Human cytomegalovirus tegument protein pUL83 inhibits IFI16-mediated DNA sensing for immune evasion.
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ABSTRACT:
SUBMITTER: Li T
PROVIDER: S-EPMC3876934 | biostudies-literature | 2013 Nov
REPOSITORIES: biostudies-literature
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Project description:The formation of multimerized protein assemblies has emerged as a core component of immune signal amplification, yet the biochemical basis of this phenomenon remains unclear for many mammalian proteins within host defense pathways. The interferon-inducible protein 16 (IFI16) is a viral DNA sensor that oligomerizes upon binding to nuclear viral DNA and induces downstream antiviral responses. Here, we identify the pyrin domain (PYD) residues that mediate IFI16 oligomerization in a charge-dependent manner. Based on structure modeling, these residues are predicted to be surface exposed within distinct α-helices. By generating oligomerization-deficient mutants, we demonstrate that IFI16 homotypic clustering is necessary for its assembly onto parental viral genomes at the nuclear periphery upon herpes simplex virus 1 (HSV-1) infection. Preventing oligomerization severely hampered the capacity of IFI16 to induce antiviral cytokine expression, suppress viral protein levels, and restrict viral progeny production. Restoring oligomerization via residue-specific charge mimics partially rescued IFI16 antiviral roles. We show that pyrin domains from PYHIN proteins are functionally interchangeable, facilitating cooperative assembly with the IFI16 HINs, highlighting an inherent role for pyrin domains in antiviral response. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10 bodies. We further discover PAF1C as an HSV-1 restriction factor. Altogether, our study uncovers intrinsic properties that govern IFI16 oligomerization, which serves as a signal amplification platform to activate innate immune responses and to recruit transcriptional regulatory proteins that suppress HSV-1 replication.IMPORTANCE The ability of mammalian cells to detect the genomes of nuclear-replicating viruses via cellular DNA sensors is fundamental to innate immunity. Recently, mounting evidence is supporting the universal role of polymerization in these host defense factors as a signal amplification strategy. Yet, what has remained unclear are the intrinsic properties that govern their immune signal transmission. Here, we uncover the biochemical basis for oligomerization of the nuclear DNA sensor, IFI16. Upon infection with herpes simplex virus 1 (HSV-1) in human fibroblasts, we characterize the contribution of IFI16 oligomerization to downstream protein interactions and antiviral functions, including cytokine induction and suppression of HSV-1 replication. Until now, the global characterization of oligomerization-dependent protein interactions for an immune receptor has never been explored. Our integrative quantitative proteomics, molecular CRISPR/Cas9-based assays, mutational analyses, and confocal microscopy shed light on the dynamics of immune signaling cascades activated against pathogens.
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Project description:Experiment Description RNA sequencing was performed on Candida albicans wild type cells (SC5314) grown to exponential phase on YNB Lactate, YNB Glucose, or YNB Glucose plus Lactate, and compared to exponential Candida albicans crz1 cells grown on YNB Glucose or YNB Glucose plus Lactate. Three independent experiments were performed.
2016-10-03 | E-MTAB-4579 | biostudies-arrayexpress
Project description:Cytomegaloviruses (CMVs), members of the herpesvirus family, have evolved a variety of mechanisms to evade the immune response to survive in infected hosts and to establish latent infection. They effectively hide infected cells from the effector mechanisms of adaptive immunity by eliminating cellular proteins (major histocompatibility Class I and Class II molecules) from the cell surface that display viral antigens to CD8 and CD4 T lymphocytes. CMVs also successfully escape recognition and elimination of infected cells by natural killer (NK) cells, effector cells of innate immunity, either by mimicking NK cell inhibitory ligands or by downregulating NK cell-activating ligands. To accomplish these immunoevasion functions, CMVs encode several proteins that function in the biosynthetic pathway by inhibiting the assembly and trafficking of cellular proteins that participate in immune recognition and thereby, block their appearance at the cell surface. However, elimination of these proteins from the cell surface can also be achieved by perturbation of their endosomal route and subsequent relocation from the cell surface into intracellular compartments. Namely, the physiological route of every cellular protein, including immune recognition molecules, is characterized by specific features that determine its residence time at the cell surface. In this review, we summarize the current understanding of endocytic trafficking of immune recognition molecules and perturbations of the endosomal system during infection with CMVs and other members of the herpesvirus family that contribute to their immune evasion mechanisms.
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Project description:Detection of pathogenic nucleic acids is essential for mammalian innate immunity. IFN-inducible protein IFI16 has emerged as a critical sensor for detecting pathogenic DNA, stimulating both type I IFN and proinflammatory responses. Despite being predominantly nuclear, IFI16 can unexpectedly sense pathogenic DNA in both the cytoplasm and the nucleus. However, the mechanisms regulating its localization and sensing ability remain uncharacterized. Here, we propose a two-signal model for IFI16 sensing. We first identify an evolutionarily conserved multipartite nuclear localization signal (NLS). Next, using FISH and immunopurification, we demonstrate that IFI16 detects HSV-1 DNA primarily in the nucleus, requiring a functional NLS. Furthermore, we establish a localization-dependent IFN-? induction mediated by IFI16 in response to HSV-1 infection or viral DNA transfection. To identify mechanisms regulating the secondary cytoplasmic localization, we explored IFI16 posttranslation modifications. Combinatorial MS analyses identified numerous acetylations and phosphorylations on endogenous IFI16 in lymphocytes, in which we demonstrate an IFI16-mediated IFN-? response. Importantly, the IFI16 NLS was acetylated in lymphocytes, as well as in macrophages. Mutagenesis and nuclear import assays showed that NLS acetylations promote cytoplasmic localization by inhibiting nuclear import. Additionally, broad-spectrum deacetylase inhibition triggered accumulation of cytoplasmic IFI16, and we identify the acetyltransferase p300 as a regulator of IFI16 localization. Collectively, these studies establish acetylation as a molecular toggle of IFI16 distribution, providing a simple and elegant mechanism by which this versatile sensor detects pathogenic DNA in a localization-dependent manner.
| S-EPMC3387042 | biostudies-literature
Project description:The rapid activation of pattern recognition receptor (PRR)-mediated type I interferon (IFN) signaling is crucial for the host response to infection. In turn, human cytomegalovirus (HCMV) must evade this potent response to establish life-long infection. Here, we reveal that the HCMV tegument protein UL35 antagonizes the activation of type I IFN transcription downstream of the DNA and RNA sensors cGAS and RIG-I, respectively. We show that ectopic expression of UL35 diminishes the type I IFN response, while infection with a recombinant HCMV lacking UL35 induces an elevated type I IFN response compared to wildtype HCMV. With a series of luciferase reporter assays and the analysis of signaling kinetics upon HCMV infection, we observed that UL35 downmodulates PRR signaling at the level of the key signaling factor TANK-binding kinase 1 (TBK1). Finally, we demonstrate that UL35 and TBK1 co-immunoprecipitate when co-expressed in HEK293T cells. In addition, we show that a previously reported cellular binding partner of UL35, O-GlcNAc transferase (OGT), post-translationally GlcNAcylates UL35, but that this modification is not required for the antagonizing effect of UL35 on PRR signaling. In summary, we have identified UL35 as the first HCMV protein to antagonize the type I IFN response at the level of TBK1, thereby enriching our understanding of how this important herpesvirus escapes host immune responses.
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Project description:Cytomegaloviruses (CMVs) are ubiquitous pathogens known to employ numerous immunoevasive strategies that significantly impair the ability of the immune system to eliminate the infected cells. Here, we report that the single mouse CMV (MCMV) protein, m154, downregulates multiple surface molecules involved in the activation and costimulation of the immune cells. We demonstrate that m154 uses its cytoplasmic tail motif, DD, to interfere with the adaptor protein-1 (AP-1) complex, implicated in intracellular protein sorting and packaging. As a consequence of the perturbed AP-1 sorting, m154 promotes lysosomal degradation of several proteins involved in T cell costimulation, thus impairing virus-specific CD8+ T cell response and virus control in vivo. Additionally, we show that HCMV infection similarly interferes with the AP-1 complex. Altogether, we identify the robust mechanism employed by single viral immunomodulatory protein targeting a broad spectrum of cell surface molecules involved in the antiviral immune response.
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Project description:Human cytomegalovirus (HCMV) tegument protein pp150 is essential for the completion of the final steps in virion maturation. Earlier studies indicated that three pp150nt (N-terminal one-third of pp150) conformers cluster on each triplex (Tri1, Tri2A and Tri2B), and extend towards small capsid proteins atop nearby major capsid proteins, forming a net-like layer of tegument densities that enmesh and stabilize HCMV capsids. Based on this atomic detail, we designed several peptides targeting pp150nt. Our data show significant reduction in virus growth upon treatment with one of these peptides (pep-CR2) with an IC50 of 1.33 μM and no significant impact on cell viability. Based on 3D modeling, pep-CR2 specifically interferes with the pp150-capsid binding interface. Cells pre-treated with pep-CR2 and infected with HCMV sequester pp150 in the nucleus, indicating a mechanistic disruption of pp150 loading onto capsids and subsequent nuclear egress. Furthermore, pep-CR2 effectively inhibits mouse cytomegalovirus (MCMV) infection in cell culture, paving the way for future animal testing. Combined, these results indicate that CR2 of pp150 is amenable to targeting by a peptide inhibitor, and can be developed into an effective antiviral.
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Project description:UnlabelledHuman cytomegalovirus (HCMV) is an enveloped double-stranded DNA virus that causes severe disease in newborns and immunocompromised patients. During infection, the host cell endosecretory system is remodeled to form the cytoplasmic virion assembly complex (cVAC). We and others previously identified the conserved, multifunctional HCMV virion tegument protein pUL103 as important for cVAC biogenesis and efficient secondary envelopment. To help define its mechanisms of action and predict additional functions, we used two complementary methods, coimmunoprecipitation (co-IP) and proximity biotinylation (BioID), to identify viral and cellular proteins that interact with pUL103. By using the two methods in parallel and applying stringent selection criteria, we identified potentially high-value interactions of pUL103 with 13 HCMV and 18 cellular proteins. Detection of the previously identified pUL103-pUL71 interaction, as well as verification of several interactions by reverse co-IP, supports the specificity of our screening process. As might be expected for a tegument protein, interactions were identified that suggest distinct roles for pUL103 across the arc of lytic infection, including interactions with proteins involved in cellular antiviral responses, nuclear activities, and biogenesis and transport of cytoplasmic vesicles. Further analysis of some of these interactions expands our understanding of the multifunctional repertoire of pUL103: we detected HCMV pUL103 in nuclei of infected cells and identified an ALIX-binding domain within the pUL103 sequence.ImportanceHuman cytomegalovirus (HCMV) is able to reconfigure the host cell machinery to establish a virion production factory, the cytoplasmic virion assembly complex (cVAC). cVAC biogenesis and operation represent targets for development of novel HCMV antivirals. We previously showed that the HCMV tegument protein pUL103 is required for cVAC biogenesis. Using pUL103 as bait, we investigated viral and cellular protein-protein interactions to identify and understand the range of pUL103 functions. We found that pUL103 interacts with cellular antiviral defense systems and proteins involved in organelle biogenesis and transport of cytoplasmic vesicles and is present in infected cell nuclei. These results expand our understanding of the functional repertoire of pUL103 to include activities that extend from the earliest stages of infection through virion assembly and egress.
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Project description:The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease.
| S-EPMC4981668 | biostudies-literature
Project description:Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells.
| S-EPMC7872266 | biostudies-literature