Analysis of the structure and interactions of the SARS-CoV-2 ORF7b accessory protein.

SARS-CoV-2 carries a sizeable number of proteins that are accessory to replication but may be essential for virus-host interactions and modulation of the host immune response. Here, we investigated the structure and interactions of the largely unknown ORF7b, a small membranous accessory membrane protein of SARS-CoV-2. We show that structural predictions indicate a transmembrane (TM) leucine zipper for ORF7b, and experimentally confirm the predominantly α-helical secondary structure within a phospholipid membrane mimetic by solid-state NMR.

Hepatitis Delta Antigen Retains the Assembly Domain as the Only Rigid Entity.

The hepatitis delta virus (HDV) S-HDAg and L-HDAg antigens are the two isoforms of the single protein encoded by the viral genome. Together with the double-stranded RNA genome they form the HDV ribonucleoprotein (RNP) complex. In the context of a divide-and-conquer approach, we used a combination of cell-free protein synthesis and proton (H)-detected fast magic angle spinning solid-state NMR at highest magnetic field to characterize S-HDAg.

Cell-Free Synthesis of Bunyavirales Proteins in View of Their Structural Characterization by Nuclear Magnetic Resonance.

The Rift Valley fever virus is one of the bunyaviruses on the WHO's priority list of pathogens that may cause future pandemics. A better understanding of disease progression and viral pathogenesis is urgently needed to develop treatments. The non-structural proteins NSs and NSm of human pathogenic bunyaviruses represent promising therapeutic targets, as they are often key virulence factors.

Do NSm Virulence Factors in the Viral Order Originate from Gn Gene Duplication?

One-third of the nine WHO shortlisted pathogens prioritized for research and development in public health emergencies belong to the order. Several species carry an NSm protein that acts as a virulence factor. We predicted the structures of these NSm proteins and unexpectedly found that in two families, their cytosolic domain was inferred to have a similar fold to that of the cytosolic domain of the viral envelope-forming glycoprotein N (Gn) encoded on the same genome fragment.

High and fast: NMR protein-proton side-chain assignments at 160 kHz and 1.2 GHz.

The NMR spectra of side-chain protons in proteins provide important information, not only about their structure and dynamics, but also about the mechanisms that regulate interactions between macromolecules. However, in the solid-state, these resonances are particularly difficult to resolve, even in relatively small proteins. We show that magic-angle-spinning (MAS) frequencies of 160 kHz, combined with a high magnetic field of 1200 MHz proton Larmor frequency, significantly improve their spectral resolution.

Structural conservation of HBV-like capsid proteins over hundreds of millions of years despite the shift from non-enveloped to enveloped life-style.

The discovery of nackednaviruses provided new insight into the evolutionary history of the hepatitis B virus (HBV): The common ancestor of HBV and nackednaviruses was non-enveloped and while HBV acquired an envelope during evolution, nackednaviruses remained non-enveloped. We report the capsid structure of the African cichlid nackednavirus (ACNDV), determined by cryo-EM at 3.7 Å resolution.

Molecular elucidation of drug-induced abnormal assemblies of the hepatitis B virus capsid protein by solid-state NMR.

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a recent class of anti-HBV antivirals. CAMs disturb proper nucleocapsid assembly, by inducing formation of either aberrant assemblies (CAM-A) or of apparently normal but genome-less empty capsids (CAM-E). Classical structural approaches have revealed the CAM binding sites on the capsid protein (Cp), but conformational information on the CAM-induced off-path aberrant assemblies is lacking.

α-Synuclein Fibril, Ribbon and Fibril-91 Amyloid Polymorphs Generation for Structural Studies.

The human α-synuclein protein, identified as one of the main markers of Parkinson's disease, is a 140-amino acid thermostable protein that can easily be overexpressed in E. coli. The purification protocol determines the ability of the protein to assemble into amyloid fibrils of well-defined structures.

Solid-State NMR Structure of Amyloid-β Fibrils.

Amyloid fibrils are involved in a number of diseases and notably play a role in neurodegeneration, where they are present in plaques in the brain. Their structure determination might help in finding ways to interfere with their formation, and ultimately prevent disease, by revealing the structure-function relationship and helping to design molecules targeting initial assembly steps and further propagation. Here, we describe the different steps in NMR protocols which allowed the 3D structure determination of amyloid-β fibrils.

Adaptation to host cell environment during experimental evolution of Zika virus.

Zika virus (ZIKV) infection can cause important developmental and neurological defects in Humans. Type I/III interferon responses control ZIKV infection and pathological processes, yet the virus has evolved various mechanisms to defeat these host responses. Here, we established a pipeline to delineate at high-resolution the genetic evolution of ZIKV in a controlled host cell environment.

[Mental Health Situation of Sex Workers in Europe: a Scoping Review].

Background: Due to the nature of their activity, sex workers often experience a lack of acceptance and stigmatization in society. This results in numerous exclusion mechanisms and stress factors, which might be associated with an increased vulnerability to mental illness.

Objective: To identify studies on the mental health situation of sex workers in Europe.

Pharmacomodulation of a ligand targeting the HBV capsid hydrophobic pocket.

Hepatitis B virus (HBV) is a small enveloped retrotranscribing DNA virus and an important human pathogen. Its capsid-forming core protein (Cp) features a hydrophobic pocket proposed to be central notably in capsid envelopment. Indeed, mutations in and around this pocket can profoundly modulate, and even abolish, secretion of enveloped virions.

Solid-State NMR Reveals Asymmetric ATP Hydrolysis in the Multidrug ABC Transporter BmrA.

The detailed mechanism of ATP hydrolysis in ATP-binding cassette (ABC) transporters is still not fully understood. Here, we employed P solid-state NMR to probe the conformational changes and dynamics during the catalytic cycle by locking the multidrug ABC transporter BmrA in prehydrolytic, transition, and posthydrolytic states, using a combination of mutants and ATP analogues. The P spectra reveal that ATP binds strongly in the prehydrolytic state to both ATP-binding sites as inferred from the analysis of the nonhydrolytic E504A mutant.

H, N and C backbone and side chain solution NMR assignments of the truncated small hepatitis delta antigen Δ60-S-HDAg.

Hepatitis D virus (HDV) is a defective virus that relies on hepatitis B virus envelope proteins to complete its replication cycle. The HDV genome contains two isoforms of hepatitis delta antigen: the small and the large hepatitis delta antigens (S- and L-HDAg). Here we report the H, C and  N backbone and side chain resonance assignments of an N-terminally truncated form of S-HDAg (S), which lacks the 1-60 oligomerization domain.

Fast Magic-Angle-Spinning NMR Reveals the Evasive Hepatitis B Virus Capsid C-Terminal Domain.

Experimentally determined protein structures often feature missing domains. One example is the C-terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulating nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR.

Phosphorylation of the Hepatitis B Virus Large Envelope Protein.

We here establish the phosphorylation sites in the human hepatitis B virus (HBV) large envelope protein (L). L is involved in several functionally important interactions in the viral life cycle, including with the HBV cellular receptor, HBV capsid, Hsc70 chaperone, and cellular membranes during fusion. We have recently shown that cell-free synthesis of the homologous L protein of duck HBV in wheat germ extract results in very similar phosphorylation events to those previously observed in animal cells.

Correction of field instabilities in biomolecular solid-state NMR by simultaneous acquisition of a frequency reference.

With the advent of faster magic-angle spinning (MAS) and higher magnetic fields, the resolution of biomolecular solid-state nuclear magnetic resonance (NMR) spectra has been continuously increasing. As a direct consequence, the always narrower spectral lines, especially in proton-detected spectroscopy, are also becoming more sensitive to temporal instabilities of the magnetic field in the sample volume. Field drifts in the order of tenths of parts per million occur after probe insertion or temperature change, during cryogen refill, or are intrinsic to the superconducting high-field magnets, particularly in the months after charging.

Experimental Characterization of the Hepatitis B Virus Capsid Dynamics by Solid-State NMR.

Protein plasticity and dynamics are important aspects of their function. Here we use solid-state NMR to experimentally characterize the dynamics of the 3.5 MDa hepatitis B virus (HBV) capsid, assembled from  240 copies of the Cp149 core protein.

Spectroscopic glimpses of the transition state of ATP hydrolysis trapped in a bacterial DnaB helicase.

The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected P,H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds.

A fusion peptide in preS1 and the human protein disulfide isomerase ERp57 are involved in hepatitis B virus membrane fusion process.

Cell entry of enveloped viruses relies on the fusion between the viral and plasma or endosomal membranes, through a mechanism that is triggered by a cellular signal. Here we used a combination of computational and experimental approaches to unravel the main determinants of hepatitis B virus (HBV) membrane fusion process. We discovered that ERp57 is a host factor critically involved in triggering HBV fusion and infection.

Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution.

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available.

Temperature-Dependent Solid-State NMR Proton Chemical-Shift Values and Hydrogen Bonding.

Temperature-dependent NMR experiments are often complicated by rather long magnetic-field equilibration times, for example, occurring upon a change of sample temperature. We demonstrate that the fast temporal stabilization of a magnetic field can be achieved by actively stabilizing the temperature of the magnet bore, which allows quantification of the weak temperature dependence of a proton chemical shift, which can be diagnostic for the presence of hydrogen bonds. Hydrogen bonding plays a central role in molecular recognition events from both fields, chemistry and biology.

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.

The highly infectious disease COVID-19 caused by the SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail.