Multiple functional domains and complexes of the two nonstructural proteins of human respiratory syncytial virus contribute to interferon suppression and cellular location

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression.     

Viral RNA N6-methyladenosine modification modulates both innate and adaptive immune responses of human respiratory syncytial virus

Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in humans. A well-known challenge in the development of a live attenuated RSV vaccine is that interferon (IFN)-mediated antiviral responses are strongly suppressed by RSV nonstructural proteins which, in turn, dampens the subsequent adaptive immune responses. Here, we discovered a novel strategy to enhance innate and adaptive immunity to RSV infection. Specifically, we found that recombinant RSVs deficient in viral RNA N⁶-methyladenosine (m⁶A) and RSV grown in m⁶A methyltransferase (METTL3)-knockdown cells induce higher expression of RIG-I, bind more efficiently to RIG-I, and enhance RIG-I ubiquitination and IRF3 phosphorylation compared to wild-type virion RNA, leading to enhanced type I IFN production. Importantly, these m⁶A-deficient RSV mutants also induce a stronger IFN response in vivo, are significantly attenuated, induce higher neutralizing antibody and T cell immune responses in mice and provide complete protection against RSV challenge in cotton rats. Collectively, our results demonstrate that inhibition of RSV RNA m⁶A methylation enhances innate immune responses which in turn promote adaptive immunity.     

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.     

Differentiation of Th Subsets Inhibited by Nonstructural Proteins of Respiratory Syncytial Virus Is Mediated by Ubiquitination

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, constitutes an important risk factor for the development of subsequent asthma. However, the mechanism underlying RSV-induced asthma is poorly understood. Viral non-structural proteins NS1 and NS2 are critically required for RSV virulence; they strongly suppress IFN-mediated innate immunity of the host cells. In order to understand the effects of NS1 and NS2 on differentiation of Th subsets, we constructed lentiviral vectors of NS1 or NS2 to infect 16 HBE and analyzed the expression of HLA-DR, CD80 and CD86 and differentiation of Th1, Th2 and Th17 by Flow Cytometric Analysis and real-time PCR. The results showed that NS1 inhibited expression of HLA-DR, CD80 and CD86 and differentiation of Th1, Th2 and Th17 lymphocytes, which could be reversed by deleting elongin C binding domain. NS2 inhibited the differentiation of Th2 and Th17, which was reversed by proteasome inhibitors of PS-341. Our results indicated that NS1 inhibited the differentiation of T lymphocytes through its mono-ubiquitination to interacted proteins, while NS2 inhibited differentiation of Th2 and Th17 through ubiquitin-proteasome pathway, which may be related with the susceptibility to asthma after RSV infection.     

TRIM56 Is an Essential Component of the TLR3 Antiviral Signaling Pathway

Members of the tripartite motif (TRIM) proteins are being recognized as important regulators of host innate immunity. However, specific TRIMs that contribute to TLR3-mediated antiviral defense have not been identified. We show here that TRIM56 is a positive regulator of TLR3 signaling. Overexpression of TRIM56 substantially potentiated extracellular dsRNA-induced expression of interferon (IFN)-β and interferon-stimulated genes (ISGs), while knockdown of TRIM56 greatly impaired activation of IRF3, induction of IFN-β and ISGs, and establishment of an antiviral state by TLR3 ligand and severely compromised TLR3-mediated chemokine induction following infection by hepatitis C virus. The ability to promote TLR3 signaling was independent of the E3 ubiquitin ligase activity of TRIM56. Rather, it correlated with a physical interaction between TRIM56 and TRIF. Deletion of the C-terminal portion of TRIM56 abrogated the TRIM56-TRIF interaction as well as the augmentation of TLR3-mediated IFN response. Together, our data demonstrate TRIM56 is an essential component of the TLR3 antiviral signaling pathway and reveal a novel role for TRIM56 in innate antiviral immunity.     

Vpu mediates depletion of interferon regulatory factor 3 during HIV infection by a lysosome-dependent mechanism

HIV has evolved sophisticated mechanisms to avoid restriction by intracellular innate immune defenses that otherwise serve to control acute viral infection and virus dissemination. Innate defenses are triggered when pattern recognition receptor (PRR) proteins of the host cell engage pathogen-associated molecule patterns (PAMPs) present in viral products. Interferon regulatory factor 3 (IRF3) plays a central role in PRR signaling of innate immunity to drive the expression of type I interferon (IFN) and interferon-stimulated genes (ISGs), including a variety of HIV restriction factors, that serve to limit viral replication directly and/or program adaptive immunity. Productive infection of T cells by HIV is dependent upon the targeted proteolysis of IRF3 that occurs through a virus-directed mechanism that results in suppression of innate immune defenses. However, the mechanisms by which HIV controls innate immune signaling and IRF3 function are not defined. Here, we examined the innate immune response induced by HIV strains identified through their differential control of PRR signaling. We identified viruses that, unlike typical circulating HIV strains, lack the ability to degrade IRF3. Our studies show that IRF3 regulation maps specifically to the HIV accessory protein Vpu. We define a molecular interaction between Vpu and IRF3 that redirects IRF3 to the endolysosome for proteolytic degradation, thus allowing HIV to avoid the innate antiviral immune response. Our studies reveal that Vpu is an important IRF3 regulator that supports acute HIV infection through innate immune suppression. These observations define the Vpu-IRF3 interface as a novel target for therapeutic strategies aimed at enhancing the immune response to HIV.     

Mitochondria and antiviral innate immunity

Mitochondria, dynamic organelles that undergo continuous cycles of fusion and fission, are the powerhouses of eukaryotic cells. Recent research indicates that mitochondria also act as platforms for antiviral immunity in vertebrates. Mitochondrial-mediated antiviral immunity depends on activation of the retinoic acid-inducible gene I (RIG-I)-like receptors signal transduction pathway and the participation of the mitochondrial outer membrane adaptor protein “mitochondrial antiviral signaling (MAVS)”. Here we discuss recent findings that suggest how mitochondria contribute to antiviral innate immunity.     

Essential role of hyperacetylated microtubules in innate immunity escape orchestrated by the EBV-encoded BHRF1 protein

Innate immunity constitutes the first line of defense against viruses, in which mitochondria play an important role in the induction of the interferon (IFN) response. BHRF1, a multifunctional viral protein expressed during Epstein-Barr virus reactivation, modulates mitochondrial dynamics and disrupts the IFN signaling pathway. Mitochondria are mobile organelles that move through the cytoplasm thanks to the cytoskeleton and in particular the microtubule (MT) network. MTs undergo various post-translational modifications, among them tubulin acetylation. In this study, we demonstrated that BHRF1 induces MT hyperacetylation to escape innate immunity. Indeed, the expression of BHRF1 induces the clustering of shortened mitochondria next to the nucleus. This “mito-aggresome” is organized around the centrosome and its formation is MT-dependent. We also observed that the α-tubulin acetyltransferase ATAT1 interacts with BHRF1. Using ATAT1 knockdown or a non-acetylatable α-tubulin mutant, we demonstrated that this hyperacetylation is necessary for the mito-aggresome formation. Similar results were observed during EBV reactivation. We investigated the mechanism leading to the clustering of mitochondria, and we identified dyneins as motors that are required for mitochondrial clustering. Finally, we demonstrated that BHRF1 needs MT hyperacetylation to block the induction of the IFN response. Moreover, the loss of MT hyperacetylation blocks the localization of autophagosomes close to the mito-aggresome, impeding BHRF1 to initiate mitophagy, which is essential to inhibiting the signaling pathway. Therefore, our results reveal the role of the MT network, and its acetylation level, in the induction of a pro-viral mitophagy.     

Dengue Virus Targets the Adaptor Protein MITA to Subvert Host Innate Immunity

Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN). We found that activation of interferon regulatory factor 3 (IRF3) triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA) but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓⁹⁶G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT) stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity.     

Poxvirus protein N1L targets the I-kappaB kinase complex, inhibits signaling to NF-kappaB by the tumor necrosis factor superfamily of receptors, and inhibits NF-kappaB and IRF3 signaling by toll-like receptors

Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways.     

IFN-induced TPR protein IFIT3 potentiates antiviral signaling by bridging MAVS and TBK1

Intracellular RNA viruses are sensed by receptors retinoic acid-inducible gene I/MDA5, which trigger formation of the mitochondrial antiviral signaling (MAVS) complex on mitochondria. Consequently, this leads to the activation of TNFR-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) and phosphorylation of IFN regulatory factor 3 (IRF3). It remains to be elucidated how MAVS activates TBK1/IRF3. In this study, we report that IFN-induced protein with tetratricopeptide repeats 3 (IFIT3) is significantly induced upon RNA virus infection. Ectopic expression or knockdown of IFIT3 could, respectively, enhance or impair IRF3-mediated gene expression. Mechanistically, the tetratrico-peptide repeat motif (E164/E165) of IFIT3 interacts with the N terminus (K38) of TBK1, thus bridging TBK1 to MAVS on the mitochondrion. Disruption of this interaction markedly attenuates the activation of TBK1 and IRF3. Furthermore, host antiviral responses are significantly boosted or crippled in the presence or absence of IFIT3. Collectively, our study characterizes IFIT3 as an important modulator in innate immunity, revealing a new function of the IFIT family proteins (IFN-induced protein with tetratricopeptide repeats).     

Inhibition of interferon regulatory factor 7 (IRF7)-mediated interferon signal transduction by the Kaposi's sarcoma-associated herpesvirus viral IRF homolog vIRF3

Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway that is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate the host IFN-mediated immune response. Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologs of the cellular IRFs, called vIRFs. Here, we report a novel immune evasion mechanism of KSHV vIRF3 to block cellular IRF7-mediated innate immunity in response to viral infection. KSHV vIRF3 specifically interacts with either the DNA binding domain or the central IRF association domain of IRF7, and this interaction leads to the inhibition of IRF7 DNA binding activity and, therefore, suppression of alpha interferon (IFN-alpha) production and IFN-mediated immunity. Remarkably, the central 40 amino acids of vIRF3, containing the double alpha helix motifs, are sufficient not only for binding to IRF7, but also for inhibiting IRF7 DNA binding activity. Consequently, the expression of the double alpha helix motif-containing peptide effectively suppresses IRF7-mediated IFN-alpha production. This demonstrates a remarkably efficient means of viral avoidance of host antiviral activity.