Monkeypox, or mpox, is a disease that has recently resurfaced and spread across the globe. Despite the availability of an FDA-approved vaccine (JYNNEOS) and an effective drug (tecovirimat), concerns remain over the possible recurrence of a viral pandemic. Like any other virus, mpox virus must overcome the immune system to replicate. Viruses have evolved various strategies to overcome both innate and adaptive immunity. Poxviruses possess an unusual nuclease, poxin, which cleaves 2'-3'-cGAMP, a cyclic dinucleotide, which is an important second messenger in the cGAS-STING signaling pathway. Here, we present the crystal structure of mpox poxin. The structure reveals a conserved, predominantly β-sheet fold and highlights the high conservation of the cGAMP binding site and of the catalytic residues His17, Tyr138, and Lys142. This research suggests that poxin inhibitors could be effective against multiple poxviruses.

• MeSH
• Humans MeSH
• Mpox, Monkeypox * MeSH
• Poxviridae * MeSH
• Drug Design MeSH
• Signal Transduction MeSH
• Monkeypox virus MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Positive-sense single-stranded RNA (+RNA) viruses have proven to be important pathogens that are able to threaten and deeply damage modern societies, as illustrated by the ongoing COVID-19 pandemic. Therefore, compounds active against most or many +RNA viruses are urgently needed. Here, we present PR673, a helquat-like compound that is able to inhibit the replication of SARS-CoV-2 and tick-borne encephalitis virus in cell culture. Using in vitro polymerase assays, we demonstrate that PR673 inhibits RNA synthesis by viral RNA-dependent RNA polymerases (RdRps). Our results illustrate that the development of broad-spectrum non-nucleoside inhibitors of RdRps is feasible.

• Keywords
• Flaviruses, RNA-dependent RNA-polymerase, SARS-CoV-2, antiviral agents, helquat-like compound,
• MeSH
• COVID-19 * MeSH
• Humans MeSH
• Pandemics MeSH
• RNA-Dependent RNA Polymerase MeSH
• SARS-CoV-2 MeSH
• Encephalitis Viruses, Tick-Borne * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA-Dependent RNA Polymerase MeSH

Osh6, a member of the oxysterol-binding protein-related protein (ORP) family, is a lipid transport protein that is involved in the transport of phosphatidylserine (PS) between the endoplasmic reticulum (ER) and the plasma membrane (PM). We used a biophysical approach to characterize its transport mechanism in detail. We examined the transport of all potential ligands of Osh6. PI4P and PS are the best described lipid cargo molecules; in addition, we showed that PIP2 can be transported by Osh6 as well. So far, it was the exchange between the two cargo molecules, PS and PI4P, in the lipid-binding pocket of Osh6 that was considered an essential driving force for the PS transport. However, we showed that Osh6 can efficiently transport PS along the gradient without the help of PI4P and that PI4P inhibits the PS transport along its gradient. This observation highlights that the exchange between PS and PI4P is indeed crucial, but PI4P bound to the protein rather than intensifying the PS transport suppresses it. We considered this to be important for the transport directionality as it prevents PS from returning back from the PM where its concentration is high to the ER where it is synthesized. Our results also highlighted the importance of the ER resident Sac1 phosphatase that enables the PS transport and ensures its directionality by PI4P consumption. Furthermore, we showed that the Sac1 activity is regulated by the negative charge of the membrane that can be provided by PS or PI anions in the case of the ER membrane.

• Keywords
• OSH6, SAC1, lipid transport, oxysterol-binding protein–related proteins, phosphatidylinositol (4, 5)-bisphosphate, phosphatidylinositol 4-phosphate, phosphatidylserine,
• Publication type
• Journal Article MeSH

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.

• Keywords
• SARS-CoV-2, capping enzyme, coronavirus, methyltransferase, nsp10, nsp14, nsp16,
• MeSH
• COVID-19 virology   MeSH
• Humans MeSH
• Methyltransferases analysis   genetics   metabolism   MeSH
• Antibodies, Monoclonal analysis   MeSH
• RNA Caps genetics   metabolism   MeSH
• RNA, Viral genetics   metabolism   MeSH
• SARS-CoV-2 chemistry   enzymology   genetics   MeSH
• Protein Transport MeSH
• Viral Nonstructural Proteins analysis   genetics   metabolism   MeSH
• Viral Regulatory and Accessory Proteins analysis   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Methyltransferases MeSH
• Antibodies, Monoclonal MeSH
• NSP10 protein, SARS-CoV-2 MeSH Browser
• NSP16 protein, SARS-CoV-2 MeSH Browser
• RNA Caps MeSH
• RNA, Viral MeSH
• Viral Nonstructural Proteins MeSH
• Viral Regulatory and Accessory Proteins MeSH

We report the crystal structure of the SARS-CoV-2 putative primase composed of the nsp7 and nsp8 proteins. We observed a dimer of dimers (2:2 nsp7-nsp8) in the crystallographic asymmetric unit. The structure revealed a fold with a helical core of the heterotetramer formed by both nsp7 and nsp8 that is flanked with two symmetry-related nsp8 β-sheet subdomains. It was also revealed that two hydrophobic interfaces one of approx. 1340 Å2 connects the nsp7 to nsp8 and a second one of approx. 950 Å2 connects the dimers and form the observed heterotetramer. Interestingly, analysis of the surface electrostatic potential revealed a putative RNA binding site that is formed only within the heterotetramer.

• Keywords
• Crystal structure, Primase, RNA, SARS-CoV-2,
• MeSH
• Betacoronavirus chemistry   MeSH
• DNA Primase chemistry   metabolism   MeSH
• Protein Conformation MeSH
• Coronavirus RNA-Dependent RNA Polymerase MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• Multiprotein Complexes MeSH
• RNA metabolism   MeSH
• SARS-CoV-2 MeSH
• Binding Sites MeSH
• Viral Nonstructural Proteins chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Primase MeSH
• Coronavirus RNA-Dependent RNA Polymerase MeSH
• Multiprotein Complexes MeSH
• NS8 protein, SARS-CoV-2 MeSH Browser
• NSP7 protein, SARS-CoV-2 MeSH Browser
• RNA MeSH
• Viral Nonstructural Proteins MeSH

Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes.

• MeSH
• Cell Membrane metabolism   MeSH
• Dimerization MeSH
• Humans MeSH
• Membrane Lipids metabolism   MeSH
• Membrane Proteins * metabolism   MeSH
• Protein Multimerization MeSH
• Fluorescence Resonance Energy Transfer * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Lipids MeSH
• Membrane Proteins * MeSH

Many picornaviruses hijack the Golgi resident Acyl-coenzyme A binding domain containing 3 (ACBD3) protein in order to recruit the phosphatidylinositol 4-kinase B (PI4KB) to viral replication organelles (ROs). PI4KB, once recruited and activated by ACBD3 protein, produces the lipid phosphatidylinositol 4-phosphate (PI4P), which is a key step in the biogenesis of viral ROs. To do so, picornaviruses use their small nonstructural protein 3A that binds the Golgi dynamics domain of the ACBD3 protein. Here, we present the analysis of the highly flexible ACBD3 proteins and the viral 3A protein in solution using small-angle X-ray scattering and computer simulations. Our analysis revealed that both the ACBD3 protein and the 3A:ACBD3 protein complex have an extended and flexible conformation in solution.

• Keywords
• ACBD3, RNA virus, coarse-grained simulations, host factor, intrinsically disordered regions, picornavirus, small-angle X-ray scattering (SAXS),
• MeSH
• Acyl Coenzyme A chemistry   metabolism   MeSH
• Adaptor Proteins, Signal Transducing chemistry   metabolism   MeSH
• Humans MeSH
• Membrane Proteins chemistry   metabolism   MeSH
• Picornaviridae chemistry   metabolism   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ACBD3 protein, human MeSH Browser
• Acyl Coenzyme A MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Membrane Proteins MeSH

Enteroviruses, members of the family of picornaviruses, are the most common viral infectious agents in humans causing a broad spectrum of diseases ranging from mild respiratory illnesses to life-threatening infections. To efficiently replicate within the host cell, enteroviruses hijack several host factors, such as ACBD3. ACBD3 facilitates replication of various enterovirus species, however, structural determinants of ACBD3 recruitment to the viral replication sites are poorly understood. Here, we present a structural characterization of the interaction between ACBD3 and the non-structural 3A proteins of four representative enteroviruses (poliovirus, enterovirus A71, enterovirus D68, and rhinovirus B14). In addition, we describe the details of the 3A-3A interaction causing the assembly of the ACBD3-3A heterotetramers and the interaction between the ACBD3-3A complex and the lipid bilayer. Using structure-guided identification of the point mutations disrupting these interactions, we demonstrate their roles in the intracellular localization of these proteins, recruitment of downstream effectors of ACBD3, and facilitation of enterovirus replication. These structures uncovered a striking convergence in the mechanisms of how enteroviruses and kobuviruses, members of a distinct group of picornaviruses that also rely on ACBD3, recruit ACBD3 and its downstream effectors to the sites of viral replication.

• MeSH
• Adaptor Proteins, Signal Transducing chemistry   genetics   metabolism   MeSH
• Phosphotransferases (Alcohol Group Acceptor) genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Host-Pathogen Interactions * MeSH
• Protein Conformation MeSH
• Crystallization MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Membrane Proteins chemistry   genetics   metabolism   MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Picornaviridae physiology   MeSH
• Virus Replication * MeSH
• Amino Acid Sequence MeSH
• Sequence Homology MeSH
• Protein Binding MeSH
• Viral Proteins chemistry   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ACBD3 protein, human MeSH Browser
• Adaptor Proteins, Signal Transducing MeSH
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• Membrane Proteins MeSH
• Viral Proteins MeSH

Zika virus is a global health threat due to significantly elevated risk of fetus malformations in infected pregnant women. Currently, neither an effective therapy nor a prophylactic vaccination is available for clinical use, desperately necessitating novel therapeutics and approaches to obtain them. Here, we present a structural model of the Zika virus RNA-dependent RNA polymerase (ZIKV RdRp) in complex with template and nascent RNAs, Mg2+ ions and accessing nucleoside triphosphate. The model allowed for docking studies aimed at effective pre-screening of potential inhibitors of ZIKV RdRp. Applicability of the structural model for docking studies was illustrated with the NITD008 artificial nucleotide that is known to effectively inhibit the function of the ZIKV RdRp. The ZIKV RdRp - RNA structural model is provided for all possible variations of the nascent RNA bases pairs to enhance its general utility in docking and modelling experiments. The developed model makes the rational design of novel nucleosides and nucleotide analogues feasible and thus provides a solid platform for the development of advanced antiviral therapy.

• MeSH
• Adenosine analogs & derivatives   chemistry   pharmacology   MeSH
• Magnesium chemistry   MeSH
• Zika Virus Infection genetics   virology   MeSH
• Protein Conformation drug effects   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Nucleosides chemistry   MeSH
• Nucleotides chemistry   MeSH
• Polyphosphates chemistry   MeSH
• Virus Replication genetics   MeSH
• RNA-Dependent RNA Polymerase chemistry   genetics   MeSH
• RNA chemistry   genetics   MeSH
• Molecular Docking Simulation MeSH
• Viral Nonstructural Proteins chemistry   genetics   MeSH
• Zika Virus chemistry   genetics   pathogenicity   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine MeSH
• Magnesium MeSH
• NITD008 MeSH Browser
• Nucleosides MeSH
• Nucleotides MeSH
• Polyphosphates MeSH
• RNA-Dependent RNA Polymerase MeSH
• RNA MeSH
• triphosphoric acid MeSH Browser
• Viral Nonstructural Proteins MeSH