Combined antiviral activity of interferon-alpha and RNA interference directed against hepatitis C without affecting vector delivery and gene silencing.
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ABSTRACT:
SUBMITTER: Pan Q
PROVIDER: S-EPMC2700866 | biostudies-other | 2009 Jul
REPOSITORIES: biostudies-other
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Project description:Disease vectors such as mosquitoes and ticks play a major role in the emergence and re-emergence of human and animal viral pathogens. Compared to mosquitoes, however, much less is known about the antiviral responses of ticks. Here we showed that Asian longhorned ticks (Haemaphysalis longicornis) produced predominantly 22-nucleotide virus-derived siRNAs (vsiRNAs) in response to severe fever with thrombocytopenia syndrome virus (SFTSV, an emerging tick-borne virus), Nodamura virus (NoV), or Sindbis virus (SINV) acquired by blood feeding. Notably, experimental acquisition of NoV and SINV by intrathoracic injection also initiated viral replication and triggered the production of vsiRNAs in H. longicornis. We demonstrated that a mutant NoV deficient in expressing its viral suppressor of RNAi (VSR) replicated to significantly lower levels than wildtype NoV in H. longicornis, but accumulated to higher levels after knockdown of the tick Dicer2-like protein identified by phylogeny comparison. Moreover, the expression of a panel of known animal VSRs in cis from the genome of SINV drastically enhanced the accumulation of the recombinant viruses. This study establishes a novel model for virus-vector-mouse experiments with longhorned ticks and provides the first in vivo evidence for an antiviral function of the RNAi response in ticks. Interestingly, comparing the accumulation levels of SINV recombinants expressing green fluorescent protein or SFTSV proteins identified the viral non-structural protein as a putative VSR. Elucidating the function of ticks' antiviral RNAi pathway in vivo is critical to understand the virus-host interaction and the control of tick-borne viral pathogens.
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Project description:Tick-borne diseases (TBDs) are the most common illnesses transmitted by ticks, and the annual number of reported TBD cases continues to increase. The Asian longhorned tick, a vector associated with at least 30 human pathogens, is native to eastern Asia and recently reached the USA as an emerging disease threat. Newly identified tick-transmitted pathogens continue to be reported, raising concerns about how TBDs occur. Interestingly, tick can harbor pathogens without being affected themselves. For viral infections, ticks have their own immune systems that protect them from infection. Meanwhile, tick-borne viruses have evolved to avoid these defenses as they establish themselves within the vector. Here, we show in detail that infecting longhorned ticks with distinct arthropod-borne RNA viruses through two approaches natural blood feeding and injection, all induce the production of vsiRNAs. Dicer2-like homolog plays a role in regulating antiviral RNAi responses as knocking down of this gene enhanced viral replication. Furthermore, we demonstrate that tick antiviral RNAi responses are inhibited through expression heterologous VSR proteins in recombinant SINV. We identify both the virus and tick factors are critical components to understanding TBDs. Importantly, our study introduces a novel, in vivo virus-vector-mouse model system for exploring TBDs in the future.
2021-11-22 | GSE159277 | GEO
Project description:Hepatitis C virus (HCV) infection is the leading cause of chronic liver-related diseases, including cirrhosis, liver failure, and hepatocellular carcinoma. Currently, no effective vaccine is available for HCV infection. Polyethylene glycol interferon-? (PegIFN-?) in combination with ribavirin (RBV) is the standard of care (SOC) for chronic hepatitis C. However, the efficacy of PegIFN-? and RBV combination therapy is less than 50% for genotype 1 HCV, which is the dominant virus in humans. In addition, IFN and RBV have several severe side effects. Therefore, strategies to improve sustained virological response (SVR) rates have been an important focus for clinical physicians. The serine protease inhibitors telaprevir and boceprevir were approved by the United States Food and Drug Administration in 2011. The addition of HCV protease inhibitors to the SOC has significantly improved the efficacy of treatments for HCV infection. Several direct-acting antiviral drugs currently in late-stage clinical trials, both with and without peg-IFN and RBV, have several advantages over the previous SOC, including higher specificity and efficacy, fewer side effects, and the ability to be administered orally, and might be optimal regimens in the future. Factors affecting the efficacy of anti-HCV treatments based on IFN-? include the HCV genotype, baseline viral load, virological response during treatment, host IL28B gene polymorphisms and hepatic steatosis. However, determining the effect of the above factors on DAA therapy is necessary. In this review, we summarize the development of anti-HCV agents and assess the main factors affecting the efficacy of antiviral treatments.
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Project description:Hepatitis C virus (HCV) infects 200 million people globally, and 60-80% of cases persist as a chronic infection that will progress to cirrhosis and liver cancer in 2-10% of patients. We recently demonstrated that HCV induces aberrant expression of two host microRNAs (miRNAs), miR-208b and miR-499a-5p, encoded by myosin genes in infected hepatocytes. These miRNAs, along with AU-rich-element-mediated decay, suppress IFNL2 and IFNL3, members of the type III interferon (IFN) gene family, to support viral persistence. In this study, we show that miR-208b and miR-499a-5p also dampen type I IFN signaling in HCV-infected hepatocytes by directly down-regulating expression of the type I IFN receptor chain, IFNAR1. Inhibition of these miRNAs by using miRNA inhibitors during HCV infection increased expression of IFNAR1. Additionally, inhibition rescued the antiviral response to exogenous type I IFN, as measured by a marked increase in IFN-stimulated genes and a decrease in HCV load. Treatment of HCV-infected hepatocytes with type I IFN increased expression of myosins over HCV infection alone. Since these miRNAs can suppress type III IFN family members, these data collectively define a novel cross-regulation between type I and III IFNs during HCV infection.
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Project description:Gamma interferon (IFN-gamma) has been shown to inhibit replication of subgenomic and genomic hepatitis C virus (HCV) RNAs in vitro and to noncytolytically suppress hepatitis B virus (HBV) replication in vivo. IFN-gamma is also known for its immunomodulatory effects and as a marker of a successful cellular immune response to HCV. Therapeutic expression of IFN-gamma in the liver may therefore facilitate resolution of chronic hepatitis C, an infection that is rarely resolved spontaneously. To analyze immunomodulatory and antiviral effects of liver-specific IFN-gamma expression in vivo, we intravenously injected two persistently HCV-infected chimpanzees twice with a recombinant, replication-deficient HBV vector and subsequently with a recombinant adenoviral vector. These vectors expressed human IFN-gamma under control of HBV- and liver-specific promoters, respectively. Gene transfer resulted in a transient increase of intrahepatic IFN-gamma mRNA, without increase in serum alanine aminotransferase levels. Ex vivo analysis of peripheral blood lymphocytes demonstrated enhanced CD16 expression on T cells and upregulation of the liver-homing marker CXCR3. Moreover, an increased frequency of HCV-specific T cells was detected ex vivo in the peripheral blood and in vitro in liver biopsy-derived, antigen-nonspecifically expanded T-cell lines. None of these immunologic effects were observed in the third chimpanzee injected with an HBV control vector. Despite these immunologic effects of the experimental vector, however, IFN-gamma gene transfer did not result in a significant and long-lasting decrease of HCV titers. In conclusion, liver-directed IFN-gamma gene delivery resulted in HCV-specific and nonspecific activation of cellular immune responses but did not result in effective control of HCV replication.
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Project description:We describe the design and characterization of a potent human respiratory syncytial virus (RSV) nucleocapsid gene-specific small interfering RNA (siRNA), ALN-RSV01. In in vitro RSV plaque assays, ALN-RSV01 showed a 50% inhibitory concentration of 0.7 nM. Sequence analysis of primary isolates of RSV showed that the siRNA target site was absolutely conserved in 89/95 isolates, and ALN-RSV01 demonstrated activity against all isolates, including those with single-mismatch mutations. In vivo, intranasal dosing of ALN-RSV01 in a BALB/c mouse model resulted in potent antiviral efficacy, with 2.5- to 3.0-log-unit reductions in RSV lung concentrations being achieved when ALN-RSV01 was administered prophylactically or therapeutically in both single-dose and multidose regimens. The specificity of ALN-RSV01 was demonstrated in vivo by using mismatch controls; and the absence of an immune stimulatory mechanism was demonstrated by showing that nonspecific siRNAs that induce alpha interferon and tumor necrosis factor alpha lack antiviral efficacy, while a chemically modified form of ALN-RSV01 lacking measurable immunostimulatory capacity retained full activity in vivo. Furthermore, an RNA interference mechanism of action was demonstrated by the capture of the site-specific cleavage product of the RSV mRNA via rapid amplification of cDNA ends both in vitro and in vivo. These studies lay a solid foundation for the further investigation of ALN-RSV01 as a novel therapeutic antiviral agent for clinical use by humans.
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Project description:Real-life data showed an increased incidence of bacterial infections in patients with advanced liver disease receiving a protease inhibitor (PI)-containing antiviral regimen against hepatitis C (HCV). However, the causes of this event are unknown. We hypothesized that PIs might impair innate immune responses through the inhibition of proteases participating in the anti-bacterial functions of neutrophils and monocytes. The aims of the study were to assess phagocytic and oxidative burst capacity in neutrophils and monocytes obtained from patients receiving a PI containing-antiviral regimen, and to determine cytokine secretion after neutrophil stimulation with flagellin. Forty patients with chronic HCV (80% with cirrhosis) were enrolled in the study, 28 received triple therapy (Group A) with pegylated-interferon and ribavirin for 4 weeks followed by the addition of a PI (telaprevir, boceprevir or simeprevir), and 12 patients received an interferon-free regimen (Group B) with simeprevir and sofosbuvir. Phagocytosis and oxidative burst capacity were analyzed by flow cytometry at baseline, week 4, and week 8 of therapy. In neutrophils from Group A patients, oxidative burst rate and oxidative enzymatic activity per cell significantly decreased throughout the study period (p = 0.014 and p = 0.010, respectively). Pairwise comparisons showed a decrease between baseline and week 4 and 8 of therapy. No differences were observed after the introduction of the PI. The oxidative enzymatic activity per cell in monocytes significantly decrease during the study period (p = 0.042) due to a decrease from baseline to week 8 of therapy (p = 0.037) in patients from Group A. None of these findings were observed in Group B patients. Cytokine secretion did not significantly change during the study in both groups. In conclusion, our data suggest that the use interferon (rather than the PI) has a deleterious effect on neutrophil and monocyte phagocytic and oxidative burst capacity in this cohort of patients with HCV-related advanced liver fibrosis.
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Project description:Hepatitis C virus (HCV) infection can result in viral chronicity or clearance. Although host genetics and particularly genetic variation in the interferon lambda (IFNL) locus are associated with spontaneous HCV clearance and treatment success, the mechanisms guiding these clinical outcomes remain unknown. Using a laser capture microdissection-driven unbiased systems virology approach, we isolated and transcriptionally profiled HCV-infected and adjacent primary human hepatocytes (PHH) approaching single cell resolution. An innate antiviral immune signature dominated the transcriptional response, but differed in magnitude and diversity between HCV-infected and adjacent cells. Molecular signatures associated with more effective antiviral control were determined by comparing donors with high and low infection frequencies. Cells from donors with clinically unfavorable IFNL genotypes were infected at a greater frequency and exhibited dampened antiviral and cell death responses. These data suggest that early virus-host interactions, particularly host genetics and induction of innate immunity, critically determine the outcome of HCV infection. Cell populations of primary human hepatocytes were collected via laser capture microdissection, their transcriptomes were amplified and analyzed via whole Illumina genome microarray. Three populations were collected: virus infected cells, cells adjacent to infected cells (“adjacent”) and mock infected cells. Cells from multiple donors were employed to address host genetic variability on our observed phenotypes. Nine different donors were assessed on 1-day post infection and three donors were assessed 3 days post infection and and four donors were assessed on 7 days post infection. For each cell population (virus infected, adjacent or mock), four biological replicate arrays were performed. In total, we analyzed 186 microarrays
2014-02-12 | E-GEOD-54648 | biostudies-arrayexpress
Project description:Antiviral RNA interference in disease vector (Asian longhorned) ticks
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Project description:FACT ( FA cilitates C hromatin T ranscription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated at lysine 674 (K674) of middle domain (MD), which involves TIP60 histone acetyltransferase. Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (EZH2, HDAC1) and affects histone marks (H3K9me3, H3K27me3 and H3ac). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, CBL0137 is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that CBL0137 also causes the remarkable activation of IFN signaling in natural killer (NK) cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of CBL0137 to treat viral infections.
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