PLP2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type I interferon production

Infections by coronaviruses such as severe acute respiratory syndrome (SARS) coronavirus (SCoV) and mouse hepatitis virus A59 (MHV-A59) result in very little type I interferon (IFN) production by host cells, which is potentially responsible for the rapid viral growth and severe immunopathology associated with SARS. However, the molecular mechanisms for the low IFN production in cells infected with coronaviruses remain unclear. Here, we provide evidence that Papain-like protease domain 2 (PLP2), a catalytic domain of the nonstructural protein 3 (nsp3) of MHV-A59, can bind to IRF3, cause its deubiquitination and prevent its nuclear translocation. As a consequence, co-expression of PLP2 strongly inhibits CARDIF-, TBK1- and IRF3-mediated IFNbeta reporter activities. In addition, we show that wild-type PLP2 but not the mutant PLP2 lacking the deubiquitinase (DUB) activity can reduce IFN induction and promote viral growth in cells infected with VSV. Thus, our study uncovered a viral DUB which coronaviruses may use to escape from the host innate antiviral responses.     

Ubiquitin-specific peptidase 20 targets TRAF6 and human T cell leukemia virus type 1 tax to negatively regulate NF-kappaB signaling

NF-κB plays a key role in innate and acquired immunity. Its activity is regulated through intricate signaling networks. Persistent or excessive activation of NF-κB induces diseases, such as autoimmune disorders and malignant neoplasms. Infection by human T cell leukemia virus type 1 (HTLV-1) causes a fatal hematopoietic malignancy termed adult T cell leukemia (ATL). The HTLV-1 viral oncoprotein Tax functions pivotally in leukemogenesis through its potent activation of NF-κB. Recent findings suggest that protein ubiquitination is crucial for proper regulation of NF-κB signaling and for Tax activity. Here, we report that ubiquitin-specific peptidase USP20 deubiquitinates TRAF6 and Tax and suppresses interleukin 1β (IL-1β)- and Tax-induced NF-κB activation. Our results point to USP20 as a key negative regulator of Tax-induced NF-κB signaling.     

Effects of interleukin 17A (IL-17A) neutralization on murine hepatitis virus (MHV-A59) infection

Mice infected with mouse hepatitis virus A59 (MHV-A59) develop hepatitis and autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH), a fact closely related to the release of alarmins such as uric acid and/or high-mobility group box protein 1 (HMGB1). We studied the effect of neutralizing monoclonal antibodies (MAb) against IL-17A in our model of mouse MHV-A59-infection. MAb anti-IL-17F and anti-IFNγ were used to complement the study. Results showed that transaminase levels markedly decreased in MHV-A59-infected mice treated with MAb anti-IL-17A whereas plasmatic Ig concentration sharply increased. Conversely, MAb anti-IL-17F enhanced transaminase liberation and did not affect Ig levels. Serum IFNγ was detected in mice infected with MHV-A59 and its concentration increased after MAb anti-IL-17A administration. Besides, MAb anti-IFNγ greatly augmented transaminase plasmatic levels. IL-17A neutralization did not affect MHV-A59-induction of HMGB1 liberation and slightly augmented plasmatic uric acid concentration. However, mice treated with the MAb failed to produce autoAb to FAH. The above results suggest a reciprocal regulation of Th1 and Th17 cells acting on the different MHV-A59 effects. In addition, it is proposed that IL-17A is involved in alarmins adjuvant effects leading to autoAb expression.     

Mouse hepatitis coronavirus A59 nucleocapsid protein is a type I interferon antagonist

The recent emergence of several new coronaviruses, including the etiological cause of severe acute respiratory syndrome, has significantly increased the importance of understanding virus-host cell interactions of this virus family. We used mouse hepatitis virus (MHV) A59 as a model to gain insight into how coronaviruses affect the type I alpha/beta interferon (IFN) system. We demonstrate that MHV is resistant to type I IFN. Protein kinase R (PKR) and the alpha subunit of eukaryotic translation initiation factor are not phosphorylated in infected cells. The RNase L activity associated with 2',5'-oligoadenylate synthetase is not activated or is blocked, since cellular RNA is not degraded. These results are consistent with lack of protein translation shutoff early following infection. We used a well-established recombinant vaccinia virus (VV)-based expression system that lacks the viral IFN antagonist E3L to screen viral genes for their ability to rescue the IFN sensitivity of the mutant. The nucleocapsid (N) gene rescued VVDeltaE3L from IFN sensitivity. N gene expression prevents cellular RNA degradation and partially rescues the dramatic translation shutoff characteristic of the VVDeltaE3L virus. However, it does not prevent PKR phosphorylation. The results indicate that the MHV N protein is a type I IFN antagonist that likely plays a role in circumventing the innate immune response.     

口蹄疫病毒结构蛋白VP0抑制I型干扰素信号通路的激活

[目的]研究口蹄疫病毒（FMDV）结构蛋白VP0对I型干扰素信号通路的影响。[方法]通过反转录PCR构建VP0真核表达载体,利用Western blotting验证VP0蛋白转染HEK-293T细胞后的表达情况;Real-time PCR检测VP0蛋白对FMDV在BHK细胞上复制的影响,检测VP0蛋白对SeV诱导的干扰素信号通路分子RIG-I、IRF3、IFN-β及下游刺激基因ISG15、ISG20表达的影响;双荧光素酶报告基因检测系统检测VP0蛋白对SeV诱导的IFN-β和NF-κB启动子激活以及对RIG-I样受体（RIG-I-like receptors,RLRs）信号通路分子激活IFN启动子的影响;免疫共沉淀检测VP0蛋白与RLRs信号通路中关键分子的相互作用。[结果]成功构建了pCAGGs-VP0真核表达载体,可以在HEK-293T细胞中表达;FMDV感染后的4-6 h,VP0蛋白显著促进FMDV在BHK细胞上的复制（P<0.01或P<0.05）;VP0蛋白明显抑制干扰素下游刺激基因的表达（P<0.01或P<0.05）。在双荧光素酶报告基因检测实验中,VP0蛋白抑制SeV诱导IFN-β和NF-κB的活化具有剂量依赖性（P<0.01）,并对RIG-I、MDA5、VISA、TBK1和IRF3介导的IFN-β产生具有抑制作用,但是对IRF7没有明显的影响。免疫共沉淀显示VP0蛋白可与IRF3发生相互作用。[结论]证实VP0蛋白可以通过与IRF3相互作用来抑制I型干扰素信号通路的激活。     

PCV2 targets cGAS to inhibit type I interferon induction to promote other DNA virus infection

Viruses use diverse strategies to impair the antiviral immunity of host in order to promote infection and pathogenesis. Herein, we found that PCV2 infection promotes the infection of DNA viruses through inhibiting IFN-β induction in vivo and in vitro. In the early phase of infection, PCV2 promotes the phosphorylation of cGAS at S278 via activation of PI3K/Akt signaling, which directly silences the catalytic activity of cGAS. Subsequently, phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS at K389, which can been served as a signal for recognizing by the ubiquitin-binding domain of histone deacetylase 6 (HDAC6), to promote the translocation of K48-ubiquitinated-cGAS from cytosol to autolysosome depending on the deacetylase activity of HDAC6, thereby eventually resulting in a markedly increased cGAS degradation in PCV2 infection-induced autophagic cells relative to Earle’s Balanced Salt Solution (EBSS)-induced autophagic cells (a typical starving autophagy). Importantly, we found that PCV2 Cap and its binding protein gC1qR act as predominant regulators to promote porcine cGAS phosphorylation and HDAC6 activation through mediating PI3K/AKT signaling and PKCδ signaling activation. Based on this finding, gC1qR-binding activity deficient PCV2 mutant (PCV2RmA) indeed shows a weakened inhibitory effect on IFN-β induction and a weaker boost effect for other DNA viruses infection compared to wild-type PCV2. Collectively, our findings illuminate a systematic regulation mechanism by which porcine circovirus counteracts the cGAS-STING signaling pathway to inhibit the type I interferon induction and promote DNA virus infection, and identify gC1qR as an important regulator for the immunosuppression induced by PCV2.     

Suppressor of cytokine signaling (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha receptor (IFNAR1)-associated tyrosine kinase Tyk2

Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN α receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2.     

Regulation of virus-triggered type I interferon signaling by cellular and viral proteins

Host pattern recognition receptors (PRRs) recognize invading viral pathogens and initiate a series of signaling cascades that lead to the expression of type I interferons (IFNs) and inflammatory cytokines. During the past decade, significant progresses have been made to characterize PRRs such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) and elucidate the molecular mechanisms of TLR- and RLR-mediated signaling. To avoid excessive and harmful immune effects caused by over-activation of these signaling pathways, host cells adopt a number of strategies to regulate them. In addition, invading viruses also employ a variety of mechanisms to inhibit the production of type I IFNs, thereby evading the supervision and clearance by the host. In this review, we briefly summarize the TLR- and RLR-mediated type I IFN signaling and then focus on the mechanisms by which host cellular and viral components regulate the expression of type I IFNs.     

HTLV-1 evades type I interferon antiviral signaling by inducing the suppressor of cytokine signaling 1 (SOCS1)

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of Adult T cell Leukemia (ATL) and the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the majority of HTLV-1-infected individuals remain asymptomatic carriers (AC) during their lifetime, 2-5% will develop either ATL or HAM/TSP, but never both. To better understand the gene expression changes in HTLV-1-associated diseases, we examined the mRNA profiles of CD4+ T cells isolated from 7 ATL, 12 HAM/TSP, 11 AC and 8 non-infected controls. Using genomic approaches followed by bioinformatic analysis, we identified gene expression pattern characteristic of HTLV-1 infected individuals and particular disease states. Of particular interest, the suppressor of cytokine signaling 1--SOCS1--was upregulated in HAM/TSP and AC patients but not in ATL. Moreover, SOCS1 was positively correlated with the expression of HTLV-1 mRNA in HAM/TSP patient samples. In primary PBMCs transfected with a HTLV-1 proviral clone and in HTLV-1-transformed MT-2 cells, HTLV-1 replication correlated with induction of SOCS1 and inhibition of IFN-α/β and IFN-stimulated gene expression. Targeting SOCS1 with siRNA restored type I IFN production and reduced HTLV-1 replication in MT-2 cells. Conversely, exogenous expression of SOCS1 resulted in enhanced HTLV-1 mRNA synthesis. In addition to inhibiting signaling downstream of the IFN receptor, SOCS1 inhibited IFN-β production by targeting IRF3 for ubiquitination and proteasomal degradation. These observations identify a novel SOCS1 driven mechanism of evasion of the type I IFN antiviral response against HTLV-1.     

The late lytic LMP-1 protein of Epstein-Barr virus can negatively regulate LMP-1 signaling

The BNLF-1 open reading frame of Epstein-Barr virus (EBV) encodes two related proteins, latent membrane protein-1 (LMP-1) and lytic LMP-1 (lyLMP-1). LMP-1 is a latent protein required for immortalization of human B cells by EBV, whereas lyLMP-1 is expressed during the lytic cycle and is found in the EBV virion. We show here that, in contrast to LMP-1, lyLMP-1 is stable, with a half-life of >20 h in tetradecanoyl phorbol acetate- and butyrate-treated B95-8 cells. Although lyLMP-1 itself has negligible effects on NF-kappaB activity, it inhibits NF-kappaB activation by LMP-1 in a dose-dependent manner. The lyLMP-1 protein does not oligomerize with LMP-1, and the negative effect of lyLMP-1 on NF-kappaB activation by LMP-1 does not result from lyLMP-1-mediated disruption of LMP-1 oligomers. Modulation of LMP-1-activated signaling pathways is the first identified biological activity associated with lyLMP-1, and this activity may contribute to the progression of EBV's lytic cycle.     

RNA recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus A59 and fusion-negative mouse hepatitis virus 2.

It has previously been shown that the murine coronavirus mouse hepatitis virus (MHV) undergoes RNA recombination at a relatively high frequency in both tissue culture and infected animals. Thus far, all of the recombination sites had been localized at the 5' half of the RNA genome. We have now performed a cross between MHV-2, a fusion-negative murine coronavirus, and a temperature-sensitive mutant of the A59 strain of MHV, which is fusion positive at the permissive temperature. By selecting fusion-positive viruses at the nonpermissive temperature, we isolated several recombinants containing multiple crossovers in a single genome. Some of the recombinants became fusion negative during the plaque purification. The fusion ability of the recombinants parallels the presence or absence of the A59 genomic sequences encoding peplomers. Several of the recombinants have crossovers within 3' end genes which encode viral structural proteins, N and E1. These recombination sites were not specifically selected with the selection markers used. This finding, together with results of previous recombination studies, indicates that RNA recombination can occur almost anywhere from the 5' end to the 3' end along the entire genome. The data also show that the replacement of A59 genetic sequences at the 5' end of gene C, which encodes the peplomer protein, with the fusion-negative MHV-2 sequences do not affect the fusion ability of the recombinant viruses. Thus, the crucial determinant for the fusion-inducing capability appears to reside in the more carboxyl portion of the peplomer protein.     

The leader proteinase of foot-and-mouth disease virus negatively regulates the type I interferon pathway by acting as a viral deubiquitinase

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) is a papain-like proteinase that plays an important role in FMDV pathogenesis. Previously, it has been shown that L(pro) is involved in the inhibition of the type I interferon (IFN) response by FMDV. However, the underlying mechanisms remain unclear. Here we demonstrate that FMDV Lb(pro), a shorter form of L(pro), has deubiquitinating activity. Sequence alignment and structural bioinformatics analyses revealed that the catalytic residues (Cys51 and His148) are highly conserved in FMDV Lb(pro) of all seven serotypes and that the topology of FMDV Lb(pro) is remarkably similar to that of ubiquitin-specific protease 14 (USP14), a cellular deubiquitylation enzyme (DUB), and to that of severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a coronaviral DUB. Both purified Lb(pro) protein and in vivo ectopically expressed Lb(pro) removed ubiquitin (Ub) moieties from cellular substrates, acting on both lysine-48- and lysine-63-linked polyubiquitin chains. Furthermore, Lb(pro) significantly inhibited ubiquitination of retinoic acid-inducible gene I (RIG-I), TANK-binding kinase 1 (TBK1), TNF receptor-associated factor 6 (TRAF6), and TRAF3, key signaling molecules in activation of type I IFN response. Mutations in Lb(pro) that ablate the catalytic activity (C51A or D163N/D164N) or disrupt the SAP (for SAF-A/B, Acinus, and PIAS) domain (I83A/L86A) abrogated the DUB activity of Lb(pro) as well as its ability to block signaling to the IFN-β promoter. Collectively, these results demonstrate that FMDV Lb(pro) possesses DUB activity in addition to serving as a viral proteinase and describe a novel mechanism evolved by FMDV to counteract host innate antiviral responses.     

Retinoic acid-induced gene-I (RIG-I) associates with nucleotide-binding oligomerization domain-2 (NOD2) to negatively regulate inflammatory signaling

Cytoplasmic caspase recruiting domain (CARD)-containing molecules often function in the induction of potent antimicrobial responses in order to protect mammalian cells from invading pathogens. Retinoic acid-induced gene-I (RIG-I) and nucleotide binding oligomerization domain 2 (NOD2) serve as key factors in the detection of viral and bacterial pathogens, and in the subsequent initiation of innate immune signals to combat infection. RIG-I and NOD2 share striking similarities in their cellular localization, both localize to membrane ruffles in non-polarized epithelial cells and both exhibit a close association with the junctional complex of polarized epithelia. Here we show that RIG-I and NOD2 not only colocalize to cellular ruffles and cell-cell junctions, but that they also form a direct interaction that is mediated by the CARDs of RIG-I and multiple regions of NOD2. Moreover, we show that RIG-I negatively regulates ligand-induced nuclear factor-κB (NF-κB) signaling mediated by NOD2, and that NOD2 negatively regulates type I interferon induction by RIG-I. We also show that the three main Crohn disease-associated mutants of NOD2 (1007fs, R702W, G908R) form an interaction with RIG-I and negatively regulate its signaling to a greater extent than wild-type NOD2. Our results show that in addition to their role in innate immune recognition, RIG-I and NOD2 form a direct interaction at actin-enriched sites within cells and suggest that this interaction may impact RIG-I- and NOD2-dependent innate immune signaling.     

Influenza virus non-structural protein 1 (NS1) disrupts interferon signaling

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.     

Epstein-Barr virus LF2: an antagonist to type I interferon

Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway, which is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Despite its association with significant human health problems, activities of Epstein-Barr virus (EBV), a human tumor-inducing herpesvirus, to evade host IFN-mediated innate immunity have not been well characterized. To search for EBV genes that block IFN signal transduction, we carried out a screening of EBV open reading frames for their abilities to block IFN-α/β-mediated luciferase expression upon Sendai virus infection. This screening demonstrates that EBV LF2 tegument protein specifically interacts with the central inhibitory association domain of IRF7, and this interaction leads to inhibition of the dimerization of IRF7, which suppresses IFN-α production and IFN-mediated immunity. This demonstrates a novel immune evasion mechanism of EBV LF2 in blocking cellular IRF7-mediated innate immunity.     

Loss of the type I interferon pathway increases vulnerability of mice to genital herpes simplex virus 2 infection

The mouse model of genital herpes relies on medoxyprogesterone treatment of female mice to render the vaginal lumen susceptible to inoculation with herpes simplex virus 2 (HSV-2). In the present study, we report that mice deficient in the A1 chain of the type I interferon receptor (CD118(-/-)) are susceptible to HSV-2 in the absence of medroxyprogesterone preconditioning. In the absence of hormone pretreatment, 2,000 PFU of a clinical isolate of HSV-2 was sufficient to establish a productive infection in the vagina of 75% ± 17% and in the spinal cord of 71% ± 14% of CD118(-/-) mice, whereas the same dose of HSV-2 replicated to detectable levels in only 13% ± 13% of vaginal samples and 0% of spinal cord samples from wild-type mice, as determined at day 5 postinfection. The susceptibility to HSV-2 infection in the CD118(-/-) mice was associated with a significant reduction in the infiltration of HSV-specific cytotoxic T lymphocytes into the vaginal tissue, the local production of gamma interferon (IFN-γ), and the expression of T cell-recruiting chemokines CCL5, CXCL9, and CXCL10. Collectively, the results underscore the significant contribution of type I IFNs in resistance to genital HSV-2 infection.