Poxvirus A51R: A microtubule maestro and virulence virtuoso.

Seo et al.1 shed light on virus-host interactions as they reveal how poxvirus A51R stabilizes microtubules in infected cells, which impacts vaccinia virus virulence in mice by potentially inhibiting reactive-oxygen-species-dependent antiviral responses in macrophages.

The Recombinant Oncolytic Virus VV-GMCSF-Lact and Chemotherapy Drugs against Human Glioma.

Virotherapy is one of the perspective technologies in the treatment of malignant neoplasms. Previously, we have developed oncolytic vaccinia virus VV-GMCSF-Lact and its high cytotoxic activity and antitumor efficacy against glioma was shown. In this work, using immortalized and patient-derived cells with different sensitivity to VV-GMCSF-Lact, we evaluated the cytotoxic effect of chemotherapy agents. Additionally, we studied the combination of VV-GMCSF-Lact with temozolomide which is the most preferred drug for glioma treatment. Experimental results indicate that first adding temozolomide and then the virus to the cells is inherently more efficient than dosing it in the reverse order. Testing these regimens in the U87 MG xenograft glioblastoma model confirmed this effect, as assessed by tumor growth inhibition index and histological analysis. Moreover, VV-GMCSF-Lact as monotherapy is more effective against U87 MG glioblastoma xenografts comparing temozolomide.

HDAC5 enhances IRF3 activation and is targeted for degradation by protein C6 from orthopoxviruses including Monkeypox virus and Variola virus.

Histone deacetylases (HDACs) regulate gene expression and innate immunity. Previously, we showed that HDAC5 is degraded during Vaccinia virus (VACV) infection and is a restriction factor for VACV and herpes simplex virus type 1. Here, we report that HDAC5 promotes interferon regulatory factor 3 (IRF3) activation downstream of Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 or Sendai virus-mediated stimulation without requiring HDAC activity. Loss of HDAC5-mediated IRF3 activation is restored by re-introduction of HDAC5 but not HDAC1 or HDAC4. The antiviral activity of HDAC5 is antagonized by VACV protein C6 and orthologs from the orthopoxviruses cowpox, rabbitpox, camelpox, monkeypox, and variola. Infection by many of these viruses induces proteasomal degradation of HDAC5, and expression of C6 alone can induce HDAC5 degradation. Mechanistically, C6 binds to the dimerization domain of HDAC5 and prevents homodimerization and heterodimerization with HDAC4. Overall, this study describes HDAC5 as a positive regulator of IRF3 activation and provides mechanistic insight into how the poxviral protein C6 binds to HDAC5 to antagonize its function.

Poxvirus A51R proteins regulate microtubule stability and antagonize a cell-intrinsic antiviral response.

Numerous viruses alter host microtubule (MT) networks during infection, but how and why they induce these changes is unclear in many cases. We show that the vaccinia virus (VV)-encoded A51R protein is a MT-associated protein (MAP) that directly binds MTs and stabilizes them by both promoting their growth and preventing their depolymerization. Furthermore, we demonstrate that A51R-MT interactions are conserved across A51R proteins from multiple poxvirus genera, and highly conserved, positively charged residues in A51R proteins mediate these interactions. Strikingly, we find that viruses encoding MT interaction-deficient A51R proteins fail to suppress a reactive oxygen species (ROS)-dependent antiviral response in macrophages that leads to a block in virion morphogenesis. Moreover, A51R-MT interactions are required for VV virulence in mice. Collectively, our data show that poxviral MAP-MT interactions overcome a cell-intrinsic antiviral ROS response in macrophages that would otherwise block virus morphogenesis and replication in animals.

Transient inhibition of neutrophil functions enhances the antitumor effect of intravenously delivered oncolytic vaccinia virus.

Oncolytic viruses (OVs) possess the unique ability to selectively replicate within tumor cells, leading to their destruction, while also reversing the immunosuppression within the tumor microenvironment and triggering an antitumor immune response. As a result, OVs have emerged as one of the most promising approaches in cancer therapy. However, the effective delivery of intravenously administered OVs faces significant challenges imposed by various immune cells within the peripheral blood, hindering their access to tumor sites. Notably, neutrophils, the predominant white blood cell population comprising approximately 50%-70% of circulating white cells in humans, show phagocytic properties. Our investigation revealed that the majority of oncolytic vaccinia viruses (VV) are engulfed and degraded by neutrophils in the bloodstream. The depletion of neutrophils using the anti-LY6G Ab (1-A8) resulted in an increased accumulation of circulating oncolytic VV in the peripheral blood and enhanced deposition at the tumor site, consequently amplifying the antitumor effect. Neutrophils heavily rely on PI3K signaling to sustain their phagocytic process. Additionally, our study determined that the inhibition of the PI3Kinase delta isoform by idelalisib (CAL-101) suppressed the uptake of oncolytic VV by neutrophils. This inhibition led to a greater presence of oncolytic VV in both the peripheral blood and at the tumor site, resulting in improved efficacy against the tumor. In conclusion, our study showed that inhibiting neutrophil functions can significantly enhance the antitumor efficacy of intravenous oncolytic VV.

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.

JAK Signaling Is Critically Important in Cytokine-Induced Viral Susceptibility of Keratinocytes.

Little is known about whether type 1 (IFNγ), 2 (IL-4/IL-13), or 3 (IL-17A/IL-22) cytokines affect the susceptibility of keratinocytes (KC) to viruses. These immune pathways predominate in various skin diseases: lupus, atopic dermatitis (AD), and psoriasis, respectively. Janus kinase inhibitors (JAKi) are approved to treat both AD and psoriasis, and are in clinical development for lupus. We evaluated whether these cytokines alter viral susceptibility of KC and determined if this effect is modulated by treatment with JAKi. Viral susceptibility to vaccinia virus (VV) or herpes simplex virus-1 (HSV-1) ± JAKi was assessed in immortalized and primary human KC pretreated with cytokines. Exposure to type 2 (IL-4 + IL-13) or the type 3 (IL-22) cytokines significantly increased KC viral susceptibility. Specifically, there was a peak increase of 12.2 ± 3.1-fold (IL-4 + IL-13) or 7.7 ± 2.8-fold (IL-22) in VV infection as measured by plaque number. Conversely, IFNγ significantly reduced susceptibility to VV (63.1 ± 64.4-fold). The IL-4 + IL-13-induced viral susceptibility was reduced (44 ± 16%) by JAK1 inhibition, while the IL-22-enhanced viral susceptibility was diminished (76 ± 19%) by TYK2 inhibition. IFNγ-mediated resistance to viral infection was reversed by JAK2 inhibition (366 ± 294% increase in infection). Cytokines expressed in AD skin (IL-4, IL-13, IL-22) increase KC viral susceptibility while IFNγ is protective. JAKi that target JAK1 or TYK2 reversed cytokine-enhanced viral susceptibility, while JAK2 inhibition reduced the protective effects of IFNγ.

Antiviral activities of two nucleos(t)ide analogs against vaccinia, mpox, and cowpox viruses in primary human fibroblasts.

Many poxviruses are significant human and animal pathogens, including viruses that cause smallpox and mpox (formerly monkeypox). Identifying novel and potent antiviral compounds is critical to successful drug development targeting poxviruses. Here we tested two compounds, nucleoside trifluridine, and nucleotide adefovir dipivoxil, for antiviral activities against vaccinia virus (VACV), mpox virus (MPXV), and cowpox virus (CPXV) in physiologically relevant primary human fibroblasts. Both compounds potently inhibited the replication of VACV, CPXV, and MPXV (MA001 2022 isolate) in plaque assays. In our recently developed assay based on a recombinant VACV expressing secreted Gaussia luciferase, they both exhibited high potency in inhibiting VACV replication with EC50s in the low nanomolar range. In addition, both trifluridine and adefovir dipivoxil inhibited VACV DNA replication and downstream viral gene expression. Our results characterized trifluridine and adefovir dipivoxil as strong poxvirus antiviral compounds and further validate the VACV Gaussia luciferase assay as a highly efficient and reliable reporter tool for identifying poxvirus inhibitors. Given that both compounds are FDA-approved drugs, and trifluridine is already used to treat ocular vaccinia, further development of trifluridine and adefovir dipivoxil holds great promise in treating poxvirus infections, including mpox.

Transcriptional reprogramming of natural killer cells by vaccinia virus shows both distinct and conserved features with mCMV.

Natural killer (NK) cells have an established role in controlling poxvirus infection and there is a growing interest to exploit their capabilities in the context of poxvirus-based oncolytic therapy and vaccination. How NK cells respond to poxvirus-infected cells to become activated is not well established. To address this knowledge gap, we studied the NK cell response to vaccinia virus (VACV) in vivo, using a systemic infection murine model. We found broad alterations in NK cells transcriptional activity in VACV-infected mice, consistent with both direct target cell recognition and cytokine exposure. There were also alterations in the expression levels of specific NK surface receptors (NKRs), including the Ly49 family and SLAM receptors, as well as upregulation of memory-associated NK markers. Despite the latter observation, adoptive transfer of VACV-expercienced NK populations did not confer protection from infection. Comparison with the NK cell response to murine cytomegalovirus (MCMV) infection highlighted common features, but also distinct NK transcriptional programmes initiated by VACV. Finally, there was a clear overlap between the NK transcriptional response in humans vaccinated with an attenuated VACV, modified vaccinia Ankara (MVA), demonstrating conservation between the NK response in these different host species. Overall, this study provides new data about NK cell activation, function, and homeostasis during VACV infection, and may have implication for the design of VACV-based therapeutics.

RACK1 Regulates Poxvirus Protein Synthesis Independently of Its Role in Ribosome-Based Stress Signaling.

Receptor for activated C kinase 1 (RACK1) is a small ribosomal subunit protein that is phosphorylated by vaccinia virus (VacV) to maximize translation of postreplicative (PR) mRNAs that harbor 5' polyA leaders. However, RACK1 is a multifunctional protein that both controls translation directly and acts as a scaffold for signaling to and from the ribosome. This includes stress signaling that is activated by ribosome-associated quality control (RQC) and ribotoxic stress response (RSR) pathways. As VacV infection activates RQC and stress signaling, whether RACK1 influences viral protein synthesis through its effects on translation, signaling, or both remains unclear. Examining the effects of genetic knockout of RACK1 on the phosphorylation of key mitogenic and stress-related kinases, we reveal that loss of RACK1 specifically blunts the activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) at late stages of infection. However, RACK1 was not required for JNK recruitment to ribosomes, and unlike RACK1 knockout, JNK inhibitors had no effect on viral protein synthesis. Moreover, reduced JNK activity during infection in RACK1 knockout cells contrasted with the absolute requirement for RACK1 in RSR-induced JNK phosphorylation. Comparing the effects of RACK1 knockout alongside inhibitors of late stage replication, our data suggest that JNK activation is only indirectly affected by the absence of RACK1 due to reduced viral protein accumulation. Cumulatively, our findings in the context of infection add further support for a model whereby RACK1 plays a specific and direct role in controlling translation of PR viral mRNAs that is independent of its role in ribosome-based stress signaling. IMPORTANCE Receptor for activated C kinase 1 (RACK1) is a multifunctional ribosomal protein that regulates translation directly and mediates signaling to and from the ribosome. While recent work has shown that RACK1 is phosphorylated by vaccinia virus (VacV) to stimulate translation of postreplicative viral mRNAs, whether RACK1 also contributes to VacV replication through its roles in ribosome-based stress signaling remains unclear. Here, we characterize the role of RACK1 in infected cells. In doing so, we find that RACK1 is essential for stress signal activation by ribotoxic stress responses but not by VacV infection. Moreover, although the loss of RACK1 reduces the level of stress-associated JNK activation in infected cells, this is an indirect consequence of RACK1's specific requirement for the synthesis of postreplicative viral proteins, the accumulation of which determines the level of cellular stress. Our findings reveal both the specific role of RACK1 and the complex downstream effects of its control of viral protein synthesis in the context of infection.

Dysregulation of Cellular VRK1, BAF, and Innate Immune Signaling by the Vaccinia Virus B12 Pseudokinase.

Poxvirus proteins remodel signaling throughout the cell by targeting host enzymes for inhibition and redirection. Recently, it was discovered that early in infection the vaccinia virus (VACV) B12 pseudokinase copurifies with the cellular kinase VRK1, a proviral factor, in the nucleus. Although the formation of this complex correlates with inhibition of cytoplasmic VACV DNA replication and likely has other downstream signaling consequences, the molecular mechanisms involved are poorly understood. Here, we further characterize how B12 and VRK1 regulate one another during poxvirus infection. First, we demonstrate that B12 is stabilized in the presence of VRK1 and that VRK1 and B12 coinfluence their respective solubility and subcellular localization. In this regard, we find that B12 promotes VRK1 colocalization with cellular DNA during mitosis and that B12 and VRK1 may be tethered cooperatively to chromatin. Next, we observe that the C-terminal tail of VRK1 is unnecessary for B12-VRK1 complex formation or its proviral activity. Interestingly, we identify a point mutation of B12 capable of abrogating interaction with VRK1 and which renders B12 nonrepressive during infection. Lastly, we investigated the influence of B12 on the host factor BAF and antiviral signaling pathways and find that B12 triggers redistribution of BAF from the cytoplasm to the nucleus. In addition, B12 increases DNA-induced innate immune signaling, revealing a new functional consequence of the B12 pseudokinase. Together, this study characterizes the multifaceted roles B12 plays during poxvirus infection that impact VRK1, BAF, and innate immune signaling. IMPORTANCE Protein pseudokinases comprise a considerable fraction of the human kinome, as well as other forms of life. Recent studies have demonstrated that their lack of key catalytic residues compared to their kinase counterparts does not negate their ability to intersect with molecular signal transduction. While the multifaceted roles pseudokinases can play are known, their contribution to virus infection remains understudied. Here, we further characterize the mechanism of how the VACV B12 pseudokinase and human VRK1 kinase regulate one another in the nucleus during poxvirus infection and inhibit VACV DNA replication. We find that B12 disrupts regulation of VRK1 and its downstream target BAF, while also enhancing DNA-dependent innate immune signaling. Combined with previous data, these studies contribute to the growing field of nuclear pathways targeted by poxviruses and provide evidence of unexplored roles of B12 in the activation of antiviral immunity.

DDX50 Is a Viral Restriction Factor That Enhances IRF3 Activation.

The transcription factors IRF3 and NF-κB are crucial in innate immune signalling in response to many viral and bacterial pathogens. However, mechanisms leading to their activation remain incompletely understood. Viral RNA can be detected by RLR receptors, such as RIG-I and MDA5, and the dsRNA receptor TLR3. Alternatively, the DExD-Box RNA helicases DDX1-DDX21-DHX36 activate IRF3/NF-κB in a TRIF-dependent manner independent of RIG-I, MDA5, or TLR3. Here, we describe DDX50, which shares 55.6% amino acid identity with DDX21, as a non-redundant factor that promotes activation of the IRF3 signalling pathway following its stimulation with viral RNA or infection with RNA and DNA viruses. Deletion of DDX50 in mouse and human cells impaired IRF3 phosphorylation and IRF3-dependent endogenous gene expression and cytokine/chemokine production in response to cytoplasmic dsRNA (polyIC transfection), and infection by RNA and DNA viruses. Mechanistically, whilst DDX50 co-immunoprecipitated TRIF, it acted independently to the previously described TRIF-dependent RNA sensor DDX1. Indeed, shRNA-mediated depletion of DDX1 showed DDX1 was dispensable for signalling in response to RNA virus infection. Importantly, loss of DDX50 resulted in a significant increase in replication and dissemination of virus following infection with vaccinia virus, herpes simplex virus, or Zika virus, highlighting its important role as a broad-ranging viral restriction factor.

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.).

The crystal structure of vaccinia virus protein E2 and perspectives on the prediction of novel viral protein folds.

The morphogenesis of vaccinia virus (VACV, family Poxviridae), the smallpox vaccine, is a complex process involving multiple distinct cellular membranes and resulting in multiple different forms of infectious virion. Efficient release of enveloped virions, which promote systemic spread of infection within hosts, requires the VACV protein E2 but the molecular basis of E2 function remains unclear and E2 lacks sequence homology to any well-characterised family of proteins. We solved the crystal structure of VACV E2 to 2.3 Å resolution, revealing that it comprises two domains with novel folds: an N-terminal annular (ring) domain and a C-terminal globular (head) domain. The C-terminal head domain displays weak structural homology with cellular (pseudo)kinases but lacks conserved surface residues or kinase features, suggesting that it is not enzymatically active, and possesses a large surface basic patch that might interact with phosphoinositide lipid headgroups. Recent deep learning methods have revolutionised our ability to predict the three-dimensional structures of proteins from primary sequence alone. VACV E2 is an exemplar 'difficult' viral protein target for structure prediction, being comprised of multiple novel domains and lacking sequence homologues outside Poxviridae. AlphaFold2 nonetheless succeeds in predicting the structures of the head and ring domains with high and moderate accuracy, respectively, allowing accurate inference of multiple structural properties. The advent of highly accurate virus structure prediction marks a step-change in structural virology and beckons a new era of structurally-informed molecular virology.

Acid Stripping after Infection Improves the Detection of Viral HLA Class I Natural Ligands Identified by Mass Spectrometry.

Identification of a natural human leukocyte antigen (HLA) ligandome is a key element to understand the cellular immune response. Advanced high throughput mass spectrometry analyses identify a relevant, but not complete, fraction of the many tens of thousands of self-peptides generated by antigen processing in live cells. In infected cells, in addition to this complex HLA ligandome, a minority of peptides from degradation of the few proteins encoded by the viral genome are also bound to HLA class I molecules. In this study, the standard immunopeptidomics strategy was modified to include the classical acid stripping treatment after virus infection to enrich the HLA ligandome in virus ligands. Complexes of HLA-B*27:05-bound peptide pools were isolated from vaccinia virus (VACV)-infected cells treated with acid stripping after virus infection. The HLA class I ligandome was identified using high throughput mass spectrometry analyses, yielding 37 and 51 natural peptides processed and presented untreated and after acid stripping treatment VACV-infected human cells, respectively. Most of these virus ligands were identified in both conditions, but exclusive VACV ligands detected by mass spectrometry detected on acid stripping treatment doubled the number of those identified in the untreated VACV-infected condition. Theoretical binding affinity prediction of the VACV HLA-B*27:05 ligands and acute antiviral T cell response characterization in the HLA transgenic mice model showed no differences between HLA ligands identified under the two conditions: untreated and under acid stripping condition. These findings indicated that acid stripping treatment could be useful to identify HLA class I ligands from virus-infected cells.

A phase 1b study of intraperitoneal oncolytic viral immunotherapy in platinum-resistant or refractory ovarian cancer.

OBJECTIVE: Our objective was to assess safety and adverse events associated with intraperitoneal Olvi-Vec virotherapy in patients with platinum-resistant or refractory ovarian cancer (PRROC). Secondary objectives included objective response rate (ORR) per RECIST 1.1 and progression-free survival (PFS). METHODS: Olvi-Vec is a modified vaccinia virus that causes oncolysis and immune activation. An open-label phase 1b trial using a 3 + 3 dose escalation was conducted. Intraperitoneal Olvi-Vec was given as monotherapy in two consecutive daily doses. Translational analyses included anti-virus antibody levels, viral shedding, circulating tumor cells (CTCs) and T cells. RESULTS: Twelve patients (median age: 69 years, range: 45-77) with median 5 prior therapies (range: 2-10) and 2 prior platinum lines (range: 1-5) were enrolled. There were three dose level cohorts: 3 × 109 (n = 6), 1 × 1010 (n = 5), and 2.5 × 1010 (n = 1) plaque forming units (PFU)/day on two consecutive days. Treatment-related adverse events (TRAEs) included G1/G2 nausea (n = 6), fever (n = 6), abdominal distention (n = 5), and abdominal pain (n = 4). There were no Grade 4 TRAEs, no dose relationship to TRAEs, and no deaths attributed to Olvi-Vec. The ORR was 9% (1/11). Stable disease (SD) was 64% (7/11), and SD ≥15 weeks was 46% (5/11). Median PFS was 15.7 weeks (95%CI: 5.7-34.5), including extended PFS in four patients (23.2, 34.5, 59.4+ and 70.8 weeks). Three patients had extended overall survival (deceased 33.6 months, and alive with disease at 54 and 59 months). CTCs diminished in 6/8 (75%) baseline-positive patients. Immune activation was demonstrated from virus-enhanced tumor infiltration of CD8+ T-cells and activation of tumor-specific T-cells in peripheral blood. CONCLUSIONS: Oncolytic viral therapy with intraperitoneal Olvi-Vec showed promising safety, clinical activities, and immune activation in patients with PRROC, warranting further clinical investigation.

Adaptive Immune Response to Vaccinia Virus LIVP Infection of BALB/c Mice and Protection against Lethal Reinfection with Cowpox Virus.

Mass vaccination has played a critical role in the global eradication of smallpox. Various vaccinia virus (VACV) strains, whose origin has not been clearly documented in most cases, have been used as live vaccines in different countries. These VACV strains differed in pathogenicity towards various laboratory animals and in reactogenicity exhibited upon vaccination of humans. In this work, we studied the development of humoral and cellular immune responses in BALB/c mice inoculated intranasally (i.n.) or intradermally (i.d.) with the VACV LIVP strain at a dose of 105 PFU/mouse, which was used in Russia as the first generation smallpox vaccine. Active synthesis of VACV-specific IgM in the mice occurred on day 7 after inoculation, reached a maximum on day 14, and decreased by day 29. Synthesis of virus-specific IgG was detected only from day 14, and the level increased significantly by day 29 after infection of the mice. Immunization (i.n.) resulted in significantly higher production of VACV-specific antibodies compared to that upon i.d. inoculation of LIVP. There were no significant differences in the levels of the T cell response in mice after i.n. or i.d. VACV administration at any time point. The maximum level of VACV-specific T-cells was detected on day 14. By day 29 of the experiment, the level of VACV-specific T-lymphocytes in the spleen of mice significantly decreased for both immunization procedures. On day 30 after immunization with LIVP, mice were infected with the cowpox virus at a dose of 46 LD50. The i.n. immunized mice were resistant to this infection, while 33% of i.d. immunized mice died. Our findings indicate that the level of the humoral immune response to vaccination may play a decisive role in protection of animals from orthopoxvirus reinfection.

Engineered Promoter-Switched Viruses Reveal the Role of Poxvirus Maturation Protein A26 as a Negative Regulator of Viral Spread.

Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MVs are further wrapped and secreted to spread infection within the host. However, late during infection, MVs accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here, we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titers. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate limiting for EV production. The A26 gene is conserved within vertebrate poxviruses but is strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfill different functions represents a novel level of complexity in mammalian viruses with major impacts on evolution, adaptation, and transmission. IMPORTANCE Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways, including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intrahost dissemination and interhost transmission, so virus maturation pathways must be tightly controlled. Here, we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation toward the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and A26 provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses, but not in those transmitted exclusively via biting arthropods, reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.

Spontaneous and Targeted Mutations in the Decapping Enzyme Enhance Replication of Modified Vaccinia Virus Ankara (MVA) in Monkey Cells.

Modified vaccinia virus Ankara (MVA) was derived by repeated passaging in chick fibroblasts, during which deletions and mutations rendered the virus unable to replicate in most mammalian cells. Marker rescue experiments demonstrated that the host range defect could be overcome by replacing DNA that had been deleted from near the left end of the genome. One virus isolate, however, recovered the ability to replicate in monkey BS-C-1 cells but not human cells without added DNA, suggesting that it arose from a spontaneous mutation. Here, we showed that variants with enhanced ability to replicate in BS-C-1 cells could be isolated by blind passaging of MVA and that in each there was a point mutation leading to an amino acid substitution in the D10 decapping enzyme. The sufficiency of these single mutations to enhance host range was confirmed by constructing recombinant viruses. The D10 mutations occurred at N- or C-terminal locations distal to the active site, suggesting an indirect effect on decapping or on another previously unknown role of D10. Although increased amounts of viral mRNA and proteins were found in BS-C-1 cells infected with the mutants compared to those with parental MVA, the increases were much less than the 1- to 2-log-higher virus yields. Nevertheless, a contributing role for diminished decapping in overcoming the host range defect was consistent with increased replication and viral protein synthesis in BS-C-1 cells infected with an MVA engineered to have active-site mutations that abrogate decapping activity entirely. Optimal decapping may vary depending on the biological context. IMPORTANCE Modified vaccinia virus Ankara (MVA) is an attenuated virus that is approved as a smallpox vaccine and is in clinical trials as a vector for other pathogens. The safety of MVA is due in large part to its inability to replicate in mammalian cells. Although host range restriction is considered a stable feature of the virus, we describe the occurrence of spontaneous mutations in MVA that increase replication considerably in monkey BS-C-1 cells but only slightly in human cells. The mutants contain single nucleotide changes that lead to amino acid substitutions in one of the two decapping enzymes. Although the spontaneous mutations are distant from the decapping enzyme active site, engineered active-site mutations also increased virus replication in BS-C-1 cells. The effects of these mutations on the immunogenicity of MVA vectors remain to be determined.

Insights into the Organization of the Poxvirus Multicomponent Entry-Fusion Complex from Proximity Analyses in Living Infected Cells.

Poxviruses are exceptional in having a complex entry-fusion complex (EFC) that is comprised of 11 conserved proteins embedded in the membrane of mature virions. However, the detailed architecture is unknown and only a few bimolecular protein interactions have been demonstrated by coimmunoprecipitation from detergent-treated lysates and by cross-linking. Here, we adapted the tripartite split green fluorescent protein (GFP) complementation system in order to analyze EFC protein contacts within living cells. This system employs a detector fragment called GFP1-9 comprised of nine GFP ß-strands. To achieve fluorescence, two additional 20-amino-acid fragments called GFP10 and GFP11 attached to interacting proteins are needed, providing the basis for identification of the latter. We constructed a novel recombinant vaccinia virus (VACV-GFP1-9) expressing GFP1-9 under a viral early/late promoter and plasmids with VACV late promoters regulating each of the EFC proteins with GFP10 or GFP11 attached to their ectodomains. GFP fluorescence was detected by confocal microscopy at sites of virion assembly in cells infected with VACV-GFP1-9 and cotransfected with plasmids expressing one EFC-GFP10 and one EFC-GFP11 interacting protein. Flow cytometry provided a quantitative way to determine the interaction of each EFC-GFP10 protein with every other EFC-GFP11 protein in the context of a normal infection in which all viral proteins are synthesized and assembled. Previous EFC protein interactions were confirmed, and new ones were discovered and corroborated by additional methods. Most remarkable was the finding that the small, hydrophobic O3 protein interacted with each of the other EFC proteins. IMPORTANCE Poxviruses are enveloped viruses with a DNA-containing core that enters cells following fusion of viral and host membranes. This essential step is a target for vaccines and therapeutics. The entry-fusion complex (EFC) of poxviruses is unusually complex and comprised of 11 conserved viral proteins. Determination of the structure of the EFC is a prerequisite for understanding the fusion mechanism. Here, we used a tripartite split green fluorescent protein assay to determine the proximity of individual EFC proteins in living cells. A network connecting components of the EFC was derived.