Filamin B restricts vaccinia virus spread and is targeted by vaccinia virus protein C4.

Vaccinia virus (VACV) is a large DNA virus that encodes scores of proteins that modulate the host immune response. VACV protein C4 is one such immunomodulator known to inhibit the activation of both the NF-κB signaling cascade and the DNA-PK-mediated DNA sensing pathway. Here, we show that the N-terminal region of C4, which neither inhibits NF-κB nor mediates interaction with DNA-PK, still contributes to virus virulence. Furthermore, this domain interacts directly and with high affinity to the C-terminal domain of filamin B (FLNB). FLNB is a large actin-binding protein that stabilizes the F-actin network and is implicated in other cellular processes. Deletion of FLNB from cells results in larger VACV plaques and increased infectious viral yield, indicating that FLNB restricts VACV spread. These data demonstrate that C4 has a new function that contributes to virulence and engages the cytoskeleton. Furthermore, we show that the cytoskeleton performs further previously uncharacterized functions during VACV infection. IMPORTANCE: Vaccinia virus (VACV), the vaccine against smallpox and monkeypox, encodes many proteins to counteract the host immune response. Investigating these proteins provides insights into viral immune evasion mechanisms and thereby indicates how to engineer safer and more immunogenic VACV-based vaccines. Here, we report that the N-terminal domain of VACV protein C4 interacts directly with the cytoskeletal protein filamin B (FLNB), and this domain of C4 contributes to virus virulence. Furthermore, VACV replicates and spreads better in cells lacking FLNB, thus demonstrating that FLNB has antiviral activity. VACV utilizes the cytoskeleton for movement within and between cells; however, previous studies show no involvement of C4 in VACV replication or spread. Thus, C4 associates with FLNB for a different reason, suggesting that the cytoskeleton has further uncharacterized roles during virus infection.

Vaccinia E5 is a major inhibitor of the DNA sensor cGAS.

The DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is not well understood. In this study, we screened 80 vaccinia genes to identify potential viral inhibitors of the cGAS/Stimulator of interferon gene (STING) pathway. We discovered that vaccinia E5 is a virulence factor and a major inhibitor of cGAS. E5 is responsible for abolishing cGAMP production during vaccinia virus (Western Reserve strain) infection of dendritic cells. E5 localizes to the cytoplasm and nucleus of infected cells. Cytosolic E5 triggers ubiquitination of cGAS and proteasome-dependent degradation via interacting with cGAS. Deleting the E5R gene from the Modified vaccinia virus Ankara (MVA) genome strongly induces type I IFN production by dendritic cells (DCs) and promotes DC maturation, and thereby improves antigen-specific T cell responses.

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.

An increase in glycoprotein concentration on extracellular virions dramatically alters vaccinia virus infectivity and pathogenesis without impacting immunogenicity.

The extracellular virion (EV) form of Orthopoxviruses is required for cell-to-cell spread and pathogenesis, and is the target of neutralizing antibodies in the protective immune response. EV have a double envelope that contains several unique proteins that are involved in its intracellular envelopment and/or subsequent infectivity. One of these, F13, is involved in both EV formation and infectivity. Here, we report that replacement of vaccinia virus F13L with the molluscum contagiosum virus homolog, MC021L, results in the production of EV particles with significantly increased levels of EV glycoproteins, which correlate with a small plaque phenotype. Using a novel fluorescence-activated virion sorting assay to isolate EV populations based on glycoprotein content we determine that EV containing either higher or lower levels of glycoproteins are less infectious, suggesting that there is an optimal concentration of glycoproteins in the outer envelope that is required for maximal infectivity of EV. This optimal glycoprotein concentration was required for lethality and induction of pathology in a cutaneous model of animal infection, but was not required for induction of a protective immune response. Therefore, our results demonstrate that there is a sensitive balance between glycoprotein incorporation, infectivity, and pathogenesis, and that manipulation of EV glycoprotein levels can produce vaccine vectors in which pathologic side effects are attenuated without a marked diminution in induction of protective immunity.

Oncolytic Vaccinia Virus Harboring Aphrocallistes vastus Lectin Inhibits the Growth of Cervical Cancer Cells Hela S3.

Aphrocallistes vastus lectin (AVL) is a C-type marine lectin produced by sponges. Our previous study demonstrated that genes encoding AVL enhanced the cytotoxic effect of oncolytic vaccinia virus (oncoVV) in a variety of cancer cells. In this study, the inhibitory effect of oncoVV-AVL on Hela S3 cervical cancer cells, a cell line with spheroidizing ability, was explored. The results showed that oncoVV-AVL could inhibit Hela S3 cells growth both in vivo and in vitro. Further investigation revealed that AVL increased the virus replication, promote the expression of OASL protein and stimulated the activation of Raf in Hela S3 cells. This study may provide insight into a novel way for the utilization of lection AVL.

Zoonotic vaccinia virus strains belonging to different genetic clades exhibit immunomodulation abilities that are proportional to their virulence.

BACKGROUND: The vaccinia virus (VACV) isolates, Guarani P1 virus (GP1V) and Passatempo virus (PSTV), were isolated during zoonotic outbreaks in Brazil. Each one of them belongs to two different VACV clades, defined by biological aspects that include virulence in mice and phylogenetic analysis. Considering that information about how vaccinia viruses from different groups elicit immune responses in animals is scarce, we investigated such responses in mice infected either by GP1V (group 2) or PSTV (group 1), using VACV Western Reserve strain (VACV-WR) as control. METHODS: The severity of the infections was evaluated in BALB/c mice considering diverse clinical signs and defined scores, and the immune responses triggered by GP1V and PSTV infections were analysed by immune cell phenotyping and intra-cytoplasmic cytokines detection. RESULTS: We detected a reduction in total lymphocytes (CD3 +), macrophages (CD14 +), and NK cells (CD3-CD49 +) in animals infected with VACV-WR or GP1V. The VACV-WR and GP1V viruses, belonging to the most virulent group in a murine model, were able to down-modulate the cell immune responses upon mice infection. In contrast, PSTV, a virus considered less virulent in a murine model, showed little ability to down-modulate the mice immune responses. Mice infected with VACV-WR and GP1V viruses presented significant weight loss and developed lesions in their spleens, as well as damage to liver and lungs whereas mice infected with PSTV developed only moderate clinical signs. CONCLUSIONS: Our results suggest that VACV immunomodulation in vivo is clade-related and is proportional to the strain's virulence upon infection. Our data corroborate the classification of the different Brazilian VACV isolates into clades 1 and 2, taking into account not only phylogenetic criteria, but also clinical and immunological data.

Vaccinia virus hijacks ESCRT-mediated multivesicular body formation for virus egress.

Poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple-membrane particles, termed intracellular enveloped virions (IEVs), are released from infected cells by fusion. Whereas the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to an RNAi screen directed against cellular membrane-trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of MVB-IEVs showed that they account for half of IEV production indicating that MVBs are a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread.

Remodeling of the tumor microenvironment using an engineered oncolytic vaccinia virus improves PD-L1 inhibition outcomes.

Immune checkpoint inhibitor (ICI) immunotherapies have vastly improved therapeutic outcomes for patients with certain cancer types, but these responses only manifest in a small percentage of all cancer patients. The goal of the present study was to improve checkpoint therapy efficacy by utilizing an engineered vaccinia virus to improve the trafficking of lymphocytes to the tumor, given that such lymphocyte trafficking is positively correlated with patient checkpoint inhibitor response rates. We developed an oncolytic vaccinia virus (OVV) platform expressing manganese superoxide dismutase (MnSOD) for use as both a monotherapy and together with anti-PD-L1. Intratumoral OVV-MnSOD injection in immunocompetent mice resulted in inflammation within poorly immunogenic tumors, thereby facilitating marked tumor regression. OVV-MnSOD administration together with anti-PD-L1 further improved antitumor therapy outcomes in models in which these monotherapy approaches were ineffective. Overall, our results emphasize the value of further studying these therapeutic approaches in patients with minimally or non-inflammatory tumors.

Golgi apparatus-synthesized sulfated glycosaminoglycans mediate polymerization and activation of the cGAMP sensor STING.

Activation of the cyclic guanosine monophosphate (GMP)-AMP (cGAMP) sensor STING requires its translocation from the endoplasmic reticulum to the Golgi apparatus and subsequent polymerization. Using a genome-wide CRISPR-Cas9 screen to define factors critical for STING activation in cells, we identified proteins critical for biosynthesis of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus. Binding of sGAGs promoted STING polymerization through luminal, positively charged, polar residues. These residues are evolutionarily conserved, and selective mutation of specific residues inhibited STING activation. Purified or chemically synthesized sGAGs induced STING polymerization and activation of the kinase TBK1. The chain length and O-linked sulfation of sGAGs directly affected the level of STING polymerization and, therefore, its activation. Reducing the expression of Slc35b2 to inhibit GAG sulfation in mice impaired responses to vaccinia virus infection. Thus, sGAGs in the Golgi apparatus are necessary and sufficient to drive STING polymerization, providing a mechanistic understanding of the requirement for endoplasmic reticulum (ER)-to-Golgi apparatus translocation for STING activation.

Oncolytic vaccinia virus reinvigorates peritoneal immunity and cooperates with immune checkpoint inhibitor to suppress peritoneal carcinomatosis in colon cancer.

BACKGROUND: Peritoneal carcinomatosis (PC) is a common and devastating manifestation of colon cancer and refractory to conventional anticancer therapeutics. During the peritoneal dissemination of colon cancer, peritoneal immunity is nullified by various mechanisms of immune evasion. Here, we employed the armed oncolytic vaccinia virus mJX-594 (JX) to rejuvenate the peritoneal antitumor immune responses in the treatment of PC. METHODS: PC model of MC38 colon cancer was generated and intraperitoneally treated with JX and/or anti-programmed cell death protein 1 (PD-1) antibody. The peritoneal tumor burden, vascular leakage, and malignant ascites formation were then assessed. Tumors and peritoneal lavage cells were analyzed by flow cytometry, multiplex tissue imaging, and a NanoString assay. RESULTS: JX treatment effectively suppressed peritoneal cancer progression and malignant ascites formation. It also restored the peritoneal anticancer immunity by activating peritoneal dendritic cells (DCs) and CD8+ T cells. Moreover, JX selectively infected and killed peritoneal colon cancer cells and promoted the intratumoral infiltration of DCs and CD8+ T cells into peritoneal tumor nodules. JX reinvigorates anticancer immunity by reprogramming immune-related transcriptional signatures within the tumor microenvironment. Notably, JX cooperates with immune checkpoint inhibitors (ICIs), anti-programmed death-1, anti-programmed death-ligand 1, and anti-lymphocyte-activation gene-3 to elicit a stronger anticancer immunity that eliminates peritoneal metastases and malignant ascites of colon cancer compared with JX or ICI alone. CONCLUSIONS: Intraperitoneal immunotherapy with JX restores peritoneal anticancer immunity and potentiates immune checkpoint blockade to suppress PC and malignant ascites in colon cancer.

Mimicry Embedding Facilitates Advanced Neural Network Training for Image-Based Pathogen Detection.

The use of deep neural networks (DNNs) for analysis of complex biomedical images shows great promise but is hampered by a lack of large verified data sets for rapid network evolution. Here, we present a novel strategy, termed "mimicry embedding," for rapid application of neural network architecture-based analysis of pathogen imaging data sets. Embedding of a novel host-pathogen data set, such that it mimics a verified data set, enables efficient deep learning using high expressive capacity architectures and seamless architecture switching. We applied this strategy across various microbiological phenotypes, from superresolved viruses to in vitro and in vivo parasitic infections. We demonstrate that mimicry embedding enables efficient and accurate analysis of two- and three-dimensional microscopy data sets. The results suggest that transfer learning from pretrained network data may be a powerful general strategy for analysis of heterogeneous pathogen fluorescence imaging data sets.IMPORTANCE In biology, the use of deep neural networks (DNNs) for analysis of pathogen infection is hampered by a lack of large verified data sets needed for rapid network evolution. Artificial neural networks detect handwritten digits with high precision thanks to large data sets, such as MNIST, that allow nearly unlimited training. Here, we developed a novel strategy we call mimicry embedding, which allows artificial intelligence (AI)-based analysis of variable pathogen-host data sets. We show that deep learning can be used to detect and classify single pathogens based on small differences.

Long-read assays shed new light on the transcriptome complexity of a viral pathogen.

Characterization of global transcriptomes using conventional short-read sequencing is challenging due to the insensitivity of these platforms to transcripts isoforms, multigenic RNA molecules, and transcriptional overlaps. Long-read sequencing (LRS) can overcome these limitations by reading full-length transcripts. Employment of these technologies has led to the redefinition of transcriptional complexities in reported organisms. In this study, we applied LRS platforms from Pacific Biosciences and Oxford Nanopore Technologies to profile the vaccinia virus (VACV) transcriptome. We performed cDNA and direct RNA sequencing analyses and revealed an extremely complex transcriptional landscape of this virus. In particular, VACV genes produce large numbers of transcript isoforms that vary in their start and termination sites. A significant fraction of VACV transcripts start or end within coding regions of neighbouring genes. This study provides new insights into the transcriptomic profile of this viral pathogen.

Effect of the Route of Administration of the Vaccinia Virus Strain LIVP to Mice on Its Virulence and Immunogenicity.

The mass smallpox vaccination campaign has played a crucial role in smallpox eradication. Various strains of the vaccinia virus (VACV) were used as a live smallpox vaccine in different countries, their origin being unknown in most cases. The VACV strains differ in terms of pathogenicity exhibited upon inoculation of laboratory animals and reactogenicity exhibited upon vaccination of humans. Therefore, each generated strain or clonal variant of VACV needs to be thoroughly studied in in vivo systems. The clonal variant 14 of LIVP strain (LIVP-14) was the study object in this work. A comparative analysis of the virulence and immunogenicity of LIVP-14 inoculated intranasally (i.n.), intradermally (i.d.), or subcutaneously (s.c.) to BALB/c mice at doses of 108, 107, and 106 pfu was carried out. Adult mice exhibited the highest sensitivity to the i.n. administered LIVP-14 strain, although the infection was not lethal. The i.n. inoculated LIVP-14 replicated efficiently in the lungs. Furthermore, this virus was accumulated in the brain at relatively high concentrations. Significantly lower levels of LIVP-14 were detected in the liver, kidneys, and spleen of experimental animals. No clinical manifestations of the disease were observed after i.d. or s.c. injection of LIVP-14 to mice. After s.c. inoculation, the virus was detected only at the injection site, while it could disseminate to the liver and lungs when delivered via i.d. administration. A comparative analysis of the production of virus-specific antibodies by ELISA and PRNT revealed that the highest level of antibodies was induced in i.n. inoculated mice; a lower level of antibodies was observed after i.d. administration of the virus and the lowest level after s.c. injection. Even at the lowest studied dose (106 pfu), i.n. or i.d. administered LIVP-14 completely protected mice against infection with the cowpox virus at the lethal dose. Our findings imply that, according to the ratio between such characteristics as pathogenicity/immunogenicity/protectivity, i.d. injection is the optimal method of inoculation with the VACV LIVP-14 strain to ensure the safe formation of immune defense after vaccination against orthopoxviral infections.

RIP1 kinase activity is critical for skin inflammation but not for viral propagation.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.

Deletion of multiple genes induces virulence reduction of vaccinia virus Tiantan strain.

The purpose of this study was to knock out two non-essential gene fragments (TC7L-TK2L and TJ2R) related to virulence, immunomodulation, and host range in the vaccinia virus Tian Tan strain (VTT), and combining with double-label screening and exogenous screening marker knockout techniques to construct attenuated strains with multiple gene deletions(rVTT-TC-TJ). The shuttle plasmids pSK-TC and pSK-TJ were constructed by designing 2 pairs of recombinant arms, combined with poxvirus early and late complex strong promoter pE/L and exogenous screening marker enhanced green fluorescent protein(EGFP). The results showed that knocking out the two gene fragments does not affect the replication ability of the virus and displays a good genetic stability. Furthermore, a series of in vivo and in vitro experiments demonstrate that although virulence of rVTT-TC-TJ is attenuated significantly, high immunogenicity was maintained. These results support the potential development of rVTT-TC-TJ as a safe viral vector or vaccine.

Genes A27L and F13L as Genetic Markers for the Isolation of Recombinant Vaccinia Virus.

After assembly in the cytosol, some Vaccinia virus particles go through a complex process that leads to virus egress and eventually cell-to-cell transmission. Intracellular particles are fully infectious, and therefore virus mutants lacking essential functions in the exit pathway are unable to form plaques but can multiply intracellularly. We isolated virus mutants in which two of the genes required for virus spread (F13L and A27L) were deleted independently or concurrently. The phenotypes of the mutant viruses were consistent with the need of A27L and F13L for intercellular virus transmission, the effect of the ΔA27L mutation being more severe than that of ΔF13L. Despite their defect in spread, ΔA27L mutant viruses could be expanded by infecting cell cultures at high multiplicity of infection, followed by the release of virions from infected cells by physical means. We developed a novel system for the isolation of recombinant Vaccinia virus in which selection is efficiently achieved by recovering plaque formation capacity after re-introduction of A27L into a ΔA27L virus. This system allowed the insertion of foreign DNA into the viral genome without the use of additional genetic markers. Furthermore, starting with a double mutant (ΔA27L-ΔF13L) virus, A27L selection was used in conjunction with F13L selection to mediate simultaneous dual insertions in the viral genome. This selection system facilitates combined expression of multiple foreign proteins from a single recombinant virus.

A vaccine based on recombinant modified Vaccinia Ankara containing the nucleoprotein from Lassa virus protects against disease progression in a guinea pig model.

Lassa fever remains the most imported viral haemorrhagic fever in Europe and is responsible for 5000 deaths per year throughout Western Africa. There is no vaccine and treatment is often ineffective. We have developed a vaccine based on modified Vaccinia Ankara expressing the nucleoprotein from Lassa virus (MVALassaNP). This study investigated the immunogenicity (in mice) and efficacy (in guinea pigs) of the MVALassaNP vaccine as a prime/boost or single vaccination regime. ELISA and ELISpot assays confirmed humoral and T-cell immunity following both a prime and prime/boost vaccination, with the prime/boost regime producing a statistically increased response compared to a prime only vaccine (P < 0.0001). The vaccine offered protection in guinea pigs against disease manifestations after challenge with virulent Lassa virus. Clinical signs, weight loss and temperature increases were observed in all animals receiving a control MVA vaccine, after challenge with Lassa virus. In contrast, no clinical signs, fever or weight loss were observed in any of the MVALassaNP vaccinated animals demonstrating that both a single immunisation, and prime/boost regime confer protection against disease progression. In conclusion, the MVALassaNP vaccine candidate elicits an immune response, demonstrates efficacy against Lassa virus disease and is suitable for further preclinical and clinical development.

Viral targeting of PDZ polarity proteins in the immune system as a potential evasion mechanism.

PDZ proteins are highly conserved through evolution; the principal function of this large family of proteins is to assemble protein complexes that are involved in many cellular processes, such as cell-cell junctions, cell polarity, recycling, or trafficking. Many PDZ proteins that have been identified as targets of viral pathogens by promoting viral replication and spread are also involved in epithelial cell polarity. Here, we briefly review the PDZ polarity proteins in cells of the immune system to subsequently focus on our hypothesis that the viral PDZ-dependent targeting of PDZ polarity proteins in these cells may alter the cellular fitness of the host to favor that of the virus; we further hypothesize that this modification of the cellular fitness landscape occurs as a common and widespread mechanism for immune evasion by viruses and possibly other pathogens.-Gutiérrez-González, L. H., Santos-Mendoza, T. Viral targeting of PDZ polarity proteins in the immune system as a potential evasion mechanism.

Working Safely with Vaccinia Virus: Laboratory Technique and Review of Published Cases of Accidental Laboratory Infections with Poxviruses.

Vaccinia virus, the prototype Orthopoxvirus, is widely used in the laboratory as a model system to study various aspects of viral biology and virus-host interactions, as a protein expression system, as a vaccine vector, and as an oncolytic agent. The ubiquitous use of vaccinia viruses in laboratories around the world raises certain safety concerns because the virus can be a pathogen in individuals with immunological and dermatological abnormalities, and on occasion can cause serious problems in normal hosts. This chapter reviews standard operating procedures when working with vaccinia virus and reviews published cases of accidental laboratory infections with poxviruses.

High-Content Analyses of Vaccinia Plaque Formation.

Vaccinia virus plaque assays are employed for quantification of virus titer through serial dilution of virus on a monolayer of cells. Once the virus titer is diluted enough to allow for only few cells of the monolayer to be infected, clonal spread of infection can be detected by observing the lesion in the cell monolayer or using virus-specific staining methods. Beyond simple titration, plaque formation bares priceless underlying information about subtle virus-host interactions and their impact on virus spread during multiple rounds of infection. These include virus infectivity, mode of virus spread, virus replication rate, and spatiotemporal spread efficacy. How this underlying information can be harnessed using a high-content imaging setup is discussed here.