Topoisomerase II inhibitors induce DNA damage-dependent interferon responses circumventing Ebola virus immune evasion

Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication

Filovirus refseq entries: Evaluation and selection of filovirus type variants, Type sequences, And names

Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information's (NCBI's) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [<virus name> (<strain>)/<isolation host-suffix>/<country of sampling>/<year of sampling>/<genetic variant designation>-<isolate designation>], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences.Other co-authors: Ralf G. Dietzgen, Norman A. Doggett, Olga Dolnik, John M. Dye, Sven Enterlein, Paul W. Fenimore, Pierre Formenty, Alexander N. Freiberg, Robert F. Garry, Nicole L. Garza, Stephen K. Gire, Jean-Paul Gonzalez, Anthony Griffiths, Christian T. Happi, Lisa E. Hensley, Andrew S. Herbert, Michael C. Hevey, Thomas Hoenen, Anna N. Honko, Georgy M. Ignatyev, Peter B. Jahrling, Joshua C. Johnson, Karl M. Johnson, Jason Kindrachuk, Hans-Dieter Klenk, Gary Kobinger, Tadeusz J. Kochel, Matthew G. Lackemeyer, Daniel F. Lackner, Eric M. Leroy, Mark S. Lever, Elke Mühlberger, Sergey V. Netesov, Gene G. Olinger, Sunday A. Omilabu, Gustavo Palacios, Rekha G. Panchal, Daniel J. Park, Jean L. Patterson, Janusz T. Paweska, Clarence J. Peters, James Pettitt, Louise Pitt, Sheli R. Radoshitzky, Elena I. Ryabchikova, Erica Ollmann Saphire, Pardis C. Sabeti, Rachel Sealfon, Aleksandr M. Shestopalov, Sophie J. Smither, Nancy J. Sullivan, Robert Swanepoel, Ayato Takada, Jonathan S. Towner, Guido van der Groen, Viktor E. Volchkov, Valentina A. Volchkova, Victoria Wahl-Jensen, Travis K. Warren, Kelly L. Warfield, and Stuart T. Nichol Output Type: Lette

Ebolavirus species-specific interferon antagonism mediated by VP24

Members of the Ebolavirus genus demonstrate a marked differences in pathogenicity in humans with Ebola (EBOV) being the most pathogenic, Bundibugyo (BDBV) less pathogenic, and Reston (RESTV) is not known to cause a disease in humans. The VP24 protein encoded by members of the Ebolavirus genus blocks type I interferon (IFN-I) signaling through interaction with host karyopherin alpha nuclear transporters, potentially contributing to virulence. Previously, we demonstrated that BDBV VP24 (bVP24) binds with lower affinities to karyopherin alpha proteins relative to EBOV VP24 (eVP24), and this correlated with a reduced inhibition in IFN-I signaling. We hypothesized that modification of eVP24-karyopherin alpha interface to make it similar to bVP24 would attenuate the ability to antagonize IFN-I response. We generated a panel of recombinant EBOVs containing single or combinations of point mutations in the eVP24-karyopherin alpha interface. Most of the viruses appeared to be attenuated in both IFN-I-competent 769-P and IFN-I-deficient Vero-E6 cells in the presence of IFNs. However, the R140A mutant grew at reduced levels even in the absence of IFNs in both cell lines, as well as in U3A STAT1 knockout cells. Both the R140A mutation and its combination with the N135A mutation greatly reduced the amounts of viral genomic RNA and mRNA suggesting that these mutations attenuate the virus in an IFN-I-independent attenuation. Additionally, we found that unlike eVP24, bVP24 does not inhibit interferon lambda 1 (IFN-λ1), interferon beta (IFN-β), and ISG15, which potentially explains the lower pathogenicity of BDBV relative to EBOV. Thus, the VP24 residues binding karyopherin alpha attenuates the virus by IFN-I-dependent and independent mechanisms

Convergence of a common solution to broad ebolavirus neutralization by glycan cap directed human antibodies

Antibodies that target the glycan cap epitope on ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization is not well-understood. Here we present cryo-electron microscopy (cryo-EM) structures of several glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLD) to the glycan cap, which we name the MLD-anchor and cradle. Antibodies that bind to the MLD-cradle share common features, including the use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form beta-hairpin structures to mimic the MLD-anchor, disrupt MLD attachment, destabilize GP quaternary structure and block cleavage events required for receptor binding. Our results collectively provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies

Experimental Infection of Mice with Avian Paramyxovirus Serotypes 1 to 9

The nine serotypes of avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds worldwide. APMV-1, also called Newcastle disease virus, was shown to be attenuated in non-avian species and is being developed as a potential vector for human vaccines. In the present study, we extended this evaluation to the other eight serotypes by evaluating infection in BALB/c mice. Mice were inoculated intranasally with a prototype strain of each of the nine serotypes and monitored for clinical disease, gross pathology, histopathology, virus replication and viral antigen distribution, and seroconversion. On the basis of multiple criteria, each of the APMV serotypes except serotype 5 was found to replicate in mice. Five of the serotypes produced clinical disease and significant weight loss in the following order of severity: 1, 2>6, 9>7. However, disease was short-lived. The other serotypes produced no evident clinical disease. Replication of all of the APMVs except APMV-5 in the nasal turbinates and lungs was confirmed by the recovery of infectious virus and by substantial expression of viral antigen in the epithelial lining detected by immunohistochemistry. Trace levels of infectious APMV-4 and -9 were detected in the brain of some animals; otherwise, no virus was detected in the brain, small intestine, kidney, or spleen. Histologically, infection with the APMVs resulted in lung lesions consistent with broncho-interstitial pneumonia of varying severity that were completely resolved at 14 days post infection. All of the mice infected with the APMVs except APMV-5 produced serotype-specific HI serum antibodies, confirming a lack of replication of APMV-5. Taken together, these results demonstrate that all APMV serotypes except APMV-5 are capable of replicating in mice with minimal disease and pathology

The Marburgvirus-Neutralizing Human Monoclonal Antibody MR191 Targets a Conserved Site to Block Virus Receptor Binding

Since their first identification 50 years ago, marburgviruses have emerged several times, with 83%–90% lethality in the largest outbreaks. Although no vaccines or therapeutics are available for human use, the human antibody MR191 provides complete protection in non-human primates when delivered several days after inoculation of a lethal marburgvirus dose. The detailed neutralization mechanism of MR191 remains outstanding. Here we present a 3.2 Å crystal structure of MR191 complexed with a trimeric marburgvirus surface glycoprotein (GP). MR191 neutralizes by occupying the conserved receptor-binding site and competing with the host receptor Niemann-Pick C1. The structure illuminates previously disordered regions of GP including the stalk, fusion loop, CX_6CC switch, and an N-terminal region of GP2 that wraps about the outside of GP1 to anchor a marburgvirus-specific “wing” antibody epitope. Virus escape mutations mapped far outside the MR191 receptor-binding site footprint suggest a role for these other regions in the GP quaternary structure

Filovirus RefSeq Entries: Evaluation and Selection of Filovirus Type Variants, Type Sequences, and Names

Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information’s (NCBI’s) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [ ()////-], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences

IgG-like bispecific antibodies with potent and synergistic neutralization against circulating SARS-CoV-2 variants of concern

Monoclonal antibodies are a promising approach to treat COVID-19, however the emergence of SARS-CoV-2 variants has challenged the efficacy and future of these therapies. Antibody cocktails are being employed to mitigate these challenges, but neutralization escape remains a major challenge and alternative strategies are needed. Here we present two anti-SARS-CoV-2 spike binding antibodies, one Class 1 and one Class 4, selected from our non-immune human single-chain variable fragment (scFv) phage library, that are engineered into four, fully-human IgG-like bispecific antibodies (BsAb). Prophylaxis of hACE2 mice and post-infection treatment of golden hamsters demonstrates the efficacy of the monospecific antibodies against the original Wuhan strain, while promising in vitro results with the BsAbs demonstrate enhanced binding and distinct synergistic effects on neutralizing activity against circulating variants of concern. In particular, one BsAb engineered in a tandem scFv-Fc configuration shows synergistic neutralization activity against several variants of concern including B.1.617.2. This work provides evidence that synergistic neutralization can be achieved using a BsAb scaffold, and serves as a foundation for the future development of broadly reactive BsAbs against emerging variants of concern

Effects of Human Respiratory Syncytial Virus, Metapneumovirus, Parainfluenza Virus 3 and Influenza Virus on CD4+ T Cell Activation by Dendritic Cells

BACKGROUND: Human respiratory syncytial virus (HRSV), and to a lesser extent human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), re-infect symptomatically throughout life without antigenic change, suggestive of incomplete immunity. One causative factor is thought to be viral interference with dendritic cell (DC)-mediated stimulation of CD4+ T cells. METHODOLOGY, PRINCIPAL FINDINGS: We infected human monocyte-derived DC with purified HRSV, HMPV, HPIV3, or influenza A virus (IAV) and compared their ability to induce activation and proliferation of autologous CD4+ T cells in vitro. IAV was included because symptomatic re-infection without antigenic change is less frequent, suggesting that immune protection is more complete and durable. We examined virus-specific memory responses and superantigen-induced responses by multiparameter flow cytometry. Live virus was more stimulatory than inactivated virus in inducing DC-mediated proliferation of virus-specific memory CD4+ T cells, suggesting a lack of strong suppression by live virus. There were trends of increasing proliferation in the order: HMPV<HRSV<HPIV3<IAV, and greater production of interferon-γ and tumor necrosis factor-α by proliferating cells in response to IAV, but differences were not significant. Exposure of DC to HRSV, HPIV3, or IAV reduced CD4+ T cell proliferation in response to secondary stimulus with superantigen, but the effect was transitory and greatest for IAV. T cell cytokine production was similar, with no evidence of Th2 or Th17 skewing. CONCLUSIONS, SIGNIFICANCE: Understanding the basis for the ability of HRSV in particular to symptomatically re-infect without significant antigenic change is of considerable interest. The present results show that these common respiratory viruses are similar in their ability to induce DC to activate CD4+ T cells. Thus, the results do not support the common model in which viral suppression of CD4+ T cell activation and proliferation by HRSV, HMPV, and HPIV3 is a major factor in the difference in re-infectability compared to IAV