Vaccinia virus subverts xenophagy through phosphorylation and nuclear targeting of p62.

Autophagy is an essential degradation program required for cell homeostasis. Among its functions is the engulfment and destruction of cytosolic pathogens, termed xenophagy. Not surprisingly, many pathogens use various strategies to circumvent or co-opt autophagic degradation. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Even though these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been extensively explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that overexpression of the xenophagy receptors p62, NDP52, and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 initially targeted cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 was dependent upon p62 NLS2 and correlated with VACV kinase mediated phosphorylation of p62 T269/S272. This suggests that VACV targets p62 during the early stages of infection to avoid destruction and further implies that poxviruses exhibit multi-layered control of autophagy to facilitate cytoplasmic replication.

Cardiac glycosides inhibit early and late vaccinia virus protein expression.

Cardiac glycosides (CGs) are natural steroid glycosides, which act as inhibitors of the cellular sodium-potassium ATPase pump. Although traditionally considered toxic to human cells, CGs are widely used as drugs for the treatment of cardiovascular-related medical conditions. More recently, CGs have been explored as potential anti-viral drugs and inhibit replication of a range of RNA and DNA viruses. Previously, a compound screen identified CGs that inhibited vaccinia virus (VACV) infection. However, no further investigation of the inhibitory potential of these compounds was performed, nor was there investigation of the stage(s) of the poxvirus lifecycle they impacted. Here, we investigated the anti-poxvirus activity of a broad panel of CGs. We found that all CGs tested were potent inhibitors of VACV replication. Our virological experiments showed that CGs did not impact virus infectivity, binding, or entry. Rather, experiments using recombinant viruses expressing reporter proteins controlled by VACV promoters and arabinoside release assays demonstrated that CGs inhibited early and late VACV protein expression at different concentrations. Lack of virus assembly in the presence of CGs was confirmed using electron microscopy. Thus, we expand our understanding of compounds with anti-poxvirus activity and highlight a yet unrecognized mechanism by which poxvirus replication can be inhibited.

Bisbenzimide compounds inhibit the replication of prototype and pandemic potential poxviruses.

We previously identified the bisbenzimide Hoechst 33342 (H42) as a potent multi-stage inhibitor of the prototypic poxvirus, the vaccinia virus (VACV), and several parapoxviruses. A recent report showed that novel bisbenzimide compounds similar in structure to H42 could prevent human cytomegalovirus replication. Here, we assessed whether these compounds could also serve as poxvirus inhibitors. Using virological assays, we show that these bisbenzimide compounds inhibit VACV spread, plaque formation, and the production of infectious progeny VACV with relatively low cell toxicity. Further analysis of the VACV lifecycle indicated that the effective bisbenzimide compounds had little impact on VACV early gene expression but inhibited VACV late gene expression and truncated the formation of VACV replication sites. Additionally, we found that bisbenzimide compounds, including H42, can inhibit both monkeypox and a VACV mutant resistant to the widely used anti-poxvirus drug TPOXX (Tecovirimat). Therefore, the tested bisbenzimide compounds were inhibitors of both prototypic and pandemic potential poxviruses and could be developed for use in situations where anti-poxvirus drug resistance may occur. Additionally, these data suggest that bisbenzimide compounds may serve as broad-activity antiviral compounds, targeting diverse DNA viruses such as poxviruses and betaherpesviruses.IMPORTANCEThe 2022 mpox (monkeypox) outbreak served as a stark reminder that due to the cessation of smallpox vaccination over 40 years ago, most of the human population remains susceptible to poxvirus infection. With only two antivirals approved for the treatment of smallpox infection in humans, the need for additional anti-poxvirus compounds is evident. Having shown that the bisbenzimide H33342 is a potent inhibitor of poxvirus gene expression and DNA replication, here we extend these findings to include a set of novel bisbenzimide compounds that show anti-viral activity against mpox and a drug-resistant prototype poxvirus mutant. These results suggest that further development of bisbenzimides for the treatment of pandemic potential poxviruses is warranted.

The vaccinia chondroitin sulfate binding protein drives host membrane curvature to facilitate fusion.

Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection.

PIM1 controls GBP1 activity to limit self-damage and to guard against pathogen infection.

Disruption of cellular activities by pathogen virulence factors can trigger innate immune responses. Interferon-γ (IFN-γ)-inducible antimicrobial factors, such as the guanylate binding proteins (GBPs), promote cell-intrinsic defense by attacking intracellular pathogens and by inducing programmed cell death. Working in human macrophages, we discovered that GBP1 expression in the absence of IFN-γ killed the cells and induced Golgi fragmentation. IFN-γ exposure improved macrophage survival through the activity of the kinase PIM1. PIM1 phosphorylated GBP1, leading to its sequestration by 14-3-3σ, which thereby prevented GBP1 membrane association. During Toxoplasma gondii infection, the virulence protein TgIST interfered with IFN-γ signaling and depleted PIM1, thereby increasing GBP1 activity. Although infected cells can restrain pathogens in a GBP1-dependent manner, this mechanism can protect uninfected bystander cells. Thus, PIM1 can provide a bait for pathogen virulence factors, guarding the integrity of IFN-γ signaling.

Coding-Complete Genome Sequences of Copenhagen and Copenhagen-Derived vP811 Strains of Vaccinia Virus Isolated from Cell Culture.

The coding-complete genomes of laboratory vaccinia virus strain Copenhagen and the Copenhagen-derived deletion strain, vP811, were determined by short-read sequencing. Relative to the NCBI reference genome M35027, seven common coding differences were revealed, including an intact copy of the vaccinia virus immunomodulator A46R in both Cop and vP811.

Poxviruses package viral redox proteins in lateral bodies and modulate the host oxidative response.

All poxviruses contain a set of proteinaceous structures termed lateral bodies (LB) that deliver viral effector proteins into the host cytosol during virus entry. To date, the spatial proteotype of LBs remains unknown. Using the prototypic poxvirus, vaccinia virus (VACV), we employed a quantitative comparative mass spectrometry strategy to determine the poxvirus LB proteome. We identified a large population of candidate cellular proteins, the majority being mitochondrial, and 15 candidate viral LB proteins. Strikingly, one-third of these are VACV redox proteins whose LB residency could be confirmed using super-resolution microscopy. We show that VACV infection exerts an anti-oxidative effect on host cells and that artificial induction of oxidative stress impacts early and late gene expression as well as virion production. Using targeted repression and/or deletion viruses we found that deletion of individual LB-redox proteins was insufficient for host redox modulation suggesting there may be functional redundancy. In addition to defining the spatial proteotype of VACV LBs, these findings implicate poxvirus redox proteins as potential modulators of host oxidative anti-viral responses and provide a solid starting point for future investigations into the role of LB resident proteins in host immunomodulation.

Vaccinia Virus Arrests and Shifts the Cell Cycle.

Modulation of the host cell cycle is a common strategy used by viruses to create a pro-replicative environment. To facilitate viral genome replication, vaccinia virus (VACV) has been reported to alter cell cycle regulation and trigger the host cell DNA damage response. However, the cellular factors and viral effectors that mediate these changes remain unknown. Here, we set out to investigate the effect of VACV infection on cell proliferation and host cell cycle progression. Using a subset of VACV mutants, we characterise the stage of infection required for inhibition of cell proliferation and define the viral effectors required to dysregulate the host cell cycle. Consistent with previous studies, we show that VACV inhibits and subsequently shifts the host cell cycle. We demonstrate that these two phenomena are independent of one another, with viral early genes being responsible for cell cycle inhibition, and post-replicative viral gene(s) responsible for the cell cycle shift. Extending previous findings, we show that the viral kinase F10 is required to activate the DNA damage checkpoint and that the viral B1 kinase and/or B12 pseudokinase mediate degradation of checkpoint effectors p53 and p21 during infection. We conclude that VACV modulates host cell proliferation and host cell cycle progression through temporal expression of multiple VACV effector proteins. (209/200.).

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.

Acrylamide inhibits vaccinia virus through vimentin-independent anti-viral granule formation.

The replication and assembly of vaccinia virus (VACV), the prototypic poxvirus, occurs exclusively in the cytoplasm of host cells. While the role of cellular cytoskeletal components in these processes remains poorly understood, vimentin-a type III intermediate filament-has been shown to associate with viral replication sites and to be incorporated into mature VACV virions. Here, we employed chemical and genetic approaches to further investigate the role of vimentin during the VACV lifecycle. The collapse of vimentin filaments, using acrylamide, was found to inhibit VACV infection at the level of genome replication, intermediate- and late-gene expression. However, we found that CRISPR-mediated knockout of vimentin did not impact VACV replication. Combining these tools, we demonstrate that acrylamide treatment results in the formation of anti-viral granules (AVGs) known to mediate translational inhibition of many viruses. We conclude that vimentin is dispensable for poxvirus replication and assembly and that acrylamide, as a potent inducer of AVGs during VACV infection, serves to bolster cell's anti-viral response to poxvirus infection.

Influenza- and MCMV-induced memory CD8 T cells control respiratory vaccinia virus infection despite residence in distinct anatomical niches.

Induction of memory CD8 T cells residing in peripheral tissues is of interest for T cell-based vaccines as these cells are located at mucosal and barrier sites and can immediately exert effector functions, thus providing protection in case of local pathogen encounter. Different memory CD8 T cell subsets patrol peripheral tissues, but it is unclear which subset is superior in providing protection upon secondary infections. We used influenza virus to induce predominantly tissue resident memory T cells or cytomegalovirus to elicit a large pool of effector-like memory cells in the lungs and determined their early protective capacity and mechanism of reactivation. Both memory CD8 T cell pools have unique characteristics with respect to their phenotype, localization, and maintenance. However, these distinct features do not translate into different capacities to control a respiratory vaccinia virus challenge in an antigen-specific manner, although differential activation mechanisms are utilized. While influenza-induced memory CD8 T cells respond to antigen by local proliferation, MCMV-induced memory CD8 T cells relocate from the vasculature into the tissue in an antigen-independent and partially chemokine-driven manner. Together these results bear relevance for the development of vaccines aimed at eliciting a protective memory CD8 T cell pool at mucosal sites.

Modulation of Early Host Innate Immune Response by an Avipox Vaccine Virus' Lateral Body Protein.

The avian pathogen fowlpox virus (FWPV) has been successfully used as a vaccine vector in poultry and humans, but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication-permissive and nonpermissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, nonessential FWPV gene knockout mutants revealed that FPV184 confers immunomodulatory capacity. We report that the FPV184-knockout virus (FWPVΔ184) induces the cellular IFN response as early as 2 h postinfection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPVΔ184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-ß promoter and IFN-α activation of the chicken Mx1 promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies, capable of suppressing IFN induction early during the next round of infection.

Author Correction: Quantitative mapping and minimization of super-resolution optical imaging artifacts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

SnapShot: Enveloped Virus Entry.

In order to initiate successful infection, viruses have to transmit and deliver their genome from one host cell or organism to another. To achieve this, enveloped viruses must first fuse their membrane with those of the target host cell. Here, we describe the sequence of events leading to the entry of representative enveloped viruses, highlighting the strategies they use to gain access to the host cell cytosol.

The zebrafish as a novel model for the in vivo study of Toxoplasma gondii replication and interaction with macrophages.

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we developed a zebrafish infection model to study the innate immune response to Toxoplasma in vivo By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision-driven automated image analysis, we reveal that Toxoplasma invades brain cells and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We also show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are cleared in vivo, we further analyzed Toxoplasma-macrophage interactions using time-lapse microscopy and three-dimensional correlative light and electron microscopy (3D CLEM). Time-lapse microscopy revealed that macrophages are recruited to the infection site and play a key role in Toxoplasma control. High-resolution 3D CLEM revealed parasitophorous vacuole breakage in brain cells and macrophages in vivo, suggesting that cell-intrinsic mechanisms may be used to destroy the intracellular niche of tachyzoites. Together, our results demonstrate in vivo control of Toxoplasma by macrophages, and highlight the possibility that zebrafish may be further exploited as a novel model system for discoveries within the field of parasite immunity.This article has an associated First Person interview with the first author of the paper.

Image-Based Quantitation of Host Cell-Toxoplasma gondii Interplay Using HRMAn: A Host Response to Microbe Analysis Pipeline.

Research on Toxoplasma gondii and its interplay with the host is often performed using fluorescence microscopy-based imaging experiments combined with manual quantification of acquired images. We present here an accurate and unbiased quantification method for host-pathogen interactions. We describe how to plan experiments and prepare, stain and image infected specimens and analyze them with the program HRMAn (Host Response to Microbe Analysis). HRMAn is a high-content image analysis method based on KNIME Analytics Platform. Users of this guide will be able to perform infection studies in high-throughput volume and to a greater level of detail. Relying on cutting edge machine learning algorithms, HRMAn can be trained and tailored to many experimental settings and questions.

Plant and insect herbivore community variation across the Paleocene-Eocene boundary in the Hanna Basin, southeastern Wyoming.

Ecosystem function and stability are highly affected by internal and external stressors. Utilizing paleobotanical data gives insight into the evolutionary processes an ecosystem undergoes across long periods of time, allowing for a more complete understanding of how plant and insect herbivore communities are affected by ecosystem imbalance. To study how plant and insect herbivore communities change during times of disturbance, we quantified community turnover across the Paleocene--Eocene boundary in the Hanna Basin, southeastern Wyoming. This particular location is unlike other nearby Laramide basins because it has an abundance of late Paleocene and Eocene coal and carbonaceous shales and paucity of well-developed paleosols, suggesting perpetually high water availability. We sampled approximately 800 semi-intact dicot leaves from five stratigraphic levels, one of which occurs late in the Paleocene-Eocene thermal maximum (PETM). Field collections were supplemented with specimens at the Denver Museum of Nature & Science. Fossil leaves were classified into morphospecies and herbivore damage was documented for each leaf. We tested for changes in plant and insect herbivore damage diversity using rarefaction and community composition using non-metric multidimensional scaling ordinations. We also documented changes in depositional environment at each stratigraphic level to better contextualize the environment of the basin. Plant diversity was highest during the mid-late Paleocene and decreased into the Eocene, whereas damage diversity was highest at the sites with low plant diversity. Plant communities significantly changed during the late PETM and do not return to pre-PETM composition. Insect herbivore communities also changed during the PETM, but, unlike plant communities, rebound to their pre-PETM structure. These results suggest that insect herbivore communities responded more strongly to plant community composition than to the diversity of species present.

Nanoscale polarization of the entry fusion complex of vaccinia virus drives efficient fusion.

To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins1. For many viruses, the clustering of fusion proteins-and their distribution on virus particles-is crucial for fusion activity2,3. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion4. However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity5. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection.

Quantitative PCR-Based Assessment of Vaccinia Virus RNA and DNA in Infected Cells.

Quantitative PCR-based methods have proven to be easy-to-use, cost-effective procedures for the quantification of viral gene expression and viral genome numbers. Quantitative PCR (qPCR) and quantitative reverse transcriptase-PCR (qRT-PCR) are rapid and sensitive approaches that can be used to pinpoint defects in viral DNA replication and transcriptional activity, respectively. Due to the significant nucleotide overlap between Poxviridae these methods can be employed across a wide range of viruses from this family. Here we provide methods for the quantification of vaccinia DNA replication by qPCR and quantification of the three classes of vaccinia gene transcription by qRT-PCR.