Virus-induced Interferon Production Research Articles

Fish ubiquitin-specific protease 8 (USP8) inhibits IFN production through autophagy-lysosomal dependent degradation of IRF7

Interferon regulatory factor 7 (IRF7) is considered the master regulator of virus-induced interferon (IFN) production. However, to avoid an autoimmune response, the expression of IRF7 must be tightly controlled. In this study, we report that zebrafish ubiquitin-specific protease 8 (USP8) promotes IRF7 degradation through an autophagy-lysosome-dependent pathway to inhibit IFN production. First, zebrafish usp8 is induced upon spring viremia of carp virus (SVCV) infection and polyinosinic/polycytidylic acid (poly I:C) stimulation. Second, overexpression of USP8 suppresses SVCV or poly I:C-mediated IFN expression. Mechanistically, USP8 interacts with IRF7 and promotes its degradation via an autophagy-lysosome-dependent pathway. Finally, USP8 significantly suppresses cellular antiviral responses and enhances SVCV proliferation. In summary, our discoveries offer a perspective on the role of zebrafish USP8 and provide additional understanding of the regulation of IRF7 in host antiviral immune response.

SARS-CoV-2 ORF6 protein does not antagonize interferon signaling in respiratory epithelial Calu-3 cells during infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths, posing a substantial threat to global public health. Viruses evolve different strategies to antagonize or evade host immune responses. While ectopic expression of SARS-CoV-2 accessory protein ORF6 blocks interferon (IFN) production and downstream IFN signaling, the role of ORF6 in IFN signaling during bona fide viral infection of respiratory cells is unclear. By comparing wild-type (WT) and ORF6-deleted (ΔORF6) SARS-CoV-2 infection and IFN signaling in respiratory cells, we found that ΔORF6 SARS-CoV-2 replicates more efficiently than WT virus and, thus, stimulates more robust immune signaling. Loss of ORF6 does not alter innate signaling in infected cells: both WT and ΔORF6 virus induce delayed IFN responses only in bystander cells. Moreover, expression of ORF6 in the context of SARS-CoV-2 infection has no effect on Sendai virus-stimulated IFN induction: robust translocation of IRF3 is observed in both SARS-CoV-2 infected and bystander cells. Furthermore, IFN pretreatment potently blocks WT and ΔORF6 virus replication similarly, and both viruses fail to suppress the induction of interferon-stimulated genes (ISGs) upon IFN-β treatment. However, upon treatment with IFN-β, only bystander cells induce STAT1 translocation during infection with WT virus, whereas ΔORF6 virus-infected cells now show translocation. This suggests that under conditions of high IFN activation, ORF6 can attenuate STAT1 activation. These data provide evidence that ORF6 is not sufficient to antagonize IFN production or IFN signaling in SARS-CoV-2-infected respiratory cells but may impact the efficacy of therapeutics that stimulate innate immune pathways. IMPORTANCE Previous studies identified several SARS-CoV-2 proteins, including ORF6, that antagonize host innate immune responses in the context of overexpression of viral proteins in non-respiratory cells. We set out to determine the role of ORF6 in IFN responses during SARS-CoV-2 infection of respiratory cells. Using a deletion strain, we observed no reduction of infection and no difference in evasion of IFN signaling, with responses limited to bystander cells. Moreover, stimulation of Sendai virus-induced IFN production or IFN-β-stimulated ISG expression was comparable between SARS-CoV-2 virus and SARS-CoV-2 lacking ORF6 virus, suggesting that ORF6 is not sufficient to counteract IFN induction or IFN signaling during viral infection.

Involvement of Virus-Induced Interferon Production in IgG Autoantibody-Mediated Anemia.

Infection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody-opsonized erythrocytes by activated macrophages. Type I interferon receptor-deficient mice show enhanced anemia, which suggests a protective effect of these cytokines, partly through the control of type II interferon production. The development of anemia requires the expression of Fcγ receptors (FcγR) I, III, and IV. Whereas LDV infection decreases FcγR III expression, it enhances FcγR I and IV expression in wild-type animals. The LDV-associated increase in the expression of FcγR I and IV is largely reduced in type I interferon receptor-deficient mice, through both type II interferon-dependent and -independent mechanisms. Thus, the regulation of the expression of FcγR I and IV, but not III, by interferons may partly explain the exacerbating effect of LDV infection on anemia that results from the enhanced phagocytosis of IgG autoantibody-opsonized erythrocytes.

Unveiling the long non-coding RNA profile of porcine reproductive and respiratory syndrome virus-infected porcine alveolar macrophages

BackgroundLong noncoding RNA (lncRNA) is highly associated with inflammatory response and virus-induced interferon production. By far the majority of studies have focused on the immune-related lncRNAs of mice and humans, but the function of lncRNAs in porcine immune cells are poorly understood. Porcine reproductive and respiratory syndrome virus (PRRSV) impairs local immune responses in the lungs of nursery and growing pigs, whereas the virus triggers the inflammatory responses. Porcine alveolar macrophage (PAM) is the primary target cell of PRRSV, thus PRRSV is used as an in vitro model of inflammation. Here, we profiled lncRNA and mRNA repertories from PRRSV-infected PAMs to explore the underlying mechanism of porcine lncRNAs in regulating host immune responses.ResultsIn this study, a total of 350 annotated lncRNAs and 1792 novel lncRNAs in PAMs were identified through RNA-seq analysis. Among them 86 differentially expressed (DE) lncRNAs and 406 DE protein-coding mRNAs were identified upon PRRSV incubation. GO category and KEGG pathway enrichment analyses revealed that these DE lncRNAs and mRNAs were mainly involved in inflammation- and pathogen infection-induced pathways. The results of dynamic correlated expression networks between lncRNAs and their predicted target genes uncovered that numerous lncRNAs, such as XLOC-022175, XLOC-019295, and XLOC-017089, were correlated with innate immune genes. Further analysis validated that these three lncRNAs were positively correlated with their predicted target genes including CXCL2, IFI6, and CD163. This study suggests that porcine lncRNAs affect immune responses against PRRSV infection through regulating their target genes in PAMs.ConclusionThis study provides both transcriptomic and epigenetic status of porcine macrophages. In response to PRRSV infection, comprehensive DE lncRNAs and mRNAs were profiled from PAMs. Co-expression analysis demonstrated that lncRNAs are emerging as the important modulators of immune gene activities through their critical influence upon PRRSV infection in porcine macrophages.

TARBP2 negatively regulates IFN-β production and innate antiviral response by targeting MAVS

MAVS as an essential receptor protein for anti-virus innate immunity plays an important role in the production of virus-induced typeⅠ interferon and regulation of interferon regulatory factor 3/7. Understanding the MAVS-mediated antiviral signaling pathway can provide detailed insights. In this study, we identify transactivation response element RNA-binding protein (TARBP2), as an inhibitor of the cellular protein kinase PKR, negatively regulates virus -induced IFN-β production by targets MAVS. Overexpression of TARBP2 inhibits virus-induced IFN-β production as well as cellular antiviral response. Then knockdown of TARBP2 inhibited virus-induced IFN-β signaling. Further studies demonstrated that TARBP2 interacted with MAVS and targeted MAVS to abrogate MAVS-RIG-I and MAVS-TRAF3 association. Our findings suggest that TARBP2 is an important non-redundant virus-mediated negative regulator of typeⅠ interferon.

FAS-associated death domain and tripartite motif-containing protein 21 negatively regulate virus-induced interferon production (P6126)

Abstract While induction of type I Interferon (IFN) during the host antiviral response is imperative for efficient viral clearance, regulation of this response is crucial for immune system homeostasis. We have reported that FADD, a well characterized apoptotic protein, is involved in negatively regulating RNA virus-induced IFN-α production through a novel interaction with the E3 ubiquitin ligase, Tripartite Motif-Containing Protein 21 (TRIM21). Interaction between FADD and TRIM21 cooperatively represses IFN-α activation in Sendai virus infected cells. This occurs through TRIM21 targeted ubiquitin-mediated degradation of transcription factor IRF7. Recently, it was also determined that FADD and TRIM21 can be found in 2 different complexes within cells: one containing FADD/TRIM21/IRF7 and a second containing FADD/TRIM21/RIP1 (receptor-interacting protein 1). We hypothesized that TRIM21 may have different functions dependent on complex composition and that its activity could be regulated via its autoubiquitination status. Interaction of TRIM21 with FADD yields increased autoubiquition while interaction with RIP1 yields decreased autoubiqtuitination suggesting a regulation mechanism. However, studies involving mutant forms of TRIM21 which lack autoubiquitination demonstrate that autoubiquitination status does not regulate TRIM21’s ability to target IRF7 for degradation. We are currently investigating the function of TRIM21 in other FADD/RIP1 dependent processes like cell death.

Mapping a dynamic innate immunity protein interaction network regulating type I interferon production (108.2)

Abstract We use co-immunoprecipitation and mass spectrometry to identify antiviral signaling networks which regulate innate immune responses. Fifty-eight baits were associated with 260 interacting proteins forming a Human Innate Immunity Interactome of 401 unique interactions; 21% of interactions were remodeled following RNA, DNA, or LPS stimulation. Overexpression and depletion analyses identified 22 novel genes which regulate NF-kB and ISRE reporter activity, viral replication, or virus-induced interferon production. The innate immune interactome provides a dynamic physical and regulatory network which serves as a foundation for mechanistic analysis of antiviral signaling and links innate immunity with other cell processes.

Mapping a Dynamic Innate Immunity Protein Interaction Network Regulating Type I Interferon Production

To systematically investigate innate immune signaling networks regulating production of type I interferon, we analyzed protein complexes formed after microbial recognition. Fifty-eight baits were associated with 260 interacting proteins forming a human innate immunity interactome for type I interferon (HI5) of 401 unique interactions; 21% of interactions were modulated by RNA, DNA, or LPS. Overexpression and depletion analyses identified 22 unique genes that regulated NF-κB and ISRE reporter activity, viral replication, or virus-induced interferon production. Detailed mechanistic analysis defined a role for mind bomb (MIB) E3 ligases in K63-linked ubiquitination of TBK1, a kinase that phosphorylates IRF transcription factors controlling interferon production. Mib genes selectively controlled responses to cytosolic RNA. MIB deficiency reduced antiviral activity, establishing the role of MIB proteins as positive regulators of antiviral responses. The HI5 provides a dynamic physical and regulatory network that serves as a resource for mechanistic analysis of innate immune signaling.

West Nile Virus-Induced Interferon Production Is Mediated by the Double-Stranded RNA-Dependent Protein Kinase PKR

Cells carry a variety of molecules, referred to as pathogen recognition receptors (PRRs), which are able to sense invading pathogens. Interaction of PRRs with viral compounds instigates a signaling pathway(s), resulting in the activation of genes, including those for type I interferon (IFN), which are critical for an effective antiviral response. Here we demonstrate that the double-stranded RNA (dsRNA)-dependent protein kinase PKR, which has been shown to function as a PRR in cells treated with the dsRNA mimetic poly(I:C), serves as a PRR in West Nile virus (WNV)-infected cells. Evidence for PKR's role as a PRR was obtained from both human and murine cells. Using mouse embryonic fibroblasts (MEFs), we demonstrated that PKR gene knockout, posttranscriptional gene silencing of PKR mRNA using small interfering RNA (siRNA), and chemical inhibition of PKR function all interfered with IFN synthesis following WNV infection. In three different human cell lines, siRNA knockdown and chemical inhibition of PKR blocked WNV-induced IFN synthesis. Using the same approaches, we demonstrated that PKR was not necessary for Sendai virus-induced IFN synthesis, suggesting that PKR is particularly important for recognition of WNV infection. Taken together, our data suggest that PKR could serve as a PRR for recognition of WNV infection.

Functional Characterization of Kaposi's Sarcoma-Associated Herpesvirus ORF45 byBacterial Artificial Chromosome-Based Mutagenesis

Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an immediate-early protein. This protein is also present in virions as a tegument protein. ORF45 protein interacts with interferon regulatory factor 7 (IRF-7) and inhibits virus-induced type I interferon production by blocking activation of IRF-7. To define further the function of ORF45 and the mechanism underlying its action, we constructed an ORF45-null recombinant virus genome (BAC-stop45) by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BAC-stop45 genomes were generated. When monolayers of 293T BAC36 and 293T BAC-stop45 cells were induced with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate, no significant difference was found between them in overall viral gene expression and lytic DNA replication, but induced 293T BAC-stop45 cells released 10-fold fewer virions to the medium than did 293T BAC36 cells. When ORF45-null virus was used to infect cells, lower infectivity was observed than for wild-type BAC36. These results suggest that KSHV ORF45 plays roles in both early and late stages of viral infection, probably in viral ingress and egress.

RIG-I: an essential regulator of virus-induced interferon production

Intracellular double-stranded RNA (dsRNA) is a chief sign of replication for many viruses. Host mechanisms detect the dsRNA and initiate antiviral responses. In this report, we identify retinoic acid inducible gene I (RIG-I), which encodes a DExD/H box RNA helicase that contains a caspase recruitment domain, as an essential regulator for dsRNA-induced signalling, as assessed by functional screening and assays. A helicase domain with intact ATPase activity was responsible for the dsRNA-mediated signalling. The caspase recruitment domain transmitted ‘downstream’ signals, resulting in the activation of transcription factors NF-kappaB and IRF-3. Subsequent gene activation by these factors induced antiviral functions, including type I interferon production. Thus, RIG-I is key in the detection and subsequent eradication of the replicating viral genomes. [Abstract reproduced by permission of Nat Immunol 2004;5:730–7]

Decreased interferon-alpha production in HIV-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors.

Peripheral blood mononuclear cells from patients with human immunodeficiency virus (HIV) infection exhibit a progressively marked decrease in the production of virus-induced interferon (IFN)-alpha, a finding that correlates with and is highly predictive of disease progression and opportunistic infections. The major IFN-alpha producing population has recently been defined as the precursor to type 2 dendritic cells (pDC2) or plasmacytoid DC (pDC). Using four-color flow cytometry, we have enumerated the pDC2 vs non-IFN-alpha producing myeloid DC1 in peripheral blood from HIV-infected patients and healthy controls and related these values to CD4 cell numbers, viral load, and functional activity. The patients had reductions in the numbers of both pDC2 (lin-/HLA-DR+/CD123(bright)) and DC1 (lin1-/HLA-DR+/CD123(dim)/CD11c+), both at an absolute level and as a percentage of cells. The decreases were most evident in patients with decreased CD4 levels. Viral load correlated with the functional frequency of the IFN producing cells but not with absolute pDC2 levels. Using intracellular flow cytometric analysis for IFN-alpha, the patients were demonstrated to have fewer pDC2, as well as a lower percentage of responding cells among those remaining. We conclude that deficient production of IFN-alpha by pDC2 from HIV-infected patients results from both selective loss of these cells and their qualitative dysfunction. Given the central role of DC, and in particular, DC2, in linking innate and adaptive immune responses, these qualitative and quantitative changes in pDC2 are likely to be key contributors to HIV pathogenesis.

Lowered yields of virus-induced interferon production in leukocyte cultures and risk of recurrent respiratory infections in children.

Objective: To study the correlation between the yield of virus-induced interferon (IFN) production in leukocyte cultures and the risk of recurrent respiratory infections. Methods: A sample of 71 consecutive children enrolled in the Finnish Otitis Media Cohort Study were selected. Children suffering from frequently recurring respiratory infections (FRRIs) were defined as the highest quintile of the entire cohort of 329 children, as regards the number of upper respiratory infections (URIs) and/or episodes of acute otitis media (AOM) during the follow-up period from 2 to 24 months. Results: In the sample of 71 children, there were 18 children with FRRI (≥9 URI and/or ≥4 AOM). Leukocyte cultures, prepared from blood drawn from these 18 children at 6 months of age, produced lower yields of IFN than those of the remaining 53 children, when stimulated with adenovirus (P<0.001), coronavirus (P<0.001) or rhinovirus (P=0.002). The difference in IFN yields was even greater (P<0.001 with all three viruses) if the comparison was made between children with FRRI and those with no or maximally one URI during the follow-up period. When the IFN production capacity induced by rhinovirus was measured at the age of 24 months, a statistically significant difference between the children with FRRI and the others was also seen (P=0.002). Influenza A virus-induced IFN production capacity did not differ between the groups at either age (P=0.209). Conclusions: Lowered IFN responses in children suffering from recurrent URIs and/or AOM may, in a subgroup of the children, be due to a genetic property of the child. However, because of the great interindividual variations, we cannot use the IFN production capacity as such for prediction of forthcoming respiratory infections and/or otitis media.

Inhibition of reovirus-stimulated murine natural killer cell cytotoxicity by cyclosporine.

Reovirus type 3 is a double-stranded RNA virus that is a potent inducer of murine natural killer (NK) cell cytotoxicity, most likely as a result of virus-induced interferon production. We determined that reovirus was an effective inducer of high levels of NK cytotoxicity. A single injection of cyclosporine (CS), administered concurrently with reovirus or delayed until three days after injecting CS, significantly inhibited NK cytotoxicity. CS significantly suppressed reovirus-induced NK cytotoxicity when added directly to the chromium-release assay. We determined that CS inhibited the ability of murine spleen cells to form conjugates with YAC-1 tumor target cells. Finally, CS was shown to directly inhibit reovirus replication in vitro. Our results demonstrate that CS is an effective inhibitor of reovirus-induced NK cytotoxicity and suggest that inhibition occurs through multiple mechanisms including direct effects on the NK cells and direct inhibition of virus replication.

Virus-induced interferon production in leukocyte cultures from children with recurrent respiratory infections. A follow-up study

Background: Lowered yields of virus-induced interferon (IFN) by leukocyte cultures were previously suggested to be associated with recurrent respiratory infections in children (Pitkäranta et al. (1993) Clin. Diagn. Virol. 1, 101–108) Objectives: To investigate if the observed lowered IFN producing capacity was secondary to the underlying disease and, consequently, would be normalized after recovery of the child from the chain of infections. Study design: Forty-eight 3–12-year-old children suffering from recurrent upper respiratory tract infections (acute otitis media included) were followed-up for 2 years. Their clinical condition and virus-induced interferon production in cultures of peripheral blood leukocytes were examined at the beginning and end of this period. Results: In 24 children the health improved strikingly during the follow-up, in 12 children a mild improvement took place, while 12 children remained constantly ill. IFN yields in cultures stimulated with corona- and respiratory syncytical viruses improved along with the clinical situation of the children. Parallel cultures induced with adeno-, influenza A or rhinoviruses did not show a similar correlation. Conclusion: These results suggest that the relationship between interferon production by leukocyte cultures and recurrent infections is complex and may be virus-specific.

Virus-Induced Interferon Production in Human Leukocytes: A Low Responder to One Virus Can Be a High Responder to Another Virus

We measured the amounts of interferon (IFN) induced by four wild-type strains and two attenuated vaccine strains of poliovirus, testing each virus in 12-58 separate batches of human peripheral blood leukocytes. There were big and consistent differences in the mean amounts of IFN formed by leukocytes from different donors in response to all these polioviruses. Almost invariably, the wild-type polioviruses gave rise to more IFN than the corresponding attenuated strains, but overall, the amounts induced by one strain of poliovirus were proportional to those induced by another. There were indications of similar correlations with six different influenza viruses and with three strains of herpes simplex virus. In contrast, there was no correlation between the IFN yields induced with a poliovirus, three other enteroviruses, and various other viruses. These results suggest that the leukocytes from a given individual have a capacity to form IFN in vitro that is determined both by the genetic make-up of that individual and the particular virus concerned. If the same phenomenon applies to IFN production by cells in vivo, it may have a role in pathogenesis of viral infections.

The Role of the Various Human Peripheral Blood Mononuclear Cells in the Production of Sendai Virus-Induced Interferon: IFN mRNA Studies

We have studied the relative contribution made to the production of interferon (IFN) in vitro in response to Sendai virus by the cells of different types present in human peripheral blood, with particular emphasis on the amounts of poly(A) plus RNA extractable from each subpopulation and its content of IFN mRNA. Peripheral blood cells were fractionated by conventional techniques, and the amounts of IFN made after induction with Sendai virus were measured. The proportion of IFN-producing cells in the various fractions was determined by immunofluorescent staining. Poly(A) plus RNA was extracted from each population and the content of IFN mRNA determined by microinjection into Xenopus laevis oocytes. Information obtained in these three ways was essentially concordant, and showed that monocytes and E rosette-negative lymphocytes predominantly contribute to IFN production.

Herpes simplex virus-induced immunespecific interferon production by cryopreserved human peripheral blood mononuclear leukocytes

Peripheral blood mononuclear leukocytes (PBML) from 21 human donors seropositive for herpes simplex virus (HSV) type 1, and from seven seronegative donors were employed to produce interferon (IFN) in response to stimulation by HSV and phytohemagglutinin (PHA). Cryopreserved PBML produced titers of IFN, including immunespecific IFN, that were not significantly different from titers produced by freshly prepared PBML. In addition, cryopreserved PBML stored under conditions designed to stimulate inter-institutional exchange of specimens produced quantities of IFN that did not vary significantly from quantities of IFN produced by cryopreserved cells that were stored under conventional conditions. Production of immunespecific IFN by cryopreserved cells represents an example of a measurement of cell-mediated immunity that is analogous in reliability and flexibility to currently available measurements of humoral immune responses.

Effects of cimetidine on various biological activities of human leucocyte interferon and on interferon production in lymphocytes.

In vitro effects of cimetidine on the antiviral, the antiproliferative and the natural killer (NK) cell stimulating activities of human leucocyte interferon (IFN-alpha (Le], and on the Sendai virus induced interferon production in lymphocytes were investigated. The antiviral activity of IFN-alpha (Le) was dosedependently enhanced by cimetidine, which alone did not demonstrate any antiviral action. The observed titer of the same IFN preparation titrated with 60 micrograms/ml of cimetidine was significantly higher (p less than 0.05) than that without cimetidine. Cimetidine slightly but dosedependently inhibited the growth of Daudi or G-361 cells. The combination effects of these two agents were revealed to be statistically additive or synergistic. The NK cell activity of peripheral blood lymphocytes (PBL) was suppressed after a 16 hr pretreatment with cimetidine. Furthermore, the interferon production in normal lymphocyte cultures was suppressed dosedependently by cimetidine. On the other hand, the augmentation of the NK cell activity by interferon was further potentiated by cimetidine.

Treatment of human peripheral blood leukocytes with proteases does not affect Sendai virus-induced interferon production.

Trypsinization of human peripheral blood leukocytes was found to have no effect on Sendai virus adsorption or on interferon induction by the virus. alpha-Chymotrypsin and papain also did not affect interferon induction, although the three proteases did remove part of the leukocyte surface material. In contrast, treatment of leukocytes with neuraminidase reduced virus adsorption and thoroughly abolished interferon induction. We conclude that protease-resistant structures on leukocyte surfaces serve as the receptor of Sendai virus for the induction of interferon.