Oncolytic vaccinia virus immunotherapy antagonizes image-guided radiotherapy in mouse mammary tumor models.

Ionizing radiation (IR) and oncolytic viruses are both used to treat cancer, and the effectiveness of both agents depends upon stimulating an immune response against the tumor. In this study we tested whether combining image guided ionizing radiation (IG-IR) with an oncolytic vaccinia virus (VACV) could yield a better therapeutic response than either treatment alone. ΔF4LΔJ2R VACV grew well on irradiated human and mouse breast cancer cells, and the virus can be combined with 4 or 8 Gy of IR to kill cells in an additive or weakly synergistic manner. To test efficacy in vivo we used immune competent mice bearing orthotopic TUBO mammary tumors. IG-IR worked well with 10 Gy producing 80% complete responses, but this was halved when the tumors were treated with VACV starting 2 days after IG-IR. VACV monotherapy was ineffective in this model. The antagonism was time dependent as waiting for 21 days after IG-IR eliminated the inhibitory effect but without yielding any further benefits over IR alone. In irradiated tumors, VACV replication was also lower, suggesting that irradiation created an environment that did not support infection as well in vivo as in vitro. A study of how four different treatment regimens affected the immune composition of the tumor microenvironment showed that treating irradiated tumors with VACV altered the immunological profiles in tumors exposed to IR or VACV alone. We detected more PD-1 and PD-L1 expression in tumors exposed to IR+VACV but adding an αPD-1 antibody to the protocol did not change the way VACV interferes with IG-IR therapy. VACV encodes many immunosuppressive gene products that may interfere with the ability of radiotherapy to induce an effective anti-tumor immune response through the release of danger-associated molecular patterns. These data suggest that infecting irradiated tumors with VACV, too soon after exposure, may interfere in the innate and linked adaptive immune responses that are triggered by radiotherapy to achieve a beneficial impact.

Monkeypox virus infection of human astrocytes causes gasdermin B cleavage and pyroptosis.

Monkeypox virus (MPXV) infections in humans cause neurological disorders while studies of MPXV-infected animals indicate that the virus penetrates the brain. Pyroptosis is an inflammatory type of regulated cell death, resulting from plasma membrane rupture (PMR) due to oligomerization of cleaved gasdermins to cause membrane pore formation. Herein, we investigated the human neural cell tropism of MPXV compared to another orthopoxvirus, vaccinia virus (VACV), as well as its effects on immune responses and cell death. Astrocytes were most permissive to MPXV (and VACV) infections, followed by microglia and oligodendrocytes, with minimal infection of neurons based on plaque assays. Aberrant morphological changes were evident in MPXV-infected astrocytes that were accompanied with viral protein (I3) immunolabelling and detection of over 125 MPXV-encoded proteins in cell lysates by mass spectrometry. MPXV- and VACV-infected astrocytes showed increased expression of immune gene transcripts (IL12, IRF3, IL1B, TNFA, CASP1, and GSDMB). However, MPXV infection of astrocytes specifically induced proteolytic cleavage of gasdermin B (GSDMB) (50 kDa), evident by the appearance of cleaved N-terminal-GSDMB (30 kDa) and C-terminal- GSDMB (18 kDa) fragments. GSDMB cleavage was associated with release of lactate dehydrogenase and increased cellular nucleic acid staining, indicative of PMR. Pre-treatment with dimethyl fumarate reduced cleavage of GSDMB and associated PMR in MPXV-infected astrocytes. Human astrocytes support productive MPXV infection, resulting in inflammatory gene induction with accompanying GSDMB-mediated pyroptosis. These findings clarify the recently recognized neuropathogenic effects of MPXV in humans while also offering potential therapeutic options.

Immunogenicity and Efficacy of TNX-1800, A Live Virus Recombinant Poxvirus Vaccine Candidate, against SARS-CoV-2 Challenge in Nonhuman Primates.

TNX-1800 is a synthetically derived live recombinant chimeric horsepox virus (rcHPXV) vaccine candidate expressing Wuhan SARS-CoV-2 spike (S) protein. The primary objective of this study was to evaluate the immunogenicity and efficacy of TNX-1800 in two nonhuman primate species challenged with USA-WA1/2020 SARS-CoV-2. TNX-1800 vaccination was well tolerated with no serious adverse events or significant changes in clinical parameters. A single dose of TNX-1800 generated humoral responses in African Green Monkeys and Cynomolgus Macaques, as measured by the total binding of anti-SARS-CoV-2 S IgG and neutralizing antibody titers against the USA-WA1/2020 strain. In addition, a single dose of TNX-1800 induced an interferon-gamma (IFN-γ)-mediated T-cell response in Cynomolgus Macaques. Following challenge with SARS-CoV-2, African Green and Cynomolgus Macaques exhibited rapid clearance of virus in the upper and lower respiratory tract. Future studies will assess the efficacy of TNX-1800 against newly emerging variants and demonstrate its safety in humans.

Immunogenicity and Tolerability of a SARS-CoV-2 TNX-1800, a Live Recombinant Poxvirus Vaccine Candidate, in Syrian Hamsters and New Zealand White Rabbits.

TNX-1800 is a preclinical stage synthetic-derived live attenuated chimeric horsepox virus vaccine engineered to express the SARS-CoV-2 spike (S) gene. The objectives of this study were to assess the safety, tolerability, and immunogenicity of TNX-1800 administration in Syrian golden hamsters and New Zealand white rabbits. Animals were vaccinated at three doses via percutaneous inoculation. The data showed that the single percutaneous administration of three TNX-1800 vaccine dose levels was well tolerated in both hamsters and rabbits. At all dose levels, rabbits were more decerning regarding vaccine site reaction than hamsters. Lastly, no TNX-1800 genomes could be detected at the site of vaccination. Post-vaccination, all animals had anti-SARS-CoV-2 spike protein IgG specific antibody responses. These data demonstrate that TNX-1800 infection was limited, asymptomatic, and cleared by the end of this study, and a single dose was able to generate immune responses.

Radiation combined with oncolytic vaccinia virus provides pronounced antitumor efficacy and induces immune protection in an aggressive glioblastoma model.

Glioblastoma (GB) is a malignant and immune-suppressed brain cancer that remains incurable despite the current standard of care. Radiotherapy is a mainstay of GB treatment, however invasive cancer cells outside the irradiated field and radioresistance preclude complete eradication of GB cells. Oncolytic virus therapy harnesses tumor-selective viruses to spread through and destroy tumors while stimulating antitumor immune responses, and thus has potential for use following radiotherapy. We demonstrate that oncolytic ΔF4LΔJ2R vaccinia virus (VACV) replicates in and induces cytotoxicity of irradiated brain tumor initiating cells in vitro. Importantly, a single 10 Gy dose of radiation combined with ΔF4LΔJ2R VACV produced considerably superior anticancer effects relative to either monotherapy when treating immune-competent orthotopic CT2A-luc mouse models-significantly extending survival and curing the majority of mice. Mice cured by the combination displayed significantly increased survival relative to naïve age-matched controls following intracranial tumor challenge, with some complete rejections. Further, the combination therapy was associated with an increased ratio of CD8+ effector T cells to regulatory T cells compared to either monotherapy. This study validates the use of radiation with an oncolytic ΔF4LΔJ2R VACV to improve treatment of this malignant brain cancer.

Single Dose of Recombinant Chimeric Horsepox Virus (TNX-801) Vaccination Protects Macaques from Lethal Monkeypox Challenge.

The ongoing global Monkeypox outbreak that started in the spring of 2022 has reinforced the importance of protecting the population using live virus vaccines based on the vaccinia virus (VACV). Smallpox also remains a biothreat and although some U.S. military personnel are immunized with VACV, safety concerns limit its use in other vulnerable groups. Consequently, there is a need for an effective and safer, single dose, live replicating vaccine against both viruses. One potential approach is to use the horsepox virus (HPXV) as a vaccine. Contemporary VACV shares a common ancestor with HPXV, which from the time of Edward Jenner and through the 19th century, was extensively used to vaccinate against smallpox. However, it is unknown if early HPXV-based vaccines exhibited different safety and efficacy profiles compared to modern VACV. A deeper understanding of HPXV as a vaccine platform may allow the construction of safer and more effective vaccines against the poxvirus family. In a proof-of-concept study, we vaccinated cynomolgus macaques with TNX-801, a recombinant chimeric horsepox virus (rcHPXV), and showed that the vaccine elicited protective immune responses against a lethal challenge with monkeypox virus (MPXV), strain Zaire. The vaccine was well tolerated and protected animals from the development of lesions and severe disease. These encouraging data support the further development of TNX-801.

SARS-CoV-2 infection downregulates myocardial ACE2 and potentiates cardiac inflammation in humans and hamsters.

Myocardial pathologies resulting from SARS-CoV-2 infections are consistently rising with mounting case rates and reinfections; however, the precise global burden is largely unknown and will have an unprecedented impact. Understanding the mechanisms of COVID-19-mediated cardiac injury is essential toward the development of cardioprotective agents that are urgently needed. Assessing novel therapeutic strategies to tackle COVID-19 necessitates an animal model that recapitulates human disease. Here, we sought to compare SARS-CoV-2-infected animals with patients with COVID-19 to identify common mechanisms of cardiac injury. Two-month-old hamsters were infected with either the ancestral (D614) or Delta variant (B.1.617.2) of SARS-CoV-2 for 2 days, 7 days, and/or 14 days. We measured viral RNA and cytokine expression at the earlier time points to capture the initial stages of infection in the lung and heart. We assessed myocardial angiotensin-converting enzyme 2 (ACE2), the entry receptor for the SARS-CoV-2 virus, and cardioprotective enzyme, as well as markers for inflammatory cell infiltration in the hamster hearts at days 7 and 14. In parallel, human hearts were stained for ACE2, viral nucleocapsid, and inflammatory cells. Indeed, we identify myocardial ACE2 downregulation and myeloid cell burden as common events in both hamsters and humans infected with SARS-CoV-2, and we propose targeting downstream ACE2 downregulation as a therapeutic avenue that warrants clinical investigation.NEW & NOTEWORTHY Cardiac manifestations of COVID-19 in humans are mirrored in the SARS-CoV-2 hamster model, recapitulating myocardial damage, ACE2 downregulation, and a consistent pattern of immune cell infiltration independent of viral dose and variant. Therefore, the hamster model is a valid approach to study therapeutic strategies for COVID-19-related heart disease.

Alzheimer's disease as an autoimmune disorder of innate immunity endogenously modulated by tryptophan metabolites.

Introduction: Alzheimer's disease (AD) is characterized by neurotoxic immuno-inflammation concomitant with cytotoxic oligomerization of amyloid beta (Aß) and tau, culminating in concurrent, interdependent immunopathic and proteopathic pathogeneses. Methods: We performed a comprehensive series of in silico, in vitro, and in vivo studies explicitly evaluating the atomistic-molecular mechanisms of cytokine-mediated and Aß-mediated neurotoxicities in AD.  Next, 471 new chemical entities were designed and synthesized to probe the pathways identified by these molecular mechanism studies and to provide prototypic starting points in the development of small-molecule therapeutics for AD. Results: In response to various stimuli (e.g., infection, trauma, ischemia, air pollution, depression), Aß is released as an early responder immunopeptide triggering an innate immunity cascade in which Aß exhibits both immunomodulatory and antimicrobial properties (whether bacteria are present, or not), resulting in a misdirected attack upon "self" neurons, arising from analogous electronegative surface topologies between neurons and bacteria, and rendering them similarly susceptible to membrane-penetrating attack by antimicrobial peptides (AMPs) such as Aß. After this self-attack, the resulting necrotic (but not apoptotic) neuronal breakdown products diffuse to adjacent neurons eliciting further release of Aß, leading to a chronic self-perpetuating autoimmune cycle.  AD thus emerges as a brain-centric autoimmune disorder of innate immunity. Based upon the hypothesis that autoimmune processes are susceptible to endogenous regulatory processes, a subsequent comprehensive screening program of 1137 small molecules normally present in human brain identified tryptophan metabolism as a regulator of brain innate immunity and a source of potential endogenous anti-AD molecules capable of chemical modification into multi-site therapeutic modulators targeting AD's complex immunopathic-proteopathic pathogenesis. Discussion:  Conceptualizing AD as an autoimmune disease, identifying endogenous regulators of this autoimmunity, and designing small molecule drug-like analogues of these endogenous regulators represents a novel therapeutic approach for AD.

The mismatched nucleotides encoded in vaccinia virus flip-and-flop hairpin telomeres serve an essential role in virion maturation.

Poxvirus genomes consist of a linear duplex DNA that ends in short inverted and complementary hairpin structures. These elements also encode loops and mismatches that likely serve a role in genome packaging and perhaps replication. We constructed mutant vaccinia viruses (VACV) where the native hairpins were replaced by altered forms and tested effects on replication, assembly, and virulence. Our studies showed that structure, not sequence, likely determines function as one can replace an Orthopoxvirus (VACV) hairpin with one copied from a Leporipoxvirus with no effect on growth. Some loops can be deleted from VACV hairpins with little effect, but VACV bearing too few mismatches grew poorly and we couldn't recover viruses lacking all mismatches. Further studies were conducted using a mutant bearing only one of six mismatches found in wild-type hairpins (SΔ1Δ3-6). This virus grew to ~20-fold lower titers, but neither DNA synthesis nor telomere resolution was affected. However, the mutant exhibited a particle-to-PFU ratio 10-20-fold higher than wild-type viruses and p4b/4b core protein processing was compromised, indicating an assembly defect. Electron microscopy showed that SΔ1Δ3-6 mutant development was blocked at the immature virus (IV) stage, which phenocopies known effects of I1L mutants. Competitive DNA binding assays showed that recombinant I1 protein had less affinity for the SΔ1Δ3-6 hairpin than the wild-type hairpin. The SΔ1Δ3-6 mutant was also attenuated when administered to SCID-NCR mice by tail scarification. Mice inoculated with viruses bearing wild-type hairpins exhibited a median survival of 30-37 days, while mice infected with SΔ1Δ3-6 virus survived >70 days. Persistent infections favor genetic reversion and genome sequencing detected one example where a small duplication near the hairpin tip likely created a new loop. These observations show that mismatches serve a critical role in genome packaging and provide new insights into how VACV "flip and flop" telomeres are arranged.

Detection and quantification of infectious severe acute respiratory coronavirus-2 in diverse clinical and environmental samples.

To explore the potential modes of Severe Acute Respiratory Coronavirus-2 (SARS-CoV-2) transmission, we collected 535 diverse clinical and environmental samples from 75 infected hospitalized and community patients. Infectious SARS-CoV-2 with quantitative burdens varying from 5 plaque-forming units/mL (PFU/mL) up to 1.0 × 106 PFU/mL was detected in 151/459 (33%) of the specimens assayed and up to 1.3 × 106 PFU/mL on fomites with confirmation by plaque morphology, PCR, immunohistochemistry, and/or sequencing. Infectious virus in clinical and associated environmental samples correlated with time since symptom onset with no detection after 7-8 days in immunocompetent hosts and with N-gene based Ct values ≤ 25 significantly predictive of yielding plaques in culture. SARS-CoV-2 isolated from patient respiratory tract samples caused illness in a hamster model with a minimum infectious dose of ≤ 14 PFU. Together, our findings offer compelling evidence that large respiratory droplet and contact (direct and indirect i.e., fomites) are important modes of SARS-CoV-2 transmission.

Vaccinia Virus Gene Acquisition through Nonhomologous Recombination.

Many of the genes encoded by poxviruses are orthologs of cellular genes. These virus genes serve different purposes, but perhaps of most interest is the way some have been repurposed to inhibit the antiviral pathways that their cellular homologs still regulate. What is unclear is how these virus genes were acquired, although it is presumed to have been catalyzed by some form(s) of nonhomologous recombination (NHR). We used transfection assays and substrates encoding a fluorescent and drug-selectable marker to examine the NHR frequency in vaccinia virus (VAC)-infected cells. These studies showed that when cells were transfected with linear duplex DNAs bearing VAC N2L gene homology, it yielded a recombinant frequency (RF) of 6.7 × 10-4. In contrast, DNA lacking any VAC homology reduced the yield of recombinants ∼400-fold (RF = 1.6 × 10-6). DNA-RNA hybrids were also substrates, although homologous molecules yielded fewer recombinants (RF = 2.1 × 10-5), and nonhomologous substrates yielded only rare recombinants (RF ≤ 3 × 10-8). NHR was associated with genome rearrangements ranging from simple insertions with flanking sequence duplications to large-scale indels that produced helper-dependent viruses. The insert was often also partially duplicated and would rapidly rearrange through homologous recombination. Most of the virus-insert junctions exhibited little or no preexiting microhomology, although a few encoded VAC topoisomerase recognition sites (C/T·CCTT). These studies show that VAC can catalyze NHR through a process that may reflect a form of aberrant replication fork repair. Although it is less efficient than classical homologous recombination, the rates of NHR may still be high enough to drive virus evolution. IMPORTANCE Large DNA viruses sometimes interfere in antiviral defenses using repurposed and mutant forms of the cellular proteins that mediate these same reactions. Such virus orthologs of cellular genes were presumably captured through nonhomologous recombination, perhaps in the distant past, but nothing is known about the processes that might promote "gene capture" or even how often these events occur over the course of an infectious cycle. This study shows that nonhomologous recombination in vaccinia virus-infected cells is frequent enough to seed a small but still significant portion of novel recombinants into large populations of newly replicated virus particles. This offers a route by which a pool of virus might survey the host genome for sequences that offer a selective growth advantage and potentially drive discontinuous virus evolution (saltation) through the acquisition of adventitious traits.

Deciphering the Immunomodulatory Capacity of Oncolytic Vaccinia Virus to Enhance the Immune Response to Breast Cancer.

Vaccinia virus (VACV) is a double-stranded DNA virus that devotes a large portion of its 200 kbp genome to suppressing and manipulating the immune response of its host. Here, we investigated how targeted removal of immunomodulatory genes from the VACV genome impacted immune cells in the tumor microenvironment with the intention of improving the therapeutic efficacy of VACV in breast cancer. We performed a head-to-head comparison of six mutant oncolytic VACVs, each harboring deletions in genes that modulate different cellular pathways, such as nucleotide metabolism, apoptosis, inflammation, and chemokine and interferon signaling. We found that even minor changes to the VACV genome can impact the immune cell compartment in the tumor microenvironment. Viral genome modifications had the capacity to alter lymphocytic and myeloid cell compositions in tumors and spleens, PD-1 expression, and the percentages of virus-targeted and tumor-targeted CD8+ T cells. We observed that while some gene deletions improved responses in the nonimmunogenic 4T1 tumor model, very little therapeutic improvement was seen in the immunogenic HER2/neu TuBo model with the various genome modifications. We observed that the most promising candidate genes for deletion were those that interfere with interferon signaling. Collectively, this research helped focus attention on the pathways that modulate the immune response in the context of VACV oncolytic virotherapy. They also suggest that the greatest benefits to be obtained with these treatments may not always be seen in "hot tumors."

Cytoplasmic factories, virus assembly, and DNA replication kinetics collectively constrain the formation of poxvirus recombinants.

Poxviruses replicate in cytoplasmic structures called factories and each factory begins as a single infecting particle. Sixty-years ago Cairns predicted that this might have effects on vaccinia virus (VACV) recombination because the factories would have to collide and mix their contents to permit recombination. We've since shown that factories collide irregularly and that even then the viroplasm mixes poorly. We've also observed that while intragenic recombination occurs frequently early in infection, intergenic recombination is less efficient and happens late in infection. Something inhibits factory fusion and viroplasm mixing but what is unclear. To study this, we've used optical and electron microscopy to track factory movement in co-infected cells and correlate these observations with virus development and recombinant formation. While the technical complexity of the experiments limited the number of cells that are amenable to extensive statistical analysis, these studies do show that intergenic recombination coincides with virion assembly and when VACV replication has declined to ≤10% of earlier levels. Along the boundaries between colliding factories, one sees ER membrane remnants and other cell constituents like mitochondria. These collisions don't always cause factory fusion, but when factories do fuse, they still entrain cell constituents like mitochondria and ER-wrapped microtubules. However, these materials wouldn't seem to pose much of a further barrier to DNA mixing and so it's likely that the viroplasm also presents an omnipresent impediment to DNA mixing. Late packaging reactions might help to disrupt the viroplasm, but packaging would sequester the DNA just as the replication and recombination machinery goes into decline and further reduce recombinant yields. Many factors thus appear to conspire to limit recombination between co-infecting poxviruses.

Extended sequencing of vaccine and wild-type capripoxvirus isolates provides insights into genes modulating virulence and host range.

The genus Capripoxvirus in the subfamily Chordopoxvirinae, family Poxviridae, comprises sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which cause the eponymous diseases across parts of Africa, the Middle East and Asia. These diseases cause significant economic losses and can have a devastating impact on the livelihoods and food security of small farm holders. So far, only live classically attenuated SPPV, GTPV and LSDV vaccines are commercially available and the history, safety and efficacy of many have not been well established. Here, we report 13 new capripoxvirus genome sequences, including the hairpin telomeres, from both pathogenic field isolates and vaccine strains. We have also updated the genome annotations to incorporate recent advances in our understanding of poxvirus biology. These new genomes and genes grouped phenetically with other previously sequenced capripoxvirus strains, and these new alignments collectively identified several recurring alterations in genes thought to modulate virulence and host range. In particular, some of the many large capripoxvirus ankyrin and kelch-like proteins are commonly mutated in vaccine strains, while the variola virus B22R-like gene homolog has also been disrupted in many vaccine isolates. Among these vaccine isolates, frameshift mutations are especially common and clearly present a risk of reversion to wild type in vaccines bearing these mutations. A consistent pattern of gene inactivation from LSDV to GTPV and then SPPV is also observed, much like the pattern of gene loss in orthopoxviruses, but, rather surprisingly, the overall genome size of ~150 kbp remains relatively constant. These data provide new insights into the evolution of capripoxviruses and the determinants of pathogenicity and host range. They will find application in the development of new vaccines with better safety, efficacy and trade profiles.

Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments.

Edward Jenner and his contemporaries believed that his variolae vaccinae originated in horses and molecular analyses show that modern vaccinia virus (VACV) strains share common ancestry with horsepox virus (HPXV). Given concerns relating to the toxicity of modern VACV vaccines, we asked whether an HPXV-based vaccine might provide a superior alternative. Since HPXV may be extinct and the only specimen of HPXV that has been identified is unavailable for investigation, we explored whether HPXV could be obtained by large-scale gene synthesis. Ten large (10-30 kb) fragments of DNA were synthesized based on the HPXV sequence along with two 157 nt VACV terminal sequences, and were recombined into a live synthetic chimeric HPXV (scHPXV) in cells infected with Shope fibroma virus (SFV). Sequencing of the 212 kbp scHPXV confirmed it encoded a faithful copy of the input DNA. We believe this is the first complete synthesis of a poxvirus using synthetic biology approaches. This scHPXV produced smaller plaques, produced less extracellular virus and exhibited less virulence in mice than VACV, but still provided vaccine protection against a lethal VACV challenge. Collectively, these findings support further development of scHPXV as a novel replication-proficient smallpox vaccine.

Mutations in the Fusion Protein of Measles Virus That Confer Resistance to the Membrane Fusion Inhibitors Carbobenzoxy-d-Phe-l-Phe-Gly and 4-Nitro-2-Phenylacetyl Amino-Benzamide.

The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein.IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.

The vaccinia virus K7 protein promotes histone methylation associated with heterochromatin formation.

It has been well established that many vaccinia virus proteins suppress host antiviral pathways by targeting the transcription of antiviral proteins, thus evading the host innate immune system. However, whether viral proteins have an effect on the host's overall cellular transcription is less understood. In this study we investigated the regulation of heterochromatin during vaccinia virus infection. Heterochromatin is a highly condensed form of chromatin that is less transcriptionally active and characterized by methylation of histone proteins. We examined the change in methylation of two histone proteins, H3 and H4, which are major markers of heterochromatin, during the course of viral infection. Using immunofluorescence microscopy and flow cytometry we were able to track the overall change in the methylated levels of H3K9 and H4K20. Our results suggest that there is significant increase in methylation of H3K9 and H4K20 during Orthopoxviruses infection compared to mock-infected cells. However, this effect was not seen when we infected cells with Leporipoxviruses. We further screened several vaccinia virus single and multi-gene deletion mutant and identified the vaccinia virus gene K7R as a contributor to the increase in cellular histone methylation during infection.

Live-Cell Imaging of Vaccinia Virus Recombination.

Recombination between co-infecting poxviruses provides an important mechanism for generating the genetic diversity that underpins evolution. However, poxviruses replicate in membrane-bound cytoplasmic structures known as factories or virosomes. These are enclosed structures that could impede DNA mixing between co-infecting viruses, and mixing would seem to be essential for this process. We hypothesize that virosome fusion events would be a prerequisite for recombination between co-infecting poxviruses, and this requirement could delay or limit viral recombination. We have engineered vaccinia virus (VACV) to express overlapping portions of mCherry fluorescent protein fused to a cro DNA-binding element. In cells also expressing an EGFP-cro fusion protein, this permits live tracking of virus DNA and genetic recombination using confocal microscopy. Our studies show that different types of recombination events exhibit different timing patterns, depending upon the relative locations of the recombining elements. Recombination between partly duplicated sequences is detected soon after post-replicative genes are expressed, as long as the reporter gene sequences are located in cis within an infecting genome. The same kinetics are also observed when the recombining elements are divided between VACV and transfected DNA. In contrast, recombination is delayed when the recombining sequences are located on different co-infecting viruses, and mature recombinants aren't detected until well after late gene expression is well established. The delay supports the hypothesis that factories impede inter-viral recombination, but even after factories merge there remain further constraints limiting virus DNA mixing and recombinant gene assembly. This delay could be related to the continued presence of ER-derived membranes within the fused virosomes, membranes that may once have wrapped individual factories.

The acidic C-terminus of vaccinia virus I3 single-strand binding protein promotes proper assembly of DNA-protein complexes.

The vaccinia virus I3L gene encodes a single-stranded DNA binding protein (SSB) that is essential for virus DNA replication and is conserved in all Chordopoxviruses. The I3 protein contains a negatively charged C-terminal tail that is a common feature of SSBs. Such acidic tails are critical for SSB-dependent replication, recombination and repair. We cloned and purified variants of the I3 protein, along with a homolog from molluscum contagiosum virus, and tested how the acidic tail affected DNA-protein interactions. Deleting the C terminus of I3 enhanced the affinity for single-stranded DNA cellulose and gel shift analyses showed that it also altered the migration of I3-DNA complexes in agarose gels. Microinjecting an antibody against I3 into vaccinia-infected cells also selectively inhibited virus replication. We suggest that this domain promotes cooperative binding of I3 to DNA in a way that would maintain an open DNA configuration around a replication site.