Tick-borne encephalitis virus capsid protein induces translational shutoff as revealed by its structural-biological analysis.

Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural in vitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV.

Correction: Krízová et al. Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein. Viruses 2021, 13, 2451.

The authors wish to make the following corrections to this paper [...].

Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein.

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

Characterization and in vitro assembly of tick-borne encephalitis virus C protein.

Tick-borne encephalitis virus (TBEV), a member of flaviviruses, represents a serious health threat by causing human encephalitis mainly in central and eastern Europe, Russia, and northeastern Asia. As no specific therapy is available, there is an urgent need to understand all steps of the TBEV replication cycle at the molecular level. One of the critical events is the packaging of flaviviral genomic RNA by TBEV C protein to form a nucleocapsid. We purified recombinant TBEV C protein and used a combination of physical-chemical approaches, such as size-exclusion chromatography, circular dichroism, NMR spectroscopies, and transmission electron microscopy, to analyze its structural stability and its ability to dimerize/oligomerize. We compared the ability of TBEV C protein to assemble in vitro into a nucleocapsid-like structure with that of dengue C protein.

PF74 and Its Novel Derivatives Stabilize Hexameric Lattice of HIV-1 Mature-Like Particles.

A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein-protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.

A simple, high-throughput stabilization assay to test HIV-1 uncoating inhibitors.

Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.

Membrane Interactions of the Mason-Pfizer Monkey Virus Matrix Protein and Its Budding Deficient Mutants.

Matrix proteins (MAs) play a key role in the transport of retroviral proteins inside infected cells and in the interaction with cellular membranes. In most retroviruses, retroviral MAs are N-terminally myristoylated. This modification serves as a membrane targeting signal and also as an anchor for membrane interaction. The aim of this work was to characterize the interactions anchoring retroviral MA at the plasma membrane of infected cell. To address this issue, we compared the structures and membrane affinity of the Mason-Pfizer monkey virus (M-PMV) wild-type MA with its two budding deficient double mutants, that is, T41I/T78I and Y28F/Y67F. The structures of the mutants were determined using solution NMR spectroscopy, and their interactions with water-soluble phospholipids were studied. Water-soluble phospholipids are widely used models for studying membrane interactions by solution NMR spectroscopy. However, this approach might lead to artificial results due to unnatural hydrophobic interactions. Therefore, we used a new approach based on the measurement of the loss of the 1H NMR signal intensity of the protein sample induced by the addition of the liposomes containing phospholipids with naturally long fatty acids. HIV-1 MA was used as a positive control because its ability to interact with liposomes has already been described. We found that in contrast to HIV-1, the M-PMV MA interacted with the liposomes differently and much weaker. In our invivo experiments, the M-PMV MA did not co-localize with lipid rafts. Therefore, we concluded that M-PMV might adopt a different membrane binding mechanism than HIV-1.

Resonance assignments of the myristoylated Y28F/Y67F mutant of the Mason-Pfizer monkey virus matrix protein.

The matrix protein (MA) of the Mason-Pfizer monkey virus (M-PMV) plays a key role in the transport and budding of immature retroviral particles from the host cell. Natural N-terminal myristoylation of MA is essential for the targeting of the particles to the plasma membrane and participates in the interaction of MA with membranes phospholipids. The mutation Y28F/Y67F in MA reduces budding and thus causes the accumulation of viral particles under the cytoplasmic membrane. To investigate the impact of Y28F/Y67F mutation on the structure of MA, we prepared this protein in amount and quality suitable for NMR spectroscopy. We report backbone, side-chain and myristoyl residue assignments of the Y28F/Y67F mutant of the M-PMV matrix protein, which will be used to study the interaction with membrane phospholipids and to determine the structure of the mutant matrix protein.

Stabilization of the ß-hairpin in Mason-Pfizer monkey virus capsid protein- a critical step for infectivity.

BACKGROUND: Formation of a mature core is a crucial event for infectivity of retroviruses such as Mason-Pfizer monkey virus (M-PMV). The process is triggered by proteolytic cleavage of the polyprotein precursor Gag, which releases matrix, capsid (CA), and nucleocapsid proteins. Once released, CA assembles to form a mature core - a hexameric lattice protein shell that protects retroviral genomic RNA. Subtle conformational changes within CA induce the transition from the immature lattice to the mature lattice. Upon release from the precursor, the initially unstructured N-terminus of CA is refolded to form a ß-hairpin stabilized by a salt bridge between the N-terminal proline and conserved aspartate. Although the crucial role of the ß-hairpin in the mature core assembly has been confirmed, its precise structural function remains poorly understood. RESULTS: Based on a previous NMR analysis of the N-terminal part of M-PMV CA, which suggested the role of additional interactions besides the proline-aspartate salt bridge in stabilization of the ß-hairpin, we introduced a series of mutations into the CA sequence. The effect of the mutations on virus assembly and infectivity was analyzed. In addition, the structural consequences of selected mutations were determined by NMR spectroscopy. We identified a network of interactions critical for proper formation of the M-PMV core. This network involves residue R14, located in the N-terminal ß-hairpin; residue W52 in the loop connecting helices 2 and 3; and residues Q113, Q115, and Y116 in helix 5. CONCLUSION: Combining functional and structural analyses, we identified a network of supportive interactions that stabilize the ß-hairpin in mature M-PMV CA.

Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.

Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures or studying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon (13)C and nitrogen (15)N. The recombinant expression of labeled proteins is simple to perform but requires quite expensive chemicals. When there is a need for special labeled chemicals, like uniformly (13)C-labeled myristic acid, the price significantly rises. Here we describe a cost-effective method for the recombinant expression of uniformly labeled myristoylated proteins in Escherichia coli demonstrated on the production of Mason-Pfizer monkey virus matrix protein. We used the ability of E. coli to naturally synthetize myristic acid. When grown in isotopically labeled medium the myristic acid will be labelled as well. Bacteria were co-transfected with plasmid carrying gene for yeast N-myristoyltransferase which ensures myristoylation of expressed protein. This process provided 1.8mg of the myristoylated, doubly labeled ((13)C/(15)N)M-PMV matrix protein from 1L of (15)N/(13)C labeled M9 medium. The price represents approximately 50% cost reduction of conventional method using commercially available [U-(13)C]myristic acid.

Re-evaluation of binding properties of recombinant lymphocyte receptors NKR-P1A and CD69 to chemically synthesized glycans and peptides.

The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-D-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.

One-step separation of myristoylated and nonmyristoylated retroviral matrix proteins.

N-terminal myristoylation of retroviral matrix proteins is essential for the targeting of the Gag polyproteins to the plasma membrane. To investigate the effect of the myristoylation on the structure and membrane binding ability of the matrix proteins, it is necessary to prepare their myristoylated forms. We present purification of myristoylated matrix proteins of the mouse mammary tumor virus and murine leukemia virus, two morphogenetically distinct retroviruses. The proteins were expressed in Escherichia coli coexpressing a yeast N-myristoyltransferase. This E. coli expression system yielded a mixture of myristoylated and nonmyristoylated matrix proteins. We established efficient one-step metal affinity purification that enabled to obtain pure myristoylated matrix proteins suitable for structural and functional studies.

Interaction of Mason-Pfizer monkey virus matrix protein with plasma membrane.

Budding is the final step of the late phase of retroviral life cycle. It begins with the interaction of Gag precursor with plasma membrane (PM) through its N-terminal domain, the matrix protein (MA). However, single genera of Retroviridae family differ in the way how they interact with PM. While in case of Lentiviruses (e.g., human immunodeficiency virus) the structural polyprotein precursor Gag interacts with cellular membrane prior to the assembly, Betaretroviruses [Mason-Pfizer monkey virus (M-PMV)] first assemble their virus-like particles (VLPs) in the pericentriolar region of the infected cell and therefore, already assembled particles interact with the membrane. Although both these types of retroviruses use similar mechanism of the interaction of Gag with the membrane, the difference in the site of assembly leads to some differences in the mechanism of the interaction. Here we describe the interaction of M-PMV MA with PM with emphasis on the structural aspects of the interaction with single phospholipids.

The structure of myristoylated Mason-Pfizer monkey virus matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in its membrane binding.

We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C(8) fatty acid chains was monitored by observation of concentration-dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in (31)P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein-phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a (13)C-filtered/(13)C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P(2) binding was not strong enough for triggering of the myristoyl-switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein.

Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for NMR and MS measurements.

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.

Oligomerization of a retroviral matrix protein is facilitated by backbone flexibility on nanosecond time scale.

The oligomerization capacity of the retroviral matrix protein is an important feature that affects assembly of immature virions and their interaction with cellular membrane. A combination of NMR relaxation measurements and advanced analysis of molecular dynamics simulation trajectory provided an unprecedentedly detailed insight into internal mobility of matrix proteins of the Mason-Pfizer monkey virus. Strong evidence have been obtained that the oligomerization capacity of the wild-type matrix protein is closely related to the enhanced dynamics of several parts of its backbone on a nanosecond time scale. Increased flexibility has been observed for two regions: the loop between α-helices α2 and α3 and the C-terminal half of α-helix α3 which accommodate amino acid residues that form the oligomerization interface. On the other hand, matrix mutant R55F that has changed structure and does not exhibit any specific oligomerization in solution was found considerably more rigid. Our results document that conformational selection mechanism together with induced fit and favorable structural preorganization play an important role in the control of the oligomerization process.

Nonmyristoylated matrix protein from the Mason-Pfizer monkey virus forms oligomers.

We studied the oligomeric properties of betaretroviral nonmyristoylated matrix protein (MA) and its R55F mutant from the Mason-Pfizer monkey virus in solution by means of chemical crosslinking and NMR spectroscopy. By analyzing crosslinked products and using concentration-dependent NMR chemical shift mapping, we have proven that the wild-type (WT) MA forms oligomers in solution. Conversely, no oligomerization was observed for the R55F mutant. Structural comparison of MAs explained their different behaviors in solution, concluding that the key residues involved in intermonomeric interaction are exposed in the WT MA but buried in the mutant, preventing the oligomerization of R55F. The final model of oligomerization of the WT MA was derived by concerted use of chemical shift mapping and diffusion-ordered spectroscopy measured on a set of protein samples with varying concentrations. We found that the Mason-Pfizer monkey virus WT MA exists in a monomer-dimer-trimer equilibrium in solution, with the corresponding dissociation constants of 2.3 and 0.24 mM, respectively. Structures of the oligomers calculated with HADDOCK software are closely related to the structures of other retroviral MA trimers.

D-retrovirus morphogenetic switch driven by the targeting signal accessibility to Tctex-1 of dynein.

Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason-Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C- and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.

The role of the S-S bridge in retroviral protease function and virion maturation.

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.

An experimental and theoretical study of stereoselectivity of furan-maleic anhydride and furan-maleimide diels-alder reactions.

The stereoselectivity of the reaction of furan (1) with maleic anhydride (2) and maleimide (3) was studied experimentally and theoretically. Although the two reactions are highly similar with regard to their preference for endo and exo steroisomers, notable differences were experimentally observed and explained on the basis of calculated reaction-free energies and transition-state barriers. The experimental values of rate constants (k(1+2endo) = (1.75 +/- 0.48) x 10(-5); mol(-1) l s(-1); k(1+2exo) = (3.10 +/- 0.55) x 10(-5); mol(-1) l s(-1); k(1+3endo) = (1.93 +/- 0.082) x 10(-5); mol(-1) l s(-1), k(1+3exo) = (1.38 +/- 0.055) x 10(-5); mol(-1) l s(-1) all at 300 K) and the observed reaction course clearly confirm that neither of these reactions are prototypical examples of Diels-Alder [4 + 2] cycloadditions, whose dominant preference is for endo isomers. However, only by comparing their energetics-calculated at the CCSD(T) level of theory-with the analogous reactions involving cyclopentadiene (8) as a diene can these observations be understood. The low thermodynamic stability of furan [4 + 2] adducts opens retro-Diels-Alder reaction channels and overrules the very small kinetic preference (calculated and measured here) of initial formation for endo stereoisomers. On a macroscopic scale "an irregular"-thermodynamically more stable-exo stereoisomer was consequently observed as a dominant species.