Abstract
Coronaviral methyltransferases (MTases), nsp10/16 and nsp14, catalyze the last two steps of viral RNA-cap creation that takes place in cytoplasm. This cap is essential for the stability of ...viral RNA and, most importantly, for the evasion of innate immune system. Non-capped RNA is recognized by innate immunity which leads to its degradation and the activation of antiviral immunity. As a result, both coronaviral MTases are in the center of scientific scrutiny. Recently, X-ray and cryo-EM structures of both enzymes were solved even in complex with other parts of the viral replication complex. High-throughput screening as well as structure-guided inhibitor design have led to the discovery of their potent inhibitors. Here, we critically summarize the tremendous advancement of the coronaviral MTase field since the beginning of COVID pandemic.
Monkeypox is a disease with pandemic potential. It is caused by the monkeypox virus (MPXV), a double-stranded DNA virus from the Poxviridae family, that replicates in the cytoplasm and must encode ...for its own RNA processing machinery including the capping machinery. Here, we present crystal structures of its 2'-O-RNA methyltransferase (MTase) VP39 in complex with the pan-MTase inhibitor sinefungin and a series of inhibitors that were discovered based on it. A comparison of this 2'-O-RNA MTase with enzymes from unrelated single-stranded RNA viruses (SARS-CoV-2 and Zika) reveals a conserved sinefungin binding mode, implicating that a single inhibitor could be used against unrelated viral families. Indeed, several of our inhibitors such as TO507 also inhibit the coronaviral nsp14 MTase.
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.
Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic ...cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.
Most single stranded plus RNA viruses hijack phosphatidylinositol 4-kinases (PI4Ks) to generate membranes highly enriched in phosphatidylinositol 4-phosphate (PI4P). These membranous compartments ...known as webs, replication factories or replication organelles are essential for viral replication because they provide protection from the innate intracellular immune response while serving as platforms for viral replication. Using purified recombinant proteins and biomimetic model membranes we show that the nonstructural viral 3A protein is sufficient to promote membrane hyper-phosphorylation given the proper intracellular cofactors (PI4KB and ACBD3). However, our bio-mimetic in vitro reconstitution assay revealed that rather than the presence of PI4P specifically, negative charge alone is sufficient for the recruitment of 3D
enzymes to the surface of the lipid bilayer. Additionally, we show that membrane tethered viral 3B protein (also known as Vpg) works in combination with the negative charge to increase the efficiency of membrane recruitment of 3D
.
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•A crystal structure of coronaviral methyltransferase (MTase) nsp14 was solved with an inhibitor STM957.•The structure revealed that STM957 is, indeed, a bisubstrate inhibitor.•The ...aromatic moiety at the 7-deaza base of STM957 replaced a water network near the adenine base.•Similar mechanism of inhibition was observed in mpox MTase VP39.•Displacement of water molecules from the vicinity of the active site might be a common mechanism of viral MTase inhibitors.
Nsp14 is an RNA methyltransferase (MTase) encoded by all coronaviruses. In fact, many viral families, including DNA viruses, encode MTases that catalyze the methylation of the RNA precap structure, resulting in fully capped viral RNA. This capping is crucial for efficient viral RNA translation, stability, and immune evasion. Our previous research identified nsp14 inhibitors based on the chemical scaffold of its methyl donor − the S-adenosyl methionine (SAM) − featuring a modified adenine base and a substituted arylsulfonamide. However, the binding mode of these inhibitors was based only on docking experiments. To uncover atomic details of nsp14 inhibition we solved the crystal structure of nsp14 bound to STM957. The structure revealed the atomic details of nsp14 inhibition such that the 7-deaza-adenine moiety of STM957 forms specific interactions with Tyr368, Ala353, and Phe367, while the arylsulfonamide moiety engages with Asn388 and Phe506. The large aromatic substituent at the 7-deaza position displaces a network of water molecules near the adenine base. Surprisingly, this was recently observed in the case of an unrelated monkeypox MTase VP39, where the 7-deaza modified SAH analogs also displaced water molecules from the vicinity of the active site.
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.
ORPs are lipid-transport proteins belonging to the oxysterol-binding protein family. They facilitate the transfer of lipids between different intracellular membranes, such as the ER and plasma ...membrane. We have solved the crystal structure of the ORP8 lipid transport domain (ORD8). The ORD8 exhibited a β-barrel fold composed of anti-parallel β-strands, with three α-helices replacing β-strands on one side. This mixed alpha-beta structure was consistent with previously solved structures of ORP2 and ORP3. A large cavity (≈1860 Å
) within the barrel was identified as the lipid-binding site. Although we were not able to obtain a lipid-bound structure, we used computer simulations based on our crystal structure to dock PS and PI4P molecules into the putative lipid-binding site of the ORD8. Comparative experiments between the short ORD8
(used for crystallography) and the full-length ORD8 (lid containing) revealed the lid's importance for stable lipid binding. Fluorescence assays revealed different transport efficiencies for PS and PI4P, with the lid slowing down transport and stabilizing cargo. Coarse-grained simulations highlighted surface-exposed regions and hydrophobic interactions facilitating lipid bilayer insertion. These findings enhance our comprehension of ORD8, its structure, and lipid transport mechanisms, as well as provide a structural basis for the design of potential inhibitors.
The role the charge sign of simple ions plays in determining their surface affinity in aqueous solutions is investigated by computer simulation methods. For this purpose, the free surface of aqueous ...solutions of fictitious salts is simulated at finite concentration both with nonpolarizable point-charge and polarizable Gaussian-charge potential models. The salts consist of monovalent cations and anions that are, apart from the sign of their charge, identical to each other. In particular, we consider the small Na+ and the large I– ions together with their charge-inverted counterparts. In an attempt to avoid the interference even between the behavior of cations and anions, we also simulate systems containing only one of the above ions, and determine the free energy profile of these ions across the liquid–vapor interface of water at infinite dilution by potential of mean force (PMF) calculations. The obtained results reveal that, in the case of small ions, the anion is hydrated considerably stronger than the cation due to the close approach of water H atoms, bearing a positive fractional charge. As a consequence, the surface affinity of a small anion is even smaller than that of its cationic counterpart. However, considering that small ions are effectively repelled from the water surface, the importance of this difference is negligible. Further, a change in the hydration energy trends of the two oppositely charged ions is observed with their increasing size. This change is largely attributed to the fact that, with increasing ion size, the factor of 2 in the magnitude of the fractional charge of the closely approaching water atoms (i.e., O around cations and H around anions) outweighs the closer approach of the H than the O atom in the hydration energy. Thus, for large ions, being already surface active themselves, the surface affinity of the anion is larger than that of its positively charged counterpart. Further, such a difference is seen even in the case when the sign of the surface potential favors the adsorption of cations.
Expansion of water vapor through a small orifice to a vacuum produces liquid or frozen clusters which in the experiment serve as model particles for atmospheric aerosols. Yet, there are controversies ...about the shape of these clusters, suggesting that the nucleation process is not fully understood. Such questions can be answered by molecular dynamics simulations; however, they require microsecond-scale runs with thousands of molecules and accurate energy conservation. The available highly parallel codes typically utilize domain decomposition and are inefficient for heterogeneous systems as clusters in a dilute gas. In this work, we present an implementation of molecular dynamics on graphics processing units based on the Verlet list and apply it to several systems for which experimental data are available. We reproduce sufficiently sized clusters but not the experimentally observed clusters of irregular shape.
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