Rhinovirus Inhibitors: Including a New Target, the Viral RNA

Rhinoviruses (RVs) are the main cause of recurrent infections with rather mild symptoms characteristic of the common cold. Nevertheless, RVs give rise to enormous numbers of absences from work and school and may become life-threatening in particular settings. Vaccination is jeopardised by the large number of serotypes eliciting only poorly cross-neutralising antibodies. Conversely, antivirals developed over the years failed FDA approval because of a low efficacy and/or side effects. RV species A, B, and C are now included in the fifteen species of the genus Enteroviruses based upon the high similarity of their genome sequences. As a result of their comparably low pathogenicity, RVs have become a handy model for other, more dangerous members of this genus, e.g., poliovirus and enterovirus 71. We provide a short overview of viral proteins that are considered potential drug targets and their corresponding drug candidates. We briefly mention more recently identified cellular enzymes whose inhibition impacts on RVs and comment novel approaches to interfere with infection via aggregation, virus trapping, or preventing viral access to the cell receptor. Finally, we devote a large part of this article to adding the viral RNA genome to the list of potential drug targets by dwelling on its structure, folding, and the still debated way of its exit from the capsid. Finally, we discuss the recent finding that G-quadruplex stabilising compounds impact on RNA egress possibly via obfuscating the unravelling of stable secondary structural elements

Catching Common Cold Virus with a Net: Pyridostatin Forms Filaments in Tris Buffer That Trap Viruses—A Novel Antiviral Strategy?

The neutrophil extracellular trap (ET) is a eukaryotic host defense machinery that operates by capturing and concentrating pathogens in a filamentous network manufactured by neutrophils and made of DNA, histones, and many other components. Respiratory virus-induced ETs are involved in tissue damage and impairment of the alveolar–capillary barrier, but they also aid in fending off infection. We found that the small organic compound pyridostatin (PDS) forms somewhat similar fibrillary structures in Tris buffer in a concentration-dependent manner. Common cold viruses promote this process and become entrapped in the network, decreasing their infectivity by about 70% in tissue culture. We propose studying this novel mechanism of virus inhibition for its utility in preventing viral infection

Cellular N-myristoyltransferases play a crucial picornavirus genus-specific role in viral assembly, virion maturation, and infectivity.

In nearly all picornaviruses the precursor of the smallest capsid protein VP4 undergoes co-translational N-terminal myristoylation by host cell N-myristoyltransferases (NMTs). Curtailing this modification by mutation of the myristoylation signal in poliovirus has been shown to result in severe assembly defects and very little, if any, progeny virus production. Avoiding possible pleiotropic effects of such mutations, we here used pharmacological abrogation of myristoylation with the NMT inhibitor DDD85646, a pyrazole sulfonamide originally developed against trypanosomal NMT. Infection of HeLa cells with coxsackievirus B3 in the presence of this drug decreased VP0 acylation at least 100-fold, resulting in a defect both early and late in virus morphogenesis, which diminishes the yield of viral progeny by about 90%. Virus particles still produced consisted mainly of provirions containing RNA and uncleaved VP0 and, to a substantially lesser extent, of mature virions with cleaved VP0. This indicates an important role of myristoylation in the viral maturation cleavage. By electron microscopy, these RNA-filled particles were indistinguishable from virus produced under control conditions. Nevertheless, their specific infectivity decreased by about five hundred fold. Since host cell-attachment was not markedly impaired, their defect must lie in the inability to transfer their genomic RNA into the cytosol, likely at the level of endosomal pore formation. Strikingly, neither parechoviruses nor kobuviruses are affected by DDD85646, which appears to correlate with their native capsid containing only unprocessed VP0. Individual knockout of the genes encoding the two human NMT isozymes in haploid HAP1 cells further demonstrated the pivotal role for HsNMT1, with little contribution by HsNMT2, in the virus replication cycle. Our results also indicate that inhibition of NMT can possibly be exploited for controlling the infection by a wide spectrum of picornaviruses

Cryo-EM structure of pleconaril-resistant rhinovirus-B5 complexed to the antiviral OBR-5-340 reveals unexpected binding site

Viral inhibitors, such as pleconaril and vapendavir, target conserved regions in the capsids of rhinoviruses (RVs) and enteroviruses (EVs) by binding to a hydrophobic pocket in viral capsid protein 1 (VP1). In resistant RVs and EVs, bulky residues in this pocket prevent their binding. However, recently developed pyrazolopyrimidines inhibit pleconaril-resistant RVs and EVs, and computational modeling has suggested that they also bind to the hydrophobic pocket in VP1. We studied the mechanism of inhibition of pleconaril-resistant RVs using RV-B5 (1 of the 7 naturally pleconaril-resistant rhinoviruses) and OBR-5-340, a bioavailable pyrazolopyrimidine with proven in vivo activity, and determined the 3D-structure of the protein-ligand complex to 3.6 Å with cryoelectron microscopy. Our data indicate that, similar to other capsid binders, OBR-5-340 induces thermostability and inhibits viral adsorption and uncoating. However, we found that OBR-5-340 attaches closer to the entrance of the pocket than most other capsid binders, whose viral complexes have been studied so far, showing only marginal overlaps of the attachment sites. Comparing the experimentally determined 3D structure with the control, RV-B5 incubated with solvent only and determined to 3.2 Å, revealed no gross conformational changes upon OBR-5-340 binding. The pocket of the naturally OBR-5-340-resistant RV-A89 likewise incubated with OBR-5-340 and solved to 2.9 Å was empty. Pyrazolopyrimidines have a rigid molecular scaffold and may thus be less affected by a loss of entropy upon binding. They interact with less-conserved regions than known capsid binders. Overall, pyrazolopyrimidines could be more suitable for the development of new, broadly active inhibitors.This work was funded by the Austrian Science Fund project #27444 (D.B.). M.P. and I.Z. were supported by Erasmus fellowships. N.M. and J. K. were supported by the Bergen Research Foundation (BFS2017TMT01). T.C.M. was supported by funds through the Behörde für Wissenschaft, Forschung, und Gleichstellung of the City of Hamburg. Data for RV-B5 and RV-A89, both incubated with OBR-5-340, were collected at the Vienna Biocenter Electron Microscopy Facility. Data collection of the control sample (RV-B5 without OBR-5-340) was funded by iNEXT Grant 5950. iNEXT (project no. 653706) was funded by the Horizon 2020 program of the European Union. This article reflects only the author's view and the European Commission is not responsible for any use that may be made of the information it contains. Czech Infrastructure for Integrative Structural Biology research infrastructure project LM2015043, funded by Ministry of Education, Youth and Sports of the Czech Republic is gratefully acknowledged for the financial support of the measurements at the Central Facility Cryo-electron Microscopy and Tomography Central European Institute of Technology, Masaryk University

DDD85646 efficiently suppresses CVB3 cell-to-cell transmission.

<p>(A) HeLa cells were infected with CBV3-eGFP at an MOI of 0.1 in absence (DMSO control) or presence of 5 μM DDD85646 and observed under the fluorescence microscope 5 h and 24 h p.i. (B) Cells remaining intact after the 24 h multi-cycle infection with CVB3-eGFP were subsequently challenged with wt CVB3 in <u>complete absence</u> of the NMT inhibitor and visualized for a CPE 24 h later. Scale bar 5 μm.</p

DDD85646 massively reduces metabolic incorporation of the Alk-12 myristate analogue into VP0.

<p>(A) Work flow for detection of Alk-12 labeled viral proteins with click reagents. HeLa cells (DDD85646- or DMSO-treated) are infected with CVB3 at MOI of 10. The myristic acid analogue Alk-12 is added 4 h p.i. and incubation continued for 3 h; during this time Alk-12 becomes transferred to viral substrate proteins by metabolic incorporation. Cells are lysed and Alk-12-bearing proteins are ligated to the fluorescent reporter 5-TAMRA-azide by click chemistry, separated by SDS-PAGE and visualized by in-gel fluorescence. (B) DDD85646 at 5 μM blocks incorporation of Alk-12 into CVB3 VP0. The gel subjected to in-gel fluorescence recording was subsequently stained with InstantBlue to visualize total proteins for verifying equal loading.</p