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

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

A cultura escolar em conflito: ensino técnico e matemática moderna em Portugal

Disponível em: http://www2.pucpr.br/reol/pb/index.php/dialogo?dd1=16276&dd99=view&dd98=pbO artigo estuda as transformações exigidas às escolas do ensino profissional português durante a reforma da matemática moderna que ocorrem a partir de finais dos anos 1960. Em primeiro lugar, traça um quadro das normas associadas às escolas técnicas portuguesas antes da reforma, recorrendo à legislação fundadora, aos manuais e a artigos de opinião de professores. Em segundo, detalha o debate que antecipou a introdução da reforma recorrendo a artigos e aopiniões expressas durante os cursos preparatórios, onde são notórias as pressões para mudanças na cultura escolar, especialmente nas suas representações, suscitadas pela nova matemática. Finalmente, observar-se como se materializou a reforma nos livros de texto da experiência

What Do Antibody Studies Tell Us about Viral Infections?

Humoral immunity is an important body defense system against virus infection and is correlated to patient health status [...

Species-Specific Evolution of Ebola Virus during Replication in Human and Bat Cells.

Ebola virus (EBOV) causes a severe, often fatal disease in humans and nonhuman primates. Within the past decade, EBOV has caused two large and difficult-to-control outbreaks, one of which recently ended in the Democratic Republic of the Congo. Bats are the likely reservoir of EBOV, but little is known of their relationship with the virus. We perform serial passages of EBOV in human and bat cells and use circular sequencing to compare the short-term evolution of the virus. Virus populations passaged in bat cells have sequence markers indicative of host RNA editing enzyme activity, including evidence for ADAR editing of the EBOV glycoprotein. Multiple regions in the EBOV genome appear to have undergone adaptive evolution when passaged in bat and human cells. Individual mutated viruses are rescued and characterized. Our results provide insight into the host species-specific evolution of EBOV and highlight the adaptive flexibility of the virus

Species-Specific Evolution of Ebola Virus during Replication in Human and Bat Cells.

Ebola virus (EBOV) causes a severe, often fatal disease in humans and nonhuman primates. Within the past decade, EBOV has caused two large and difficult-to-control outbreaks, one of which recently ended in the Democratic Republic of the Congo. Bats are the likely reservoir of EBOV, but little is known of their relationship with the virus. We perform serial passages of EBOV in human and bat cells and use circular sequencing to compare the short-term evolution of the virus. Virus populations passaged in bat cells have sequence markers indicative of host RNA editing enzyme activity, including evidence for ADAR editing of the EBOV glycoprotein. Multiple regions in the EBOV genome appear to have undergone adaptive evolution when passaged in bat and human cells. Individual mutated viruses are rescued and characterized. Our results provide insight into the host species-specific evolution of EBOV and highlight the adaptive flexibility of the virus

Staufen1 Interacts with Multiple Components of the Ebola Virus Ribonucleoprotein and Enhances Viral RNA Synthesis

Ebola virus (EBOV) is a negative-strand RNA virus with significant public health importance. Currently, no therapeutics are available for Ebola, which imposes an urgent need for a better understanding of EBOV biology. Here we dissected the virus-host interplay between EBOV and host RNA-binding proteins. We identified novel EBOV host factors, including Staufen1, which interacts with multiple viral factors and is required for efficient viral RNA synthesis.Ebola virus (EBOV) genome and mRNAs contain long, structured regions that could hijack host RNA-binding proteins to facilitate infection. We performed RNA affinity chromatography coupled with mass spectrometry to identify host proteins that bind to EBOV RNAs and identified four high-confidence proviral host factors, including Staufen1 (STAU1), which specifically binds both 3′ and 5′ extracistronic regions of the EBOV genome. We confirmed that EBOV infection rate and production of infectious particles were significantly reduced in STAU1-depleted cells. STAU1 was recruited to sites of EBOV RNA synthesis upon infection and enhanced viral RNA synthesis. Furthermore, STAU1 interacts with EBOV nucleoprotein (NP), virion protein 30 (VP30), and VP35; the latter two bridge the viral polymerase to the NP-coated genome, forming the viral ribonucleoprotein (RNP) complex. Our data indicate that STAU1 plays a critical role in EBOV replication by coordinating interactions between the viral genome and RNA synthesis machinery

Phosphorylated VP30 of marburg virus is a repressor of transcription

The filoviruses Marburg virus (MARV) and Ebola virus (EBOV) cause hemorrhagic fever in humans and nonhuman primates, with high case fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here, we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling the full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions, mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation of a cluster of amino acids at positions 46 through 53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, coimmunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule, 1E7-03, targeting a noncatalytic site of the enzyme that previously was shown to increase EBOV VP30 phosphorylation was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at a cluster of phosphorylated amino acids from Ser-46 to Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35, and dramatically reduced replication of infectious MARV particles. Thus, MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds. IMPORTANCE The largest outbreak of MARV occurred in Angola in 2004 to 2005 and had a 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires a fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of Filoviridae family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of the MARV genome demonstrated differences from those of EBOV. The identified phosphorylation sites appeared to inhibit transcription and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of the MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility developing antivirals based on compounds targeting PP1