Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible Velcro-like manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.
Abstract In response to the COVID-19 pandemic, multiple vaccines were developed using platforms such as viral vectors and mRNA technology. Here, we report humoral and cellular immunogenicity data from human phase 1 clinical trials investigating two recombinant Modified Vaccinia virus Ankara vaccine candidates, MVA-SARS-2-S and MVA-SARS-2-ST, encoding the native and the prefusion-stabilized SARS-CoV-2 spike protein, respectively. MVA-SARS-2-ST was more immunogenic than MVA-SARS-2-S, but both were less immunogenic compared to licensed mRNA- and ChAd-based vaccines in SARS-CoV-2 naïve individuals. In heterologous vaccination, previous MVA-SARS-2-S vaccination enhanced T cell functionality and MVA-SARS-2-ST boosted the frequency of T cells and S1-specific IgG levels when used as a third vaccination. While the vaccine candidate containing the prefusion-stabilized spike elicited predominantly S1-specific responses, immunity to the candidate with the native spike was skewed towards S2-specific responses. These data demonstrate how the spike antigen conformation, using the same viral vector, directly affects vaccine immunogenicity in humans.
Ebola virus (EBOV) is responsible for outbreaks with case-fatality rates of up to 90% and for an epidemic in West Africa with more than ten thousand deaths. EBOV glycoprotein (EBOV-GP) is the only viral surface protein and is responsible for viral entry into cells. It has been suggested to play a role in the cytopathic effects induced by the virus. Here we uncover a critical role for sphingolipids in inhibiting viral entry and virus-mediated cytotoxicity. Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). The administration of the SphK1 activator, K6PC-5, or S1P, or the overexpression of SphK1 consistently exhibited striking inhibitory effects in EBOV-GP-driven entry in EA.hy926 endothelial-like cells. Furthermore, K6PC-5 inhibited the subsequent necrotic cell death. Finally, K6PC-5 markedly reduced the EBOV titer in infected cells. These data present K6PC‑5 as efficient tool to inhibit EBOV infection in endothelial cells, and suggest further studies to evaluate its systemic effects.
Ebola virus VP30 is an essential activator of viral transcription. In viral particles, VP30 is closely associated with the nucleocapsid complex. A conspicuous structural feature of VP30 is an unconventional zinc-binding Cys(3)-His motif comprising amino acids 68 to 95. By using a colorimetric zinc-binding assay we found that the VP30-specific Cys(3)-His motif stoichiometrically binds zinc ions in a one-to-one relationship. Substitution of the conserved cysteines and the histidine within the motif led to a complete loss of the capacity for zinc binding. Functional analyses revealed that none of the tested mutations of the proposed zinc-coordinating residues influenced binding of VP30 to nucleocapsid-like particles but, concerning its role in activating viral transcription, all resulted in a protein that was inactive.
ABSTRACT We have conducted an international quality assurance study of filovirus, Lassa virus, and orthopox virus PCR with 24 participants. Of the participating laboratories, 45.8 and 66.7% detected virus in all plasma samples, which contained ≥5,000 and ≥100,000 copies per ml, respectively. Sensitivity levels were not significantly different between viruses. False-negative results were attributable to a lack of sensitivity.
Acute kidney injury is associated with mortality in COVID-19 patients. However, host cell changes underlying infection of renal cells with SARS-CoV-2 remain unknown and prevent understanding of the molecular mechanisms that may contribute to renal pathology. Here, we carried out quantitative translatome and whole-cell proteomics analyses of primary renal proximal and distal tubular epithelial cells derived from human donors infected with SARS-CoV-2 or MERS-CoV to disseminate virus and cell type-specific changes over time. Our findings revealed shared pathways modified upon infection with both viruses, as well as SARS-CoV-2-specific host cell modulation driving key changes in innate immune activation and cellular protein quality control. Notably, MERS-CoV infection-induced specific changes in mitochondrial biology that were not observed in response to SARS-CoV-2 infection. Furthermore, we identified extensive modulation in pathways associated with kidney failure that changed in a virus- and cell type-specific manner. In summary, we provide an overview of the effects of SARS-CoV-2 or MERS-CoV infection on primary renal epithelial cells revealing key pathways that may be essential for viral replication.
Abstract The severe Ebola virus disease epidemic occurring in West Africa stems from a single zoonotic transmission event to a 2‐year‐old boy in Meliandou, Guinea. We investigated the zoonotic origins of the epidemic using wildlife surveys, interviews, and molecular analyses of bat and environmental samples. We found no evidence for a concurrent outbreak in larger wildlife. Exposure to fruit bats is common in the region, but the index case may have been infected by playing in a hollow tree housing a colony of insectivorous free‐tailed bats ( Mops condylurus ). Bats in this family have previously been discussed as potential sources for Ebola virus outbreaks, and experimental data have shown that this species can survive experimental infection. These analyses expand the range of possible Ebola virus sources to include insectivorous bats and reiterate the importance of broader sampling efforts for understanding Ebola virus ecology.
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