Negative charge and membrane-tethered viral 3B cooperate to recruit viral RNA dependent RNA polymerase 3D pol

Abstract 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 3Dpol 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 3Dpol

Structural analysis of the SARS-CoV-2 methyltransferase complex involved in RNA cap creation bound to sinefungin

SARS-CoV-2 expresses a 2′-O RNA methyltransferase (MTase) that is involved in the viral RNA cap formation and therefore a target for antiviral therapy. Here the authors provide the structure of nsp10-nsp16 with the panMTase inhibitor sinefungin and report that the development of MTase inhibitor therapies that target multiple coronoaviruses is feasible

High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates

Structure of SARS-CoV-2 MTase nsp14 with the inhibitor STM957 reveals inhibition mechanism that is shared with a poxviral MTase VP39

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

Phosphatidylinositol 4-kinase IIIβ (PI4KB) forms highly flexible heterocomplexes that include ACBD3, 14-3-3, and Rab11 proteins

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a key enzyme of the Golgi system because it produces its lipid hallmark - the phosphatidylinositol 4-phosphate (PI4P). It is recruited to Golgi by the Golgi resident ACBD3 protein, regulated by 14-3-3 proteins and it also serves as an adaptor because it recruits the small GTPase Rab11. Here, we analyzed the protein complexes formed by PI4KB in vitro using small angle x-ray scattering (SAXS) and we discovered that these protein complexes are highly flexible. The 14-3-3:PI4KB:Rab11 protein complex has 2:1:1 stoichiometry and its different conformations are rather compact, however, the ACBD3:PI4KB protein complex has both, very compact and very extended conformations. Furthermore, in vitro reconstitution revealed that the membrane is necessary for the formation of ACBD3:PI4KB:Rab11 protein complex at physiological (nanomolar) concentrations

Relation between energetic and geometric properties, obtained by numerical minimization of the energy functional Eq. (6) for fixed parameters and .

<p>(A) Minimum energy rescaled by the membrane bending rigidity as a function of the ESCRT binding energy rescaled by the area of the lipid-segregated domain. (B) Minimum energy as a function of the cosine of the membrane tangent angle along the lines of longitude at the boundary of the ESCRT-coated domain. (C) as a function of the binding energy rescaled by the area of the lipid-segregated domain . (D) as a function of the area fraction , where is the area of the ESCRT-rich domain .</p

ESCRT protein assemblies (blue) facilitate the sorting of ubiquitinated membrane proteins (green) and formation of intralumenal vesicles (ILV).

<p>The ESCRT proteins assemble on the membrane side opposite the vesicle bud (C) and do not enter the ILV lumen for possible recycling (D). Energy input from ATP hydrolysis accelerates disassembly of the ESCRT machinery (D), but is not required in steps (A–C).</p

Membrane morphology diagram from the analytical solution obtained for the spherical cap approximation (lines) and from numerical energy minimizations (symbols).

<p>Results are shown for a Gaussian bending modulus fixed at . The green squares represent the flat membrane state. The magenta triangles correspond to ESCRT-coated membrane buds. The orange circles represent bare lipid buds with ESCRTs localized in the bud neck. The black solid lines are the coexistence lines, and the black dotted line represents the spinodal at which the energetic barrier to bud formation vanishes. Exemplary membrane shapes are shown for indicated points. The red and blue lines represent lipid domains and ESCRT-rich domains, respectively.</p

Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition

Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4‑Kinase IIIβ (PI4KB)

The lipid kinase phosphatidylinositol 4-kinase IIIβ (PI4KB) is an essential host factor for many positive-sense single-stranded RNA (+RNA) viruses including human pathogens hepatitis C virus (HCV), Severe acute respiratory syndrome (SARS), coxsackie viruses, and rhinoviruses. Inhibitors of PI4KB are considered to be potential broad-spectrum virostatics, and it is therefore critical to develop a biochemical understanding of the kinase. Here, we present highly potent and selective fluorescent inhibitors that we show to be useful chemical biology tools especially in determination of dissociation constants. Moreover, we show that the coumarin-labeled inhibitor can be used to image PI4KB in cells using fluorescence-lifetime imaging microscopy (FLIM) microscopy