Denis E. Kainov

Atomic Snapshots of an RNA Packaging Motor Reveal Conformational Changes Linking ATP Hydrolysis to RNA Translocation

Many viruses package their genome into preformed capsids using packaging motors powered by the hydrolysis of ATP. The hexameric ATPase P4 of dsRNA bacteriophage phi12, located at the vertices of the icosahedral capsid, is such a packaging motor. We have captured crystallographic structures of P4 for all the key points along the catalytic pathway, including apo, substrate analog bound, and product bound. Substrate and product binding have been observed as both binary complexes and ternary complexes with divalent cations. These structures reveal large movements of the putative RNA binding loop, which are coupled with nucleotide binding and hydrolysis, indicating how ATP hydrolysis drives RNA translocation through cooperative conformational changes. Two distinct conformations of bound nucleotide triphosphate suggest how hydrolysis is activated by RNA binding. This provides a model for chemomechanical coupling for a prototype of the large family of hexameric helicases and oligonucleotide translocating enzymes.

Emerging cellular targets for influenza antiviral agents

At the global level, influenza A virus (IAV) is considered a major health threat because it causes significant morbidity. Different treatment and prevention options have been developed; however, these are insufficient in the face of recent IAV outbreaks. In particular, available antiviral agents have limited effectiveness owing to IAV resistance to these virus-directed drugs. Recent advances in understanding of IAV replication have revealed a number of cellular drug targets that counteract viral drug resistance. This review summarizes current knowledge on IAV replication with a focus on emerging cellular drug targets. Interestingly, for many of these targets, compounds for which safety testing has been carried out in humans are available. It is possible that some of these compounds, such as inhibitors of heat shock protein 90, proteasome, importin α5 or protein kinase C, will be used for treatment of IAV infections after careful evaluation in human primary cells and severely ill flu patients.

Functional visualization of viral molecular motor by hydrogen-deuterium exchange reveals transient states

Molecular motors undergo cyclical conformational changes and convert chemical energy into mechanical work. The conformational dynamics of a viral packaging motor, the hexameric helicase P4 of dsRNA bacteriophage φ8, was visualized by hydrogen-deuterium exchange and high-resolution mass spectrometry. Concerted changes of exchange kinetics revealed a cooperative unit that dynamically links ATP-binding sites and the central RNA-binding channel. The cooperative unit is compatible with a structure-based model in which translocation is mediated by a swiveling helix. Deuterium labeling also revealed the transition state associated with RNA loading, which proceeds via opening of the hexameric ring. The loading mechanism is similar to that of other hexameric helicases. Hydrogen-deuterium exchange provides an important link between time-resolved spectroscopic observations and high-resolution structural snapshots of molecular machines.

Conserved Intermediates on the Assembly Pathway of Double-stranded RNA Bacteriophages

Double-stranded RNA (dsRNA) viruses are complex RNA processing machines that sequentially perform packaging, replication and transcription of their genomes. In order to characterize the assembly intermediates of such a machine we have developed an efficient in vitro assembly system for the procapsid of bacteriophage phi8. The major structural protein P1 is a stable and soluble tetramer. Three tetramers associate with a P2 monomer (RNA-dependent RNA polymerase) to form the nucleation complex. This complex is further stabilized by a P4 hexamer (packaging motor). Further assembly proceeds via rapid addition of individual building blocks. The incorporation of the packaging and replication machinery is under kinetic control. The in vitro assembled procapsids perform packaging, replication and transcription of viral RNA. Comparison with another dsRNA phage, phi6, indicates conservation of key assembly intermediates in the absence of sequence homology and suggests that a general assembly mechanism for the dsRNA virus lineage may exist.

Inhibition of influenza A virus infection in vitro by saliphenylhalamide- loaded porous silicon nanoparticles

Influenza A viruses (IAVs) cause recurrent epidemics in humans, with serious threat of lethal worldwide pandemics. The occurrence of antiviral-resistant virus strains and the emergence of highly pathogenic influenza viruses have triggered an urgent need to develop new anti-IAV treatments. One compound found to inhibit IAV, and other virus infections, is saliphenylhalamide (SaliPhe). SaliPhe targets host vacuolar-ATPase and inhibits acidification of endosomes, a process needed for productive virus infection. The major obstacle for the further development of SaliPhe as antiviral drug has been its poor solubility. Here, we investigated the possibility to increase SaliPhe solubility by loading the compound in thermally hydrocarbonized porous silicon (THCPSi) nanoparticles. SaliPhe-loaded nanoparticles were further investigated for the ability to inhibit influenza A infection in human retinal pigment epithelium and Madin-Darby canine kidney cells, and we show that upon release from THCPSi, SaliPhe inhibited IAV infection in vitro and reduced the amount of progeny virus in IAV-infected cells. Overall, the PSi-based nanosystem exhibited increased dissolution of the investigated anti-IAV drug SaliPhe and displayed excellent in vitro stability, low cytotoxicity, and remarkable reduction of viral load in the absence of organic solvents. This proof-of-principle study indicates that PSi nanoparticles could be used for efficient delivery of antivirals to infected cells.

Hexameric molecular motors: P4 packaging ATPase unravels the mechanism

Abstract.Genome packaging into an empty capsid is an essential step in the assembly of many complex viruses. In double-stranded RNA (dsRNA) bacteriophages of the Cystoviridae family this step is performed by a hexameric helicase P4 which is one of the simplest packaging motors found in nature. Biochemical and structural studies of P4 proteins have led to a surprising finding that these proteins bear mechanistic and structural similarities to a variety of the pervasive RecA/F1-ATPase-like motors that are involved in diverse biological functions. This review describes the role of P4 proteins in assembly, transcription and replication of dsRNA bacteriophages as it has emerged over the past decade while focusing on the most recent structural studies. The P4 mechanism is compared with the models proposed for the related hexameric motors.

Obatoclax, saliphenylhalamide and gemcitabine inhibit influenza A virus infection

Background: Novel options should be developed for treatment of IAV infections. Results: Obatoclax, saliphenylhalamide, and gemcitabine target host factors and inhibit IAV and several other viruses infections. Conclusion: These compounds represent potent antiviral agents. Significance: These compounds could be exploited in treatment of severe viral infections. Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.

The proton translocation domain of cellular vacuolar ATPase provides a target for the treatment of influenza A virus infections

BACKGROUND AND PURPOSE Cellular vacuolar ATPases (v‐ATPase) play an important role in endosomal acidification, a critical step in influenza A virus (IAV) host cell infection. We investigated the antiviral activity of the v‐ATPase inhibitor saliphenylhalamide (SaliPhe) and compared it with several older v‐ATPase inhibitors concanamycin A, bafilomycin A1, (BafA) and archazolid B targeting the subunit c of the V0 sector.

Structural Basis of Mechanochemical Coupling in a Hexameric Molecular Motor.

The P4 protein of bacteriophage φ12 is a hexameric molecular motor closely related to superfamily 4 helicases. P4 converts chemical energy from ATP hydrolysis into mechanical work, to translocate single-stranded RNA into a viral capsid. The molecular basis of mechanochemical coupling, i.e. how small ∼1 Å changes in the ATP-binding site are amplified into nanometer scale motion along the nucleic acid, is not understood at the atomic level. Here we study in atomic detail the mechanochemical coupling using structural and biochemical analyses of P4 mutants. We show that a conserved region, consisting of superfamily 4 helicase motifs H3 and H4 and loop L2, constitutes the moving lever of the motor. The lever tip encompasses an RNA-binding site that moves along the mechanical reaction coordinate. The lever is flanked by γ-phosphate sensors (Asn-234 and Ser-252) that report the nucleotide state of neighboring subunits and control the lever position. Insertion of an arginine finger (Arg-279) into the neighboring catalytic site is concomitant with lever movement and commences ATP hydrolysis. This ensures cooperative sequential hydrolysis that is tightly coupled to mechanical motion. Given the structural conservation, the mutated residues may play similar roles in other hexameric helicases and related molecular motors.

Akt inhibitor MK2206 prevents influenza pH1N1 virus infection in vitro

ABSTRACT The influenza pH1N1 virus caused a global flu pandemic in 2009 and continues manifestation as a seasonal virus. Better understanding of the virus-host cell interaction could result in development of better prevention and treatment options. Here we show that the Akt inhibitor MK2206 blocks influenza pH1N1 virus infection in vitro. In particular, at noncytotoxic concentrations, MK2206 alters Akt signaling and inhibits endocytic uptake of the virus. Interestingly, MK2206 is unable to inhibit H3N2, H7N9, and H5N1 viruses, indicating that pH1N1 evolved specific requirements for efficient infection. Thus, Akt signaling could be exploited further for development of better therapeutics against pH1N1 virus.