Essay: Review: Manipulation of Type I Interferon Signaling by HIV and AIDS-Associated Viruses



Review: Manipulation of Type I Interferon Signaling by HIV and AIDS-Associated Viruses

Buyuan He*, James T. Tran* and David Jesse Sanchez

Department of Pharmaceutical Sciences

College of Pharmacy

Western University of Health Sciences

*Authors contributed equally to this work

Correspondences should be addressed to DJS at sanchezd@westernu.edu

 

Introduction

  Type I Interferons (IFN) were first described for their ability to interfere with viral infection in the 1950s.  The ability of many types of cells to induce IFN in response to viral infection, and the subsequent ability of IFN stimulation to block viral infection has given IFN an exciting place as an all-encompassing antiviral therapeutic.  However, almost seventy years of research have shown that with all its power as an antiviral therapeutic, pathogenic viruses seem to all encode countermeasures that subvert the IFN response.  In particular, chronic viral infections seem to exist in microenvironments that produce relatively high levels of IFN yet still persist.Here we focus on subversion of IFN signaling during HIV infection and AIDS.  

IFN and Clinical HIV Infection

The most effective regimen to treat against HIV is highly active antiretroviral treatment (HAART) with physicians prescribing combination antiretroviral therapy (cART) to limit replication of HIV down to undetectable levels.  IFN-I is another type of antiviral treatment that can activate the antiviral state in the host during the process of HIV invasion.  Nevertheless, there had been many controversies on whether IFN-I is an effective treatment for HIV due to its positive effect on acute viral infection and negative effect on chronic infection.  Cheng et al. showed that while cART inhibited HIV replication but failed to completely stop elevated ISG expression, implying a sustained IFN-I induction [1].  Sustained IFN-I is believed to be partially responsible for immunological exhaustion that could lead to diminished T cell function in chronic HIV infection.  To tackle this problem, they developed a monoclonal antibody to block IFN-α/β receptor (IFNAR) in humanized mice infected with HIV-1.  The anti-IFNAR1 mAb suppressed ISG expression and subsequently rescued anti–HIV-1 T cell function.  This strategy can provide a novel therapeutic approach to treat patients living with HIV-1-infection with sustained IFN-I level during cART [1].  Another study showed the elevated IFN-I signaling during chronic HIV infection is the main cause for underlying chronic inflammation, immune activation, and CD8+ T cell exhaustion [2].  They showed that the combination of ART and IFNAR blockade during chronic HIV infection increased viral inhibition and ultimate reduction in the reactivatable HIV reservoir.  The study highlights the importance of IFN-I during viral infection and supports the ideas that IFN-I may act on both sides of the table during chronic HIV infection, fueling persistent immune activation and viral dissemination [2].  

Persistent immune activation in HIV infection is linked to elevated interferon signaling.  Others have shown that there is a decrease in gene expression level of IFN-λ1, IFN-β and RANTES in HIV-1 patients after primary cells are transfected with foreign DNA compared to cells from uninfected patients.  This implies that in patients that are living with HIV-1 and that have undetectable (<50 copies/ml) viral loads, there is a lower innate responses through cytosolic DNA sensing system.  This attenuation of innate immune responses may be due to persistent immune activation [3].

HIV-specific antibodies (Abs) can also have an effect on IFN that is produced by plasmacytoid dendritic cells (pDCs).  Monoclonal Abs that interact with HIV gp120-CD4 binding suppresses the IFN response, suggesting that gp120-CD4 interaction is critical for IFN production by pDCs [4].  As a result, vaccination or other treatments that can interrupt HIV gp120 at the CD4-binding site relative to binding of other HIV envelope epitopes may have therapeutic potential in reducing immune activation.  That finding also suggests that selection of mAb, on pDC production of IFN, should be considered carefully for clinical trials because they can increase immune activation [4].  

In contrast to the negative effect of IFN-I during chronic HIV infection, stimulation by IFN-I is necessary to inhibit HIV spreading during acute infection.  The production of IFN-I is stimulated by many pattern-recognition receptors that sense HIV-1.  Activated CD4+ T-cells recognize HIV-1 infection through cytosolic DNA sensor cGAS and induce IFN-I response.  This response is regulated by two viral accessory proteins Vpr, as it increases HIV-1 sensing, and Vpu, as it suppresses cGAS-dependent IFN-I induction, in CD4+ T cells [5].  In other cases of HIV infection, T-cells were shown to have a defect in DNA signaling machinery, which result in DNA sensing that does not lead to the activation of innate response.  Subsequent lack of expression of ISGs, IFN-I, and pro-inflammatory cytokine lead to the failure to induce an antiviral state that is sufficient to suppress HIV spread from infected cells.  The data pose a question forward of why DNA sensing machinery is defected in T-cells but functional in other cell types [6].  

Of all of the interferon subtypes, IFN-α2a had been tested in many clinical trials to test its safety and effectiveness.  One clinical trial had eleven volunteers in 12 weeks of therapy with pegylated interferon alfa-2a [7].  The median plasma viral load reduce in CD4+ T-cell counts at week 12 was 0.61 log(10) copies/mL and -44 cells/µL.  This showed that IFN-α2a was tolerated and portrayed significant anti-HIV-1 activity in HIV-1 infected patients [7].  Another clinical trial with 9 participants living HIV-1 infection that were treated also treated with pegylated interferon alfa-2a [8].  A subset of ISGs (23 of 47) increased compared to the baseline by week 6, while 10 ISGs inversely correlated with responses to virus.  The results indicated the HIV virologic response only by pegylated interferon alfa-2a only includes a specific ISG subset [8].  In one other clinical test, viral suppression was detected in 9 of 20 patients who took pegylated interferon alfa-2a monotherapy for 12 weeks [9].  Patients who had a viral load of <400 copies/mL had reduced levels of integrated HIV DNA compare to those who experienced end-point failure.  The data was further supported by control of HIV replication and HIV-1 integration reduction due to pegylated interferon alfa-2a monotherapy [9].  Although IFN-I has been used to treat HIV, it is also crucial to considered which specific subtypes of IFN-I to used treat different patients with different viral infections.  One study demonstrated the increased potent anti-HIV-1 response of human IFN-α14 subtype in humanized mice compare to IFN-α2 [10].  The finding suggests that although IFN-α2 is currently approved as therapy of HBV and HCV, it is critical to determine if IFN-α2 is the most effective and safe subtype for other viruses [10].  

HIV Blocks to IFN Induction During Infection

Why is there so much inconsistency when dissecting the efficacy of IFN-I during therapy for HIV-I infection?   Although IFN-I treatment is normally essential for viral clearance, HIV-1 has evolved several mechanisms to bypass or suppress the effect of IFN-I in multiple situations.  Numerous studies have been conducted to determine the mechanisms that HIV utilizes to reduce the effectiveness of both endogenous and therapeutic IFN-α, which leads to less control over HIV infection.  HIV is able to use many of its accessory proteins to interrupt different mechanisms to suppress and evade the host immune system.  The protein Vif of HIV has been proposed to play a role for its catalysis the ubiquitination and proteasomal degradation on STAT1 and STAT3 proteins, of the JAK/STAT pathway, for degradation in monocytic cell lines, which allows for HIV-1 to block the anti-virals effects of IFN-I.  More specifically, Vif-mediated STAT1 and STAT3 inhibition and reduce IFN-α induction of ISG15 [11].  On the other hand other work has shown that Vpu and Neg are able to block the phosphorylation of Nef without degradation of STAT1 in T cell lines [12].   These proposed mechanisms show multiple mechanism that may explain the therapeutic failure of interferon on HIV infection.  

HIV Blocks to IFN Induction During Infection

HIV-1 also is able to block type I and III IFN induction in human dendritic cells and macrophages.  To do this, HIV-1 specifically inhibits the phosphorylation of TANK-binding kinase 1 (TBK1).  Deletion of Vpr and Vif, two viral proteins, from the viral genome lead to detectable IFN-I induction.  Vpr and Vif were shown to bind to TBK1 and disrupt the process of TBK1 trans-autophosphorylation, subsequent IRF3 phosphorylation, nuclear translocation, and induction of IFN-I and IFN-III gene expression (Harman et al.).  

Vpu proteins of HIV-1 group M strains was demonstrated to disrupt the restriction factor tetherin, which suppress virus release from infected cells.  A study introduced mutations of vpu genes of HIV-1 group M and N strains to interrupt their function to antagonize tetherin.  This decreased the ability of the Vpu protein and resulted in less virus production and release from CD4+ T-cells, from fivefold to twofold, with higher levels of IFN-I released.  This suggests the essential role of the Vpu protein in counteracting the human tetherin during viral infection and controlling IFN release (Kmiec et al.).

HIV Disruption to pDC-induced IFN

The majority of IFN released during viral infection is produced by plasmacytoid dendritic cells (pDCs).  Other viruses, such as Influenza or HSV, induce IFN-α production by pDCs in 4 hours to maximal levels.  On the other hand, IFN-α induction was delayed by 24 hours by HIV infection, and the maximal level was at least 10-fold less than other viruses.  Looking closer, SYK phosphorylation at numerous tyrosine sites was observed after the exposure to HIV and gp120.  This indicated that HIV may hijack BDCA-2 signaling pathway, which then lead to the inhibition of IFN production in pDCs (Lo et al.).  Gp120, an HIV-1 envelope protein, also plays an essential role in inhibition of IFN-α secretion in pDCs.  Gp120 was observed to interact with Toll-like receptor 9 (TLR9) in pDCs and subsequently obstruct the induction of IFN-α.  Furthermore, natural killer (NK) cells that were activated by pDCs to kill target cells, were found to portray decreased cytolytic activity after TLR9 agonist (CpG) treated pDCs were exposed to gp120 (Martinelli et al.).   

HIV Targets IFN-Induced ISGs

Another mechanism that HIV utilizes to avoid IFN-I therapy is to downregulate a number of IFN-stimulated genes (ISGs).  HIV was found to suppress multiple ISGs, including AXL, OAS1, XAF1, with a fold change greater than 1.5. This phenomenon demonstrates how the virus is still able to downregulate many antiviral ISGs transcription despite the fact that the virus replication is suppressed by IFN pretreatment (Wie et al.).  The infection of HIV on T-cells and macrophages often does not trigger the innate immune system to produce IFN-I.  One component that assist HIV to evade the innate immune system is the cytoplasmic exonuclease TREX1.  The data were shown in macrophages and CD4+ T-cells, HIV infection lead to IFN-I production when TREX1 is suppress by RNA interference (RNAi).  This suggested that TREX1 interacted and digested excess cytosolic HIV DNA that would generally stimulate IFN expression.  The results also demonstrated the signaling cascade through STING, TBK1, and IRF3 to induce IFN expression (Yan et al.).  More interestingly, in other scenario, HIV is able to use IFN-I to the virus’s advantage to further damage the host immune system.  B cell-activating factor (BAFF) expression and secretion had been observed to be upregulated, in human monocytes, which induced by HIV-1.  More specifically, HIV-1 had been shown to induce IFN-I production by plasmacytoid dendritic cells (pDCs), which result in increased production of BAFF.  High expression of BAFF often lead to B cell dysfunctions, including hypergammaglobulinemia and nonspecific B cell activation.  These findings highlight a mechanism for the enhanced BAFF levels during HIV-1 infection and the importance of pDC and monocyte crosstalk to stimulate BAFF secretion (Gomez et al.).  

Gargan et al. EBioMedicine. 2018 Apr; 30: 203–216.   Published online 2018 Mar 9. doi:  10.1016/j.ebiom.2018.03.006  PMCID: PMC5952252  PMID: 29580840 HIV-1 Promotes the Degradation of Components of the Type 1 IFN JAK/STAT Pathway and Blocks Anti-viral ISG Induction

Gomez et al. J Immunol March 1, 2015, 194 (5) 2300-2308; DOI: https://doi.org/10.4049/jimmunol.1402147 HIV-1–Triggered Release of Type I IFN by Plasmacytoid Dendritic Cells Induces BAFF Production in Monocytes

Harman et al. Journal of Virology Apr 2015, JVI.00889-15; DOI: 10.1128/JVI.00889-15. HIV Blocks Interferon Induction in Human Dendritic Cells and Macrophages by Dysregulation of TBK1

Lo et al. PLoS One. 2012; 7(5): e37052. Published online 2012 May 31. doi:  10.1371/journal.pone.0037052. HIV Delays IFN-α Production from Human Plasmacytoid Dendritic Cells and Is Associated with SYK Phosphorylation

Wie et al. J Interferon Cytokine Res. 2013 Feb; 33(2): 90–95. doi:  10.1089/jir.2012.0052. HIV Downregulates Interferon-Stimulated Genes in Primary Macrophages

Kmiec et al. mBio. 2016 Jul-Aug; 7(4): e00934-16. Published online 2016 Aug 16. doi:  10.1128/mBio.00934-16. Vpu-Mediated Counteraction of Tetherin Is a Major Determinant of HIV-1 Interferon Resistance

Gelais et al. PLoS One. 2012;7(3):e34521. doi: 10.1371/journal.pone.0034521. Epub 2012 Mar 30. HIV-1 Nef enhances dendritic cell-mediated viral transmission to CD4+ T cells and promotes T-cell activation.

Apps et al.  Cell Host Microbe. 2016 May 11;19(5):686-95. doi: 10.1016/j.chom.2016.04.005. HIV-1 Vpu Mediates HLA-C Downregulation

Martinelli et al. Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3396-401. Epub 2007 Feb 20. HIV-1 gp120 inhibits TLR9-mediated activation and IFN-α secretion in plasmacytoid dendritic cells

Yan et al. Nat Immunol. 2010 Nov; 11(11): 1005–1013. Published online 2010 Sep 26. doi:  10.1038/ni.1941. The cytosolic exonuclease TREX1 inhibits the innate immune response to HIV-1

HTLV and IFN-1

The human T-lymphotropic viruses (HTLV), type I and II, are another class of retroviruses that affect T-cells.  Usually, there is no signs or symptoms that can be observed, but some affected people may develop adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP).  Many types of treatment, including interferon, were tested to understand the pathology of this virus and to determine the right therapy for patients.  HTLV-1 encounter different types of dendritic cell (DC) that are in blood, intestinal, genital mucosa during blood or sexual transmission.  These differences can alter HTLV-1 ability to infect DCs and transfer to T-cells.  A few studies emphasized the idea that DCs are more susceptible to be infected by HTLV-1 than other cell types.  Higher proviral load in monocyte-derived dendritic cells (MDDCs) compare to lymphocytes, that got exposed to viral biofilm.  Higher expression of neuropilin-1 was also observed in MDDCs than activated T lymphocytes.  Furthermore, MDDCs could transfer virus to lymphocytes efficiently (Alais et al.).  Another study had similar results as they showed DCs exposed to HTLV-1 can efficiently induce transmission of virus to autologous primary CD4+ T-cells.  Neuropilin-1 is involved in the process of DC-mediate transfer of HTLV-1 that lead to efficient infection of CD4+ T-cells (Jones et al.).  The susceptibility of DCs to HTLV-1 infection was further examined to comprehend the mechanism of viral interaction with DCs.  DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) was found to be a critical DC antigen receptor.  DC-SIGN was showed to mediate HTLV-1 transmission from DCs to T-cells.  Increase of virus-induced interleukin-4 production and DC-SIGN expression lead to the success of HTLV-1 infection of MDDCs in blood myeloid DCs.  These data reveal the essential role of DC-SIGN in HTLV-1 infection and transmission and provide potential target for antiviral therapy development (Jain et al.).  A study demonstrated that IFN-α DCs restrict HTLV-1 infection significantly compare to monocyte-derived IL-4 DCs and TGF-β DCs despite their enhanced ability to capture HTLV-1 virions.  This was not because of IFN-I antiviral activity but related to distinct trafficking route of HTLV-1 in IFN-α DCs compare to other DCs (Rizkallah et al.).  IFN-I is one of the important effectors of the innate immune response.  IFN-I was reported by multiple research to work in a variety of ways with different mechanisms that contribute to the inhibition of HTLV-1.  HTLV-1 mRNA and proteins in HTLV-1 infected cells were demonstrated to get reduce when coculture with human epithelial-like cells (HEK293T) or mouse embryo fibroblasts (NIH 3T3).  The positive effect from these cocultures was due to IFN-I induction, which got detected by IFN-beta promoter activation and ISGs upregulation.  Furthermore, the suppression of HTLV-1, mediate by HEK293T and NIH 3T3, can also be inhibit by antibodies to human IFN-α/β receptors or mouse IFN-β.  The results suggest that the innate immune may inhibits HTLV-1 expression, in vivo, through IFN-I (Kinpara et al.).  Recently, the combination of IFN-α and zidovudine (AZT) often had shown the most therapeutic effects in ATL patients compare to others treatment.  One study found the level of HTLV-1 p19, a major core viral protein encoded by the gag gene, in the supernatant of IL-2 dependent HTLV-1 infected T-cells (ILTs) reduced in three days after IFN-α stimulation.  Moreover, the amount of intracellular Tax viral proteins, in 6 of 7 ILT lines tested, was observed to decrease after 24 hours following IFN-α stimulation.  The treatment of AZT alone did not influence HTLV-1 gene expression, NF-κB activities, and cell viability.  However, the treatment of AZT and IFN-α generate p53 signaling and promote cell apoptosis in these cells.  These data suggest that the susceptibility of HTVL-1-infected cells to IFN-I response is at the IL-2-dependent stage, and partially reveal the therapeutic effects of AZT/IFN-α in ATL (Kinpara et al.).  A clinical study measured the activity of HTLV-1 reverse transcriptase (RT) and other viral components, by quantitative real-time PCR, in samples from cultures of peripheral blood mononuclear cells (PBMCs) that got collected from 7 ATL patients before and after AZT and IFN treatment.  HTVL-1 tax/rex expression in PBMC cultures from 4 patients was variably inhibited compare to pretreatment samples.  P19 production decreased by 75% to 88% when measure from PBMC cultures supernatant from 5 patients.  And most importantly, RT activity was significantly suppressed in samples collected after AZT/IFN therapy in 5 patients (Macchi et al.).  Nevertheless, multiple researches reported that HTLV-1 utilizes many different viral proteins, with different mechanisms, to regulate IFN response.  Tax was observed to interrupt TBK1 kinase that phosphorylates IRF3 that lead to the inhibition of IFN-I production, in Jurkat or HEK293 cells (Yuen et al.).  Furthermore, Tax was shown to be recruited in cellular immunocomplexes with TANK Binding Kinase 1 (TBK1) and I kappa B kinase (IKKɛ) that lead to the phosphorylation of interferon regulatory factors that stimulate IFN-I gene expression.  IFN-β promoter activity was increased with the expression of Tax in the presence of TBK1 and IKKɛ.  This proposed a mechanism that Tax was recruited as a scaffold protein between IFN-β signaling factors and the kinase complexes, which allow TRAF3 to interact with TBK1/IKKɛ complex and activate IFN-β promoter (Diani et al.).  HTLV-1 bZIP factor (HBZ), another HTLV-1 gene, also plays an essential role in viral pathogenesis.  HBZ was shown to upregulate IRF7 induced ISRE promoter activities and IFN-α that can offset the inhibitory effect of Tax1 on IFN-α.  On the other hand, the combination of HBZ and Tax1 synergistically impede IFN and ISRE promoters’ induction that lead to IFN production.  Furthermore, HBZ was demonstrated to regulate, positively or negatively, TBK1 and IKK  activation of IRF7 and IRF3.  These results suggest that the variety of regulation from HTLV-1 on IFN response may be the cause that contribute to aberrant IFN signaling, immune evasion, and viral pathogenesis (Narulla et al.).  All these data present different views on whether type I interferon is efficiently enough to treat HTLV-1 and if HTLV-1 is able to bypass the effect of IFN-I therapy, either alone or in combination with other components.  Further research needs to be conducted to investigate and comprehend the mechanisms behind the role of both IFN-I and HTLV-1 viral proteins in order to produce the most sufficient and effective therapy for HTLV-1.

Rizkallah et al. PLoS Pathog. 2017 Apr 20;13(4):e1006353. doi: 10.1371/journal.ppat.1006353. eCollection 2017 Apr. Dendritic cell maturation, but not type I interferon exposure, restricts infection by HTLV-1, and viral transmission to T-cells.

Alais et al. J Virol. 2015 Oct;89(20):10580-90. doi: 10.1128/JVI.01799-15. Epub 2015 Aug 12. Viral Source-Independent High Susceptibility of Dendritic Cells to Human T-Cell Leukemia Virus Type 1 Infection Compared to That of T Lymphocytes.

Jones et al. Nat Med. 2008 Apr;14(4):429-36. doi: 10.1038/nm1745. Epub 2008 Mar 23. Cell-free HTLV-1 infects dendritic cells leading to transmission and transformation of CD4(+) T cells.

Jain et al. J Virol. 2009 Nov;83(21):10908-21. doi: 10.1128/JVI.01054-09. Epub 2009 Aug 19. DC-SIGN mediates cell-free infection and transmission of human T-cell lymphotropic virus type 1 by dendritic cells.

Yuen et al. J Virol. 2016 Mar 28;90(8):3902-3912. doi: 10.1128/JVI.00129-16. Print 2016 Apr. Suppression of Type I Interferon Production by Human T-Cell Leukemia Virus Type 1 Oncoprotein Tax through Inhibition of IRF3 Phosphorylation.

Kinpara et al. Retrovirology. 2013 May 20;10:52. doi: 10.1186/1742-4690-10-52. Interferon-α (IFN-α) suppresses HTLV-1 gene expression and cell cycling, while IFN-α combined with zidovudine induces p53 signaling and apoptosis in HTLV-1-infected cells.

Narulla et al. J Virol. 2017 Oct 15; 91(20): e00853-17. Published online 2017 Sep 27. Prepublished online 2017 Aug 2. doi: 10.1128/JVI.00853-17 Positive and Negative Regulation of Type I Interferons by the Human T Cell Leukemia Virus Antisense Protein HBZ

Kinpara et al. J Virol. 2009 May;83(10):5101-8. doi: 10.1128/JVI.02564-08. Epub 2009 Mar 4. Stromal cell-mediated suppression of human T-cell leukemia virus type 1 expression in vitro and in vivo by type I interferon.

Macchi et al. Blood Adv. 2017 May 9; 1(12): 748–752. Published online 2017 May 5. doi:  10.1182/bloodadvances.2016001370.  Quantification of HTLV-1 reverse transcriptase activity in ATL patients treated with zidovudine and interferon-α

Diani et al. Virology. 2015 Feb;476:92-99. doi: 10.1016/j.virol.2014.12.005. Epub 2014 Dec 19.  HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression.

HCV and IFN-1

Hepatitis C virus (HCV) is a single-stranded RNA virus belonging to Flaviviridae, which infects about 3.9 million people in United States and establish chronic infection in about 2.7 million people, while persistent infection of HCV can be the cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma[13]. IFN-I therapy has been used to treat Hepatitis C with a decrease of HCV RNA levels in serum being detected [14]. However, therapy with only interferon has limited success and here we discuss how HCV applies mechanisms to counter interferon treatment.

Several HCV proteins have been showed to inhibit interferon signaling. HCV encodes a non-structural-protein 5A (NS5A) that is shown to disrupt the function of several interferon stimulated genes(ISGs)[15]. PKR is a well-studied ISG, with activation of PKR leading to the phosphorylation of eukaryotic initiation factor 2α (eIF2α) and a subsequent block to the translation of viral mRNAs [16]. Gale MJ et al demonstrated that NS5A could down-regulate PKR by directly interacting with its protein kinase catalytic domain and thus represses PKR functions [17]. They also found that an interferon sensitivity-determining region (ISDR), as well as 26 amino acids carboxyl to the ISDR is needed for NS5A/PKR interaction [18]. Meanwhile, Sugiyama R, et al used a recombined JFH-1 virus that has HCV ISDR and proved its essential role to inhibit interferon signaling [19]. Since the proinflammatory chemokine IL-8 has been shown to interfere the interferon-mediated pathway such as a reduced 2'-5' oligoadenylate synthases(OAS) activities[20], the ability of NS5A to induce IL-8 production could be another mechanism inhibiting the antiviral activities of interferon[21]. The up-regulation of IL-8 by NS5A is also showed by Girard S, et al using microarray assays[22].

Other HCV proteins have also been shown to inhibit PKR activity. Since PKR phosphorylates eIF2α, HCV envelope 2 (E2) protein contains a sequence homologous to the eIF2α phosphorylation site. Taylor DR, et al performed an in vitro binding assay and proved E2's binding to PKR, which could be the mechanism of E2 inhibiting PKR activities[23].

Interferon activates the transcription of ISGs through JAK/STAT pathway [24], and HCV core has been widely demonstrated to regulate JAK/STAT pathway. Hosui A, et al found that in CL2 cells expressing HCV core protein, the phosphorylation of JAK1, JAK2 and STAT3 were all lower compared with mock CL2 cells, and the expression of HCV core protein does not significantly affect the expression level of these proteins[25]. Lin W, et al described HCV's ability to inhibit interferon signaling by degrading STAT1 and inhibiting its phosphorylation[26]. Also, they explained this mechanism as they discovered the N-terminal of HCV core protein's interaction with STAT1 SH2 domain[27]. Furthermore, HCV core protein is reported to up-regulate miR-93-5p, which blocks interferon signaling by directly targeting the interferon receptor IFNAR1 [28].

Besides miR-93-5p, other microRNAs also regulate interferon signaling. Since miR-373 expression is induced by HCV infection, Mukherjee A, et al found that miR-373 expression inhibits JAK and IRF9 while also blocks STAT1 phosphorylation. And in contrast, knockdown of miR-373 brings an enhancement of interferon signaling proteins and a reduction of HCV growth [29].

The activation of Ras/Raf/MEK pathway is reported to be the cause of a large proportion of cancers [30]. Zhang Q, et al addressed the correlation between Ras/Raf/MEK pathway and HCV infection. HCV infection is found to activate this pathway, while the Ras/Raf/MEK activation blocks the expression of IFNAR1/2, the phosphorylation of STAT1/2 and thus inhibits the JAK/STAT pathway which induce the ISGs [31].

Additionally, other factors also affect HCV's inhibition of interferon signaling. Recently extracellular matrix(ECM) is shown to affect interferon signaling in hepatitis cells. Kuwashiro T, et al compared HCV infected human hepatoma cells cultured on ECM-coated dishes or non-coated dishes. In cells grew on ECM-coated dishes, IFN stimulated response element(ISRE) luciferase activities were lower, while HCV-RNA and viral proteins amount were higher. Also, the antibodies targeting the cell-matrix interaction were able to restore the ISRE luciferase and reduce viral RNA/protein amount, showing ECM's role in affecting the interferon signaling [32].

KSHV and IFN-1

Kaposi's Sarcoma-associated Herpesvirus(KSHV), also known as Human Herpesviruses 8, is firstly identified in Kaposi's Sarcoma(KS) lesions. KSHV is later shown to cause lymphoproliferative disorders including Primary effusion lymphoma diseases (PEL) and Multicentric castleman's disease (MCD) [33].

KSHV genome encodes a group of genes homologous to human interferon regulatory factors(IRFs), among which vIRF1, vIRF2 and vIRF3 are known for blocking Type I IFN genes or ISGs [34]. Several studies reported that vIRF1 is able to disrupt interferon signaling by blocking the ISG promoters including ISG-15 and ISG-54 [35]. vIRF3 is previously shown to interact with IRF3, IRF5 and IRF7. For example, Wies E, et al described that vIRF3's ability to interact with IRF5 and inhibit ISG transcription through ISRE elements [36]. vIRF3 is also found to inhibit the PKR activated phosphorylation of eIF2α and PKR induced inhibition of protein synthesis, thus impairs the antiviral ability of PKR [37].

vIRF2, encoded by ORF K11 AND K11.1, is able to interfere multiple site of interferon signaling. Through ISRE luciferase assay, Fuld S, et al showed the full length of vIRF2 inhibits ISRE signaling induced by IFN-α, IL-28A, IL-29 and also IRF1[38]. During interferon signaling, STAT1 and STAT2 bind to the ISRE with IRF9 and form a complex called ISGF3, which facilitate transcription of ISGs. vIRF2 is shown to inhibit ISGF3 complex by targeting STAT1 and IRF9 [39]. Additionally, vIRF2 also interacts with PKR and blocks its auto- phosphorylation or phosphorylation of eIF2α[40].

KSHV viral IL-6 is another well-known KSHV viral homologous gene. described that vIL6 blocks the phosphorylation of Tyk2 and leads to an inhibition on the forming of ISGF3 complex [41]. RIF, encoded by KSHV ORF10, is also able to attenuate interferon signaling by similar mechanism. RIP is found to inhibit downstream signaling of IFNAR by associating with JAK1, STAT2 and Tyk2. Besides, RIF is also shown to interact with both IFNAR1 and IFNAR2 units, forming an inhibitory complex. And therefore, RIF blocks the phosphorylation of STAT1 and STAT2, impairing the form of ISGF3 complex [42].

During latency, KSHV encodes 12 pre-microRNAs, which are processed to at least 25 miRNAs [43, 44]. KSHV miRNAs including miR-K6-5, miR-K8, miR-K9 are found to repress Ser/Thr kinases and thus down-regulate STAT3 [44-46]. KSHV miRNAs are also shown to inhibit several targets associated in STAT3 signaling network, including upstream components such as IRAK1, PKCδ , EPOR and MET, or downstream components like BIRC5 and GADD45B [44]. In addition, another study showed that miR-17 could also target Jak1, down-regulating its mRNA level as well as protein level, thus impairs interferon signaling [47].

Epstein Barr virus and IFN-1

Epstein Barr virus (EBV) is another species of γ-herpesvirus, which is reported to be carried by 90% of the human adults asymptomatically and persistently [48]. In order to persist in host body for the entire life, certain strategies to escape from the immune detection is needed.

Latent membrane protein-1 (LMP-1) is a latent oncoprotein essential for EBV persistency in B cells [49], which shares many signaling intermediates with TLRs and also activates NF-κB [50]. Geiger TR & Martin JM demonstrated LMP-1's ability to interact with Tyk2 and inhibit the phosphorylation of Tyk2 and STAT2, thus blocking the activation of ISRE. And they found the interaction does not require CTAR1 or the 155 residues of the C terminus encoding CTAR2 and CTAR3. Also higher level of LMP-1 is observed in EBV infected lymphoblastoid cells cultured in interferon, suggesting LMP-1's function in resisting antiproliferative pressure [51]. However, other studies also showed LMP-1's ability to induce STAT1 expression by its C-terminal activating region 1 (CTAR-1) [52]. Moreover, the C-terminal activator regions of LMP-1 is also reported to inducing interferon [53]. This contradiction may explains LMP-1's multiple roles in maintaining cell survival but also inhibiting immune responses that are threatening to latent virus.

Latent membrane protein-2 (LMP-2) is designated as LMP2A and LMP2B. LMP2A, being a viral mimic of B-cell receptor has been described to promote viral latency and cell survival [54, 55]. In addition, in EBV infected endothelial cells, LMP2A is also shown to inhibit both STAT signaling and NF-κB signaling. Previously it has been reported that LMP2B is a negative modulator of LMP2A activities [56]. However, LMP2B is also shown to cooperate together with LMP1A to inhibit interferon signaling. Both LMP2A and LMP2B are found to inhibit IFN-induced ISRE activity by blocking JAK/STAT1 phosphorylation. And consequently they attenuate ISGs transcription, which is found to be "globally" [57].

After a screening of EBV open reading frames by Wu L, et al, the tegument protein LF2 is found to the ISRE activation induced by specifically cellular IRF7 but not IRF3. Moreover, LF2 does not affect IRF7 level but blocks the IFN signaling by binding to the central inhibitory association domain of IRF7, which causes an inhibition of the dimerization of IRF7 [58].

EBV encodes two non-polyadenylated RNAs (EBERs), among which EBER1 is found to disrupt anti-viral effects of IFN-α and IFN-γ by interacting with PKR and blocking its function [59]. EBV has also been shown to encode more than 40 microRNAs [60]. Within a cluster of 10 EBV BART miRNAs impairing interferon responses, miR-BART16 is identified to be the major inhibitor of the IFN-induced ISRE activity, while other miRNAs also contribute to this repressive impact. Furthermore, CBP is identified to be the target of miR-BART16, which consequently attenuates the antiproliferative effect of IFN-α[61].

Suppressor of cytokine signalling (SOCS) is a family of cellular proteins that inhibits cytokine signalling pathways, inhibiting interferon signaling by negative feedbacks [62]. Besides EBV viral proteins and RNAs, EBV infection also induce the activation of SOCS3, which suppress interferon signaling by blocking JAK/STAT pathway [63].