heptad repeat 1 and 2 (HR1 and HR2) regions of the HIV-1 envelope glycoprotein gp41 provided crucial information on the viral fusogenic mechanism. We used a similar approach to study the fusogenic mechanism of severe-acute-respiratory-syndrome-associated coronavirus (SARS-CoV). Methods We tested the inhibitory activity against infection of two sets of peptides corresponding to sequences of SARS-CoV spike protein HR1 and HR2 regions and investigated the interactions between the HR1 and HR2 peptides by surface plasmon resonance, sedimentation equilibration analysis, circular dichroism, native polyacrylamide-gel electrophoresis, size exclusion high-performance liquid chromatography, and computer-aided homology modelling and molecule docking analysis. Findings One peptide, CP-1, derived from the HR2 region, inhibited SARS-CoV infection in the micromolar range. CP-1 bound with high affinity to a peptide from the HR1 region, NP-1. CP-1 alone had low -helicity and self-associated to form a trimer in phosphate buffer (pH 7·2). CP-1 and NP-1 mixed in equimolar concentrations formed a six-helix bundle, similar to the fusogenic core structure of HIV-1 gp41. Interpretation After binding to the target cell, the transmembrane spike protein might change conformation by association between the HR1 and HR2 regions to form an oligomeric structure, leading to fusion between the viral and target-cell membranes. At the prefusion intermediate state, CP-1 could bind to the HR1 region and interfere with the conformational changes, resulting in inhibition of SARS-CoV fusion with the target cells. CP-1 might be modifiable to increase its anti-SARS-CoV activity and could be further developed as an antiviral agent for treatment or prophylaxis of SARS-CoV infection.

PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients

Programmed death 1 (PD‐1) and its ligand (PD‐L1) play pivotal roles in regulating host immune responses. However, the inhibitory effects of this pathway on the function of cytotoxic CD8+ T lymphocytes, the main effector cells in hepatocellular carcinoma (HCC) patients, are not well defined. In this study, we characterized circulating and intratumor PD‐1/PD‐L1 expression and analyzed their association with disease progression in a cohort of hepatitis B virus‐infected patients, including 56 with HCC, 20 with liver cirrhosis (LC) and 20 healthy controls (HC). The frequency of circulating PD‐1+CD8+ T cells increased with disease progression from LC to HCC patients versus HC. Furthermore, tumor‐infiltrating effector CD8+ T cells showed a drastic increase in PD‐1 expression. These increases in circulating and intratumor PD‐1+CD8+ T cells could predict poorer disease progression and postoperative recurrence. Immunohistochemical staining showed that PD‐L1 expressing hepatoma cells and apoptotic infiltrating CD8+ T cells were both enriched in tumor sections. In vitro, CD8+ T cells induced PD‐L1 expression on hepatoma cells in an IFN‐γ–dependent manner, which in turn promoted CD8+ T cells apoptosis, and blocking PD‐L1 reversed this effect. Therefore, this study extends our knowledge of the role of the PD‐1/PD‐L1 pathway in tumor evasion and provides evidence for a new therapeutic target in HCC patients.

Functional impairment in circulating and intrahepatic NK cells and relative mechanism in hepatocellular carcinoma patients.

Functional defects in natural killer (NK) cells have been proposed to be responsible for the failure of anti-tumor immune responses. Whether and how NK cells are impaired in hepatocellular carcinoma (HCC) patients remain unknown. In this study, we found that HCC patients displayed a dramatic reduction in peripheral CD56(dim)CD16(pos) NK subsets compared with healthy subjects. A significant reduction of CD56(dim)CD16(pos) NK subsets was also found in tumor regions compared with non-tumor regions in the livers of these HCC patients. Both these peripheral and tumor-infiltrating NK cells exhibited poorer capacity to produce IFN-gamma and kill K562 targets, which was further found to be associated with increased CD4(+)CD25(+) T regulatory cells as we previously-described in HCC patients. Addition of Tregs from HCC patients efficiently inhibited the anti-tumor ability of autologous NK cells in vitro. These findings are helpful for understanding the mechanism of NK cell-mediated anti-tumor immune responses in HCC patients.

Circulating microRNAs in hepatitis B virus-infected patients.

Summary.  MicroRNAs (miRNAs) are stably present in human serum. The relationship between circulating miRNAs and hepatitis B virus (HBV) infected liver disease has not been previously reported. Applied Biosystems array‐based miRNA expression profiling was performed on pooled sera obtained from identified groups of chronic asymptomatic carriers (ASC), patients with chronic hepatitis B (CHB) and HBV‐associated acute‐on‐chronic liver failure (ACLF), as well as healthy controls (HC). Nine miRNAs were verified in more clinical samples by RT‐PCR. The correlation between miRNAs expression and the relationship between miRNA levels and clinical characteristics was analysed. Results showed that circulating miRNAs were detected in all disease and control samples, and their numbers increased with symptom severity, from 37 in HC, 77 in ASC, 101 in CHB, to 135 in ACLF. The expression levels of most miRNAs were also up‐regulated in HBV‐infected patients when compared to HC. Expression of the liver‐specific miR‐122 was significantly up‐regulated in HBV‐infected patients. Concomitant regulation of miRNAs not in clusters was disrupted by HBV infection. However, such disruption was not observed for miRNAs in paralogous clusters. Furthermore, the level of miRNAs in the CHB serum was up‐regulated most in hepatitis B e antigen–positive patients. The expression levels of miR‐122 and miR‐194 correlated negatively with the age of patients with CHB or ACLF. Functional analysis showed that miR‐122 could inhibit HBV replication in Huh7 and HepG2 cells. In all, our study revealed that a number of miRNAs were differentially expressed during HBV infection and underscored the potential importance of miR‐122 in the infection process.

Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase

The N7-methylguanosine (m7G) cap is the defining structural feature of eukaryotic mRNAs. Most eukaryotic viruses that replicate in the cytoplasm, including coronaviruses, have evolved strategies to cap their RNAs. In this report, we used a yeast genetic system to functionally screen for the cap-forming enzymes encoded by severe acute respiratory syndrome (SARS) coronavirus and identified the nonstructural protein (nsp) 14 of SARS coronavirus as a (guanine-N7)-methyltransferase (N7-MTase) in vivo in yeast cells and in vitro using purified enzymes and RNA substrates. Interestingly, coronavirus nsp14 was previously characterized as a 3′-to-5′ exoribonuclease, and by mutational analysis, we mapped the N7-MTase domain to the carboxy-terminal part of nsp14 that shows features conserved with cellular N7-MTase in structure-based sequence alignment. The exoribonuclease active site was dispensable but the exoribonuclease domain was required for N7-MTase activity. Such combination of the 2 functional domains in coronavirus nsp14 suggests that it may represent a novel form of RNA-processing enzymes. Mutational analysis in a replicon system showed that the N7-MTase activity was important for SARS virus replication/transcription and can thus be used as an attractive drug target to develop antivirals for control of coronaviruses including the deadly SARS virus. Furthermore, the observation that the N7-MTase of RNA life could function in lieu of that in DNA life provides interesting evolutionary insight and practical possibilities in antiviral drug screening.

Negative regulation of MDA5- but not RIG-I-mediated innate antiviral signaling by the dihydroxyacetone kinase

Viral infection leads to activation of the transcription factors interferon regulatory factor-3 and NF-κB, which collaborate to induce type I IFNs. The RNA helicase proteins RIG-I and MDA5 were recently identified as two cytoplasmic viral RNA sensors that recognize different species of viral RNAs produced during viral replication. In this study, we identified DAK, a functionally unknown dihydroacetone kinase, as a specific MDA5-interacting protein. DAK was associated with MDA5, but not RIG-I, under physiological conditions. Overexpression of DAK inhibited MDA5- but not RIG-I- or TLR3-mediated IFN-β induction. Overexpression of DAK also inhibited cytoplasmic dsRNA and SeV-induced activation of the IFN-β promoter, whereas knockdown of endogenous DAK by RNAi activated the IFN-β promoter, and increased cytoplasmic dsRNA- or SeV-triggered activation of the IFN-β promoter. In addition, overexpression of DAK inhibited MDA5- but not RIG-I-mediated antiviral activity, whereas DAK RNAi increased cytoplasmic dsRNA-triggered antiviral activity. These findings suggest that DAK is a physiological suppressor of MDA5 and specifically inhibits MDA5- but not RIG-I-mediated innate antiviral signaling.

Regulation of virus-triggered signaling by OTUB1- and OTUB2-mediated deubiquitination of TRAF3 and TRAF6.

Ubiquitination and deubiquitination have emerged as critical post-translational regulatory mechanisms for activation or attenuation of the virus-triggered type I interferon (IFN)2 induction pathways. In this study, we identified two deubiquitinating enzymes, OTUB1 and OTUB2, as negative regulators of virus-triggered type I IFN induction. Overexpression of OTUB1 and OTUB2 inhibited virus-induced activation of IRF3 and NF-κB, transcription of the IFNB1 gene as well as cellular antiviral response, whereas knockdown of OTUB1 and OTUB2 had opposite effects. Coimmunoprecipitations indicated OTUB1 and -2 interacted with TRAF3 and TRAF6, two E3 ubiquitin ligases required for virus-triggered IRF3 and NF-κB activation, respectively. Furthermore, we found that OTUB1 and OTUB2 mediated virus-triggered deubiquitination of TRAF3 and -6. These findings suggest that OTUB1 and OTUB2 negatively regulate virus-triggered type I IFN induction and cellular antiviral response by deubiquitinating TRAF3 and -6.

Analysis of synonymous codon usage in classical swine fever virus

Using the complete genome sequences of 35 classical swine fever viruses (CSFV) representing all three genotypes and all three kinds of virulence, we analyzed synonymous codon usage and the relative dinucleotide abundance in CSFV. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in CSFV. Furthermore, we observed that the relative abundance of dinucleotides in CSFV is independent of the overall base composition but is still the result of differential mutational pressure, which also shapes codon usage. In addition, other factors, such as the subgenotypes and aromaticity, also influence the codon usage variation among the genomes of CSFV. This study represents the most comprehensive analysis to date of CSFV codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias.

Myostatin antisense RNA-mediated muscle growth in normal and cancer cachexia mice.

Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to muscle growth. Here we have used modified RNA oligonucleotides targeting the myostatin mRNA and examined the therapeutic potential in normal and cancer cachexia mouse models. We found that the RNA oligonucleotides could suppress the myostatin expression in vivo, leading to the increase in muscle growth both in normal and cachectic mice. We also established that the effect of myostatin inhibition caused by the RNA oligonucleotides may be through the MyoD pathway, as evidenced by a significant upregulation of MyoD expression. Taken together, these results demonstrate the feasibility using antisense strategy for the treatment of muscle wasting conditions.