Vaccinia Virus Protein Research Articles

Molecular mimicry of NF-κB by vaccinia virus protein enables selective inhibition of antiviral responses.

Infection of mammalian cells with viruses activates NF-κB to induce the expression of cytokines and chemokines and initiate an antiviral response. Here, we show that a vaccinia virus protein mimics the transactivation domain of the p65 subunit of NF-κB to inhibit selectively the expression of NF-κB-regulated genes. Using co-immunoprecipitation assays, we found that the vaccinia virus protein F14 associates with NF-κB co-activator CREB-binding protein (CBP) and disrupts the interaction between p65 and CBP. This abrogates CBP-mediated acetylation of p65, after which it reduces promoter recruitment of the transcriptional regulator BRD4 and diminishes stimulation of NF-κB-regulated genes CXCL10 and CCL2. Recruitment of BRD4 to the promoters of NFKBIA and CXCL8 remains unaffected by either F14 or JQ1 (a competitive inhibitor of BRD4 bromodomains), indicating that BRD4 recruitment is acetylation-independent. Unlike other viral proteins that are general antagonists of NF-κB, F14 is a selective inhibitor of NF-κB-dependent gene expression. An in vivo model of infection demonstrated that F14 promotes virulence. Molecular mimicry of NF-κB may be conserved because other orthopoxviruses, including variola, monkeypox and cowpox viruses, encode orthologues of F14.

Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis

Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to eliminate infected cells. During infection, vaccinia virus (VACV) protein E3 prevents death signaling by competing for Z-form RNA through an N-terminal Zα domain. In the absence of this E3 domain, Z-form RNA accumulates during the early phase of VACV infection, triggering ZBP1 to recruit receptor interacting protein kinase (RIPK)3 and execute necroptosis. The C-terminal E3 double-strand RNA-binding domain must be retained to observe accumulation of Z-form RNA and induction of necroptosis. Substitutions of Zα from either ZBP1 or the RNA-editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 yields fully functional E3 capable of suppressing virus-induced necroptosis. Overall, our evidence reveals the importance of Z-form RNA generated during VACV infection as a pathogen-associated molecular pattern (PAMP) unleashing ZBP1/RIPK3/MLKL-dependent necroptosis unless suppressed by viral E3.

Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection.

ABSTRACTViral infection both activates stress signaling pathways and redistributes ribosomes away from host mRNAs to translate viral mRNAs. The intricacies of this ribosome shuffle from host to viral mRNAs are poorly understood. Here, we uncover a role for the ribosome-associated quality control (RQC) factor ZNF598 during vaccinia virus mRNA translation. ZNF598 acts on collided ribosomes to ubiquitylate 40S subunit proteins uS10 (RPS20) and eS10 (RPS10), initiating RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection enhances uS10 ubiquitylation, indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells that either lack ZNF598 or express a ubiquitylation-deficient version of uS10. Using SILAC-based proteomics and concurrent RNA-seq analysis, we determine that translation, but not transcription of vaccinia virus mRNAs is compromised in cells with deficient RQC activity. Additionally, vaccinia virus infection reduces cellular RQC activity, suggesting that co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.

Vaccinia Virus Protein B18R: Influence on mRNA Immunogenicity and Translation upon Non-Viral Delivery in Different Ocular Cell Types.

In the last few years, interest has grown in the use of nucleic acids as an ocular therapy for retinal genetic diseases. Recently, our research group has demonstrated that mRNA delivery could result in effective protein expression in ocular cells following subretinal injection. Yet, although mRNA therapy comes with many advantages, its immunogenicity resulting in hampered mRNA translation delays development to the clinic. Therefore, several research groups investigate possible strategies to reduce this innate immunity. In this study, we focus on B18R, an immune inhibitor to suppress the mRNA-induced innate immune responses in two ocular cell types. We made use of retinal pigment epithelial (RPE) cells and Müller cells both as immortalized cell lines and primary bovine cells. When cells were co-incubated with both B18R and mRNA-MessengerMAX lipoplexes we observed an increase in transfection efficiency accompanied by a decrease in interferon-β production, except for the Müller cells. Moreover, uptake efficiency and cell viability were not hampered. Taken together, we showed that the effect of B18R is cell type-dependent but remains a possible strategy to improve mRNA translation in RPE cells.

NMR assignments of vaccinia virus protein A28: an entry-fusion complex component.

Vaccinia virus (VACV) belonging to the poxvirus family enters the host cell via two different entry pathways; either endocytosis or virus/host cell membrane fusion. With respect to the virus/host cell membrane fusion, there are eleven viral membrane proteins forming a complicated entry-fusion complex (EFC), including A28, A21, A16, F9, G9, G3, H2, J5, L5, L1 and O3, to conduct the fusion function. These EFC components are highly conserved in all poxviruses and each of them is essential and necessary for the fusion activity. So far, with the exceptions of L1 and F9 whose crystal structures were reported, the structural information about other EFC components remains largely unclear. We aim to conduct a structural and functional investigation of VACV virus-entry membrane protein A28. In this work, we expressed and purified a truncated form of A28 (14kDa; residues 38-146, abbreviated as tA28 hereinafter), with deletion of its transmembrane domain (residues 1-22) and a hydrophobic segment (residues 23-37). And the assignments of its backbone and side chain 1H, 13C and 15N chemical shifts of tA28 are reported. The secondary structure propensity from TALOS+ indicates that tA28 does contain three α-helices, six β-strands and connecting loops. Aside from this, we demonstrated that tA28 does interact with fusion suppressor viral protein A26 (residues 351-500) by the 1H-15N HSQC spectrum. We interpret that A28 binding to A26 deactivates EFC fusion activity. The current study provides a valuable framework towards further structural analyses of this protein and for better understanding virus/host cell membrane fusion mechanism in association with virus entry.

Surprisingly Effective Priming of CD8+ T Cells by Heat-Inactivated Vaccinia Virus Virions.

Robust priming of CD8+ T cells by viruses is considered to require infection and de novo expression of viral antigens. A corollary of this is that inactivated viruses are thought of as being inevitably poor vaccines for eliciting these responses. In contrast to this dogma, we found that some antigens present in vaccinia virus (VACV) virions prime strong CD8+ T cell responses when the virus was rendered noninfectious by heat. More surprisingly, in some cases these responses were similar in magnitude to those primed by infectious virus administered at an equivalent dose. Next, we tested whether this was a special property of particular antigens and their epitopes and found that foreign epitopes tagged onto three different VACV virion proteins were able to elicit CD8+ T cell responses irrespective of whether the virus was viable or heat killed. Further, the polyfunctionality and cytotoxic ability of the CD8+ T cells primed by these VACVs was equivalent irrespective of whether they were administered to mice as inactivated or live viruses. Finally, we used these VACVs in prime-boost combinations of inactivated and live virus and found that priming with dead virus before a live booster was the most immunogenic regime. We conclude that VACV virions can be efficient vectors for targeting antigens to dendritic cells for effective priming of CD8+ T cells, even when rendered noninfectious and speculate that this might also be the case for other viruses.IMPORTANCE The design of viral vectored vaccines is often considered to require a trade-off between efficacy and safety. This is especially the case for vaccines that aim to induce killer (CD8+) T cells, where there is a well-established dogma that links infection in vaccinated individuals with effective induction of immunity. However, we found that some proteins of vaccinia virus generate strong CD8+ T cell responses even when the virus preparation was inactivated by heat prior to administration as a vaccine. We took advantage of this finding by engineering a new vaccine vector virus that could be used as an inactivated vaccine. These results suggest that vaccinia virus may be a more versatile vaccine vector than previously appreciated and that in some instances safety can be prioritized by the complete elimination of viral replication without a proportional loss of immunogenicity.

Identification of protective T-cell antigens for smallpox vaccines

Background aimsE3L is an immediate-early protein of vaccinia virus (VV) that is detected within 0.5 h of infection, potentially before the many immune evasion genes of vaccinia can exert their protective effects. E3L is highly conserved among orthopoxviruses and hence could provide important protective T-cell epitopes that should be retained in any subunit or attenuated vaccine. We have therefore evaluated the immunogenicity of E3L in healthy VV-vaccinated donors. MethodsPeripheral blood mononuclear cells from healthy volunteers (n = 13) who had previously received a smallpox vaccine (Dryvax) were activated and expanded using overlapping E3L peptides and their function, specificity and antiviral activity was analyzed. E3L-specific T cells were expanded from 7 of 12 (58.3%) vaccinated healthy donors. Twenty-five percent of these produced CD8+ T-cell responses and 87.5% produced CD4+ T cells. We identified epitopes restricted by HLA-B35 and HLA-DR15. ResultsE3L-specific T cells killed peptide-loaded target cells as well as vaccinia-infected cells, but only CD8+ T cells could prevent the spread of infectious virus in virus inhibition assays. The epitopes recognized by E3L-specific T cells were shared with monkeypox, and although there was a single amino acid change in the variola epitope homolog, it was recognized by vaccinia-specific T-cells. ConclusionsIt might be important to include E3L in any deletion mutant or subunit vaccine and E3L could provide a useful antigen to monitor protective immunity in humans.

Stable Isotope-Triggered Offset Fragmentation Allows Massively Multiplexed Target Profiling on Quadrupole-Orbitrap Mass Spectrometers

Parallel-reaction monitoring (PRM) using high resolution, accurate mass (HR/AM) analysis on quadrupole-Orbitrap mass spectrometers, like the Q Exactive, is one of the most promising approaches for targeted protein analysis. However, PRM has a limited multiplexing capacity, which depends heavily on the reproducibility of peptide retention times. To overcome these limitations, we aimed to establish an easily applicable data acquisition mode that allows retention-time-independent massive multiplexing on Q Exactive mass spectrometers. The presented method is based on data-dependent acquisition and is called pseudo-PRM. In principle, high-intensity stable isotope-labeled peptides are used to trigger the repeated fragmentation of the corresponding light peptides. In this way, pseudo-PRM data can be analyzed like normal PRM data. We tested pseudo-PRM for the target detection from yeast, human cells, and serum, showing good reproducibility and sensitivities comparable to normal PRM. We demonstrated further that pseudo-PRM can be used for accurate and precise quantification of target peptides, using both precursor and fragment ion areas. Moreover, we showed multiplexing of more than 1000 targets in a single run. Finally, we applied pseudo-PRM to quantify vaccinia virus proteins during infection, verifying that pseudo-PRM presents an alternative method for multiplexed target profiling on Q Exactive mass spectrometers.

Degradation of Herpes Simplex Virus-1 Viral miRNA H11 by Vaccinia Virus Protein VP55 Attenuates Viral Replication

Among 29 distinct miRNAs expressed by the herpes simplex virus-1 (HSV-1) during lytic infection, miR-H11, together with miR-H1 to miR-H8 are reported to locate in the RNA-induced silencing complex (RISC). miR-H11 is encoded within viral origins of replication and lies entirely within the origins of replication. However, the roles of this miRNA derived from lytic infection with HSV-1 remain unclear. Using the advantage of vaccinia virus protein VP55 (VP55)-mediated degradation of miRNAs, we constructed a recombinant virus expressing VP55 (R5502) to demonstrate that: (1) accumulation of miR-H11 from R5502 was reduced by 540-fold versus that in cells infected with wild-type HSV-1, but miR-H1 to miR-H8 which also located in the RISC were not reduced significantly from R5502 compare with wild-type HSV-1; (2) downregulation of miR-H11 from R5502 infected cells results in markedly lower viral DNA synthesis compared with wild-type HSV-1; and (3) downregulation of miR-H11 also restricted viral spreading, and resulted in low accumulation of representative viral proteins and viral yields. The findings were confirmed through either using of a miR-H11 inhibitor or pre-transfection of a plasmid expressing VP55. These data suggest that miR-H11 plays a currently unidentified role in maintaining sufficient viral DNA synthesis during the course of viral infection.

Magnitude and diversity of immune response to vaccinia virus is dependent on route of administration

Historic observations suggest that survivors of smallpox maintained lifelong immunity and protection to subsequent infection compared to vaccinated individuals. Although protective immunity by vaccination using a related virus (vaccinia virus (VACV) strains) was the key for smallpox eradication, it does not uniformly provide long term, or lifelong protective immunity (Heiner et al., 1971). To determine differences in humoral immune responses, mice were inoculated with VACV either systemically, using intranasal inoculation (IN), or locally by an intradermal (ID) route. We hypothesized that sub-lethal IN infections may mimic systemic or naturally occurring infection and lead to an immunodominance reaction, in contrast to localized ID immunization. The results demonstrated systemic immunization through an IN route led to enhanced adaptive immunity to VACV-expressed protein targets both in magnitude and in diversity when compared to an ID route using a VACV protein microarray. In addition, cytokine responses, assessed using a Luminex® mouse cytokine multiplex kit, following IN infection was greater than that stemming from ID infection. Overall, the results suggest that the route of immunization (or infection) influences antibody responses. The greater magnitude and diversity of response in systemic infection provides indirect evidence for anecdotal observations made during the smallpox era that survivors maintain lifelong protection. These findings also suggest that systemic or disseminated host immune induction may result in a superior response, that may influence the magnitude of, as well as duration of protective responses.

Competition between E3 and ZBP1 for Z-RNA Dictates Susceptibility to Vaccinia Virus-Induced Necroptosis

Necroptosis mediated by Z-nucleic acid binding protein (ZBP)1 (also known as DAI and DLM1) provides innate host defense against viruses. Unless blocked by vaccinia virus (VACV) protein E3, ZBP1 triggers RIP-homotypic interaction motif (RHIM)-dependent activation of RIPK3 to execute cell death, cut short replication and limit pathogenesis. Here, Z-form RNA is shown to be sequestered in the cytoplasm dependent on the Z-nucleic acid binding domain of E3. Mutating the Zα motif within this domain permits accumulation of free Z-RNA during the early phase of VACV infection that triggers ZBP1-mediated VACV-induced necroptosis. Zα from either ZBP1 or the RNA editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 are functionally interchangeable with E3 Zα and suffice to block virus-induced necroptosis as a component of chimeric viruses. Overall, the evidence shows that Z-form RNA generated during VACV infection unleashes ZBP1-RIPK3-dependent necroptosis and unveils the mechanism through which viral E3 suppresses sensing of this PAMP.

The Vaccinia Virus (VACV) B1 and Cellular VRK2 Kinases Promote VACV Replication Factory Formation through Phosphorylation-Dependent Inhibition of VACV B12.

Comparative examination of viral and host protein homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and viral origin. The vaccinia virus B1 protein kinase is involved in promoting multiple facets of the virus life cycle and is a homolog of three conserved cellular enzymes called vaccinia virus-related kinases (VRKs). Recent evidence indicates that B1 and VRK2 mediate a common pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vaccinia virus protein B12, which otherwise impedes an early event in the viral lifecycle. Herein, we characterize the B1/VRK2 signaling axis to better understand their shared functions. First, we demonstrate that vaccinia virus uniquely requires VRK2 for viral replication in the absence of B1, unlike other DNA viruses. Employing loss-of-function analysis, we demonstrate that vaccinia virus's dependence on VRK2 is only observed in the presence of B12, suggesting that B1 and VRK2 share a pathway controlling B12. Moreover, we substantiate a B1/VRK2/B12 signaling axis by examining coprecipitation of B12 by B1 and VRK2. Employing execution point analysis, we reveal that virus replication proceeds normally through early protein translation and uncoating but stalls at replication factory formation in the presence of B12 activity. Finally, structure/function analyses of B1 and VRK2 demonstrate that enzymatic activity is essential for B1 or VRK2 to inhibit B12. Together, these data provide novel insights into B1/VRK signaling coregulation and support a model in which these enzymes modulate B12 in a phosphorylation-dependent manner.IMPORTANCE Constraints placed on viral genome size require that these pathogens must employ sophisticated, yet parsimonious mechanisms to effectively integrate with host cell signaling pathways. Poxviruses are no exception and employ several methods to balance these goals, including encoding single proteins that impact multiple downstream pathways. This study focuses on the vaccinia virus B1 protein kinase, an enzyme that promotes virus replication at multiple phases of the viral lifecycle. Herein, we demonstrate that in addition to its previously characterized functions, B1 inhibits vaccinia virus B12 protein via a phosphorylation-dependent mechanism and that this function of B1 can be complemented by the cellular B1 homolog VRK2. Combined with previous data implicating functional overlap between B1 and an additional cellular B1 homolog, VRK1, these data provide evidence of how poxviruses can be multifaceted in their mimicry of cellular proteins through the consolidation of functions of both VRK1 and VRK2 within the viral B1 protein kinase.

Asparagine Is a Critical Limiting Metabolite for Vaccinia Virus Protein Synthesis during Glutamine Deprivation.

Viruses actively interact with host metabolism because viral replication relies on host cells to provide nutrients and energy. Vaccinia virus (VACV; the prototype poxvirus) prefers glutamine to glucose for efficient replication to the extent that VACV replication is hindered in glutamine-free medium. Remarkably, our data show that VACV replication can be fully rescued from glutamine depletion by asparagine supplementation. By global metabolic profiling, as well as genetic and chemical manipulation of the asparagine supply, we provide evidence demonstrating that the production of asparagine, which exclusively requires glutamine for biosynthesis, accounts for VACV's preference of glutamine to glucose rather than glutamine's superiority over glucose in feeding the tricarboxylic acid (TCA) cycle. Furthermore, we show that sufficient asparagine supply is required for efficient VACV protein synthesis. Our study highlights that the asparagine supply, the regulation of which has been evolutionarily tailored in mammalian cells, presents a critical barrier to VACV replication due to a high asparagine content of viral proteins and a rapid demand of viral protein synthesis. The identification of asparagine availability as a critical limiting factor for efficient VACV replication suggests a new direction of antiviral strategy development.IMPORTANCE Viruses rely on their infected host cells to provide nutrients and energy for replication. Vaccinia virus, the prototypic member of the poxviruses, which comprise many significant human and animal pathogens, prefers glutamine to glucose for efficient replication. Here, we show that the preference is not because glutamine is superior to glucose as the carbon source to fuel the tricarboxylic acid cycle for vaccinia virus replication. Rather interestingly, the preference is because the asparagine supply for efficient viral protein synthesis becomes limited in the absence of glutamine, which is necessary for asparagine biosynthesis. We provide further genetic and chemical evidence to demonstrate that asparagine availability plays a critical role in efficient vaccinia virus replication. This discovery identifies a weakness of vaccinia virus and suggests a possible direction to intervene in poxvirus infection.

Histone deacetylase 4 promotes type I interferon signaling, restricts DNA viruses, and is degraded via vaccinia virus protein C6

Interferons (IFNs) represent an important host defense against viruses. Type I IFNs induce JAK-STAT signaling and expression of IFN-stimulated genes (ISGs), which mediate antiviral activity. Histone deacetylases (HDACs) perform multiple functions in regulating gene expression and some class I HDACs and the class IV HDAC, HDAC11, influence type I IFN signaling. Here, HDAC4, a class II HDAC, is shown to promote type I IFN signaling and coprecipitate with STAT2. Pharmacological inhibition of class II HDAC activity, or knockout of HDAC4 from HEK-293T and HeLa cells, caused a defective response to IFN-α. This defect in HDAC4-/- cells was rescued by reintroduction of HDAC4 or catalytically inactive HDAC4, but not HDAC1 or HDAC5. ChIP analysis showed HDAC4 was recruited to ISG promoters following IFN stimulation and was needed for binding of STAT2 to these promoters. The biological importance of HDAC4 as a virus restriction factor was illustrated by the observations that (i) the replication and spread of vaccinia virus (VACV) and herpes simplex virus type 1 (HSV-1) were enhanced in HDAC4-/- cells and inhibited by overexpression of HDAC4; and (ii) HDAC4 is targeted for proteasomal degradation during VACV infection by VACV protein C6, a multifunctional IFN antagonist that coprecipitates with HDAC4 and is necessary and sufficient for HDAC4 degradation.

Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

BTB–Kelch proteins are substrate-specific adaptors for cullin-3 (Cul3) RING-box–based E3 ubiquitin ligases, mediating protein ubiquitylation for subsequent proteasomal degradation. Vaccinia virus encodes three BTB–Kelch proteins: A55, C2, and F3. Viruses lacking A55 or C2 have altered cytopathic effects in cultured cells and altered pathology in vivo. Previous studies have shown that the ectromelia virus orthologue of A55 interacts with Cul3 in cells. We report that the N-terminal BTB-BACK (BB) domain of A55 binds directly to the Cul3 N-terminal domain (Cul3–NTD), forming a 2:2 complex in solution. We solved the structure of an A55BB/Cul3–NTD complex from anisotropic crystals diffracting to 2.3/3.7 Å resolution in the best/worst direction, revealing that the overall interaction and binding interface closely resemble the structures of cellular BTB/Cul3–NTD complexes, despite low sequence identity between A55 and cellular BTB domains. Surprisingly, despite this structural similarity, the affinity of Cul3–NTD for A55BB was stronger than for cellular BTB proteins. Glutamate substitution of the A55 residue Ile-48, adjacent to the canonical φX(D/E) Cul3-binding motif, reduced affinity of A55BB for Cul3–NTD by at least 2 orders of magnitude. Moreover, Ile-48 and the φX(D/E) motif are conserved in A55 orthologues from other poxviruses, but not in the vaccinia virus proteins C2 or F3. The high-affinity interaction between A55BB and Cul3–NTD suggests that, in addition to directing the Cul3–RING E3 ligase complex to degrade cellular/viral target proteins that are normally unaffected, A55 may also sequester Cul3 from cellular adaptor proteins, thereby protecting substrates of these cellular adaptors from ubiquitylation and degradation.

Structural mechanism of vaccinia virus protein C6 mediated inactivation of transcription factors IRF3 and IRF7 via inhibition of TBK1 mediated scaffold protein‐protein interactions

Interferons (IFNs) are signaling proteins with potent antiviral and immune stimulatory effects. IFN signaling is initiated after host cell pattern recognition receptors (PRRs) recognize pathogen‐associated molecular patterns (PAMPs) present in many viral proteins. This event in turn generates a complex signaling cascade involving numerous proteins, activation of various transcription factors (TF), such as IFN‐regulatory factors (IRF) 3 and 7. These TFs subsequently translocate to the nucleus to elevate the transcription of type I IFNs, cytokines, and chemokines. Viruses such as Vaccinia virus (VACV) have evolved many strategies to interfere with the IFNs or their mechanism of action. Specifically, VACV encodes a small 16kDa protein, C6, that inhibits the activation of IRF3/7 via binding with an upstream kinase, TBK1, and its adaptor proteins TANK, NAP1, and SINTBAD in order to interfere with the production of IFNβ. Binding with these adaptor proteins, C6 prevents TBK1/IKKɛ‐dependent activation of IRF3 and IRF7 allowing VACV to evade immune recognition. To elucidate the structural mechanism of how C6 mediated inhibition of these adaptor protein/TBK1 interactions can block the activation of IRF3 and IRF7, we have chosen C6 and TANK as canonical binding partners. We have identified the binding regions between C6 and TANK by utilizing co‐expression/co‐purification studies of C6 and TANK proteins, and co‐crystallization trials are ongoing. The immune evasive strategy exploited by VACV can be used to develop novel therapies, such as C6 mimetics to provide TBK1 inhibition. The development of therapeutics depends immensely on a detailed structural analysis of the interactions between C6 and adaptor proteins. Our structural studies of C6 and TBK1 adaptor proteins will provide useful insights in developing superior therapies against malignancies, infectious diseases, and autoimmune disorders.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of IFN-gamma signalling by vaccinia virus protein C6: a multi-functional interferon antagonist

Vaccinia virus (VACV) encodes multiple proteins that function to inhibit both the production and downstream effects of interferons, which are critical modulators of the antiviral response. One such protein, VACV C6, inhibits both the production of type I IFN (by blocking IRF3 signalling) and the downstream effects of type I IFN (by blocking JAK-STAT signalling). Further to this, our findings suggest C6 can also inhibit the downstream effects of type II IFN of which IFN-gamma is the sole member. The observation that C6 co-immunoprecipitates with STAT1, a protein crucial in activating the downstream functions of IFN-gamma, might explain its inhibitory activity.

NF-κB activation is a turn on for vaccinia virus phosphoprotein A49 to turn off NF-κB activation

Vaccinia virus protein A49 inhibits NF-κB activation by molecular mimicry and has a motif near the N terminus that is conserved in IκBα, β-catenin, HIV Vpu, and some other proteins. This motif contains two serines, and for IκBα and β-catenin, phosphorylation of these serines enables recognition by the E3 ubiquitin ligase β-TrCP. Binding of IκBα and β-catenin by β-TrCP causes their ubiquitylation and thereafter proteasome-mediated degradation. In contrast, HIV Vpu and VACV A49 are not degraded. This paper shows that A49 is phosphorylated at serine 7 but not serine 12 and that this is necessary and sufficient for binding β-TrCP and antagonism of NF-κB. Phosphorylation of A49 S7 occurs when NF-κB signaling is activated by addition of IL-1β or overexpression of TRAF6 or IKKβ, the kinase needed for IκBα phosphorylation. Thus, A49 shows beautiful biological regulation, for it becomes an NF-κB antagonist upon activation of NF-κB signaling. The virulence of viruses expressing mutant A49 proteins or lacking A49 (vΔA49) was tested. vΔA49 was attenuated compared with WT, but viruses expressing A49 that cannot bind β-TrCP or bind β-TrCP constitutively had intermediate virulence. So A49 promotes virulence by inhibiting NF-κB activation and by another mechanism independent of S7 phosphorylation and NF-κB antagonism. Last, a virus lacking A49 was more immunogenic than the WT virus.

Proteomics Analysis Reveals That Structural Proteins of the Virion Core and Involved in Gene Expression Are the Main Source for HLA Class II Ligands in Vaccinia Virus-Infected Cells.

Protective cellular and humoral immune responses require previous recognition of viral antigenic peptides complexed with human leukocyte antigen (HLA) class II molecules on the surface of the antigen presenting cells. The HLA class II-restricted immune response is important for the control and the clearance of poxvirus infection including vaccinia virus (VACV), the vaccine used in the worldwide eradication of smallpox. In this study, a mass spectrometry analysis was used to identify VACV ligands bound to HLA-DR and -DP class II molecules present on the surface of VACV-infected cells. Twenty-six naturally processed viral ligands among the tens of thousands of cell peptides bound to HLA class II proteins were identified. These viral ligands arose from 19 parental VACV proteins: A4, A5, A18, A35, A38, B5, B13, D1, D5, D7, D12, D13, E3, E8, H5, I2, I3, J2, and K2. The majority of these VACV proteins yielded one HLA ligand and were generated mainly, but not exclusively, by the classical HLA class II antigen processing pathway. Medium-sized and abundant proteins from the virion core and/or involved in the viral gene expression were the major source of VACV ligands bound to HLA-DR and -DP class II molecules. These findings will help to understand the effectiveness of current poxvirus-based vaccines and will be important in the design of new ones.

DNA-PK Is Targeted by Multiple Vaccinia Virus Proteins to Inhibit DNA Sensing

SummaryVirus infection is sensed by pattern recognition receptors (PRRs) detecting virus nucleic acids and initiating an innate immune response. DNA-dependent protein kinase (DNA-PK) is a PRR that binds cytosolic DNA and is antagonized by vaccinia virus (VACV) protein C16. Here, VACV protein C4 is also shown to antagonize DNA-PK by binding to Ku and blocking Ku binding to DNA, leading to a reduced production of cytokines and chemokines in vivo and a diminished recruitment of inflammatory cells. C4 and C16 share redundancy in that a double deletion virus has reduced virulence not seen with single deletion viruses following intradermal infection. However, non-redundant functions exist because both single deletion viruses display attenuated virulence compared to wild-type VACV after intranasal infection. It is notable that VACV expresses two proteins to antagonize DNA-PK, but it is not known to target other DNA sensors, emphasizing the importance of this PRR in the response to infection in vivo.