UNCOATING OF RHINOVIRUS RNA: STRUCTURE & PROTEIN INTERACTIONS

We propose analyzing the RNA genome of a common cold virus with respect to its structural changes and its dynamic interactions with the inner capsid surface during the early steps in infection. This encompasses transition of the native virion to the expanded A-particle upon arrival in the acidic endosome, and RNA exit from within the protein shell into the cytosol. The analyses will be conducted by using UV-induced crosslinking, chemical modification, fluorescence correlation spectroscopy, mass spectrometry, massive parallel sequencing, and cryo-electron microscopy 3D image reconstruction. The proposed experiments will identify the nature and locations of contacts between amino acid residues of the protein shell and nucleotides of the viral genome and their changes during uncoating. They will reveal the secondary structure of the RNA when in solution, inside the native shell, and inside the A- particle and indicate whether folding is chaperoned. We shall further study the directionality of RNA release from virions inside infected cells and purified virus-containing endosomes and compare the results with existing in vitro data. Along these lines we aim at identifying uncoating facilitators to explain why RNA egress trigged by in vitro acidification occurs only with low efficiency. The results of these studies will yield a complete picture of viral uncoating inside the cell and further our understanding of the role of RNA dynamics, folding, and protein contacts in Enterovirus infection.

Picornaviruses are small infectious particles harboring a single-stranded ribonucleic acid (ssRNA) genome tightly packaged within a spherical capsid formed from 60 copies of usually 4 viral proteins, VP1, VP2, VP3, and VP4. Their many representatives such as rhino-, polio-, coxsackie- and foot-and-mouth disease virus can cause illnesses ranging from a mild common cold to cases of substantial morbidity and mortality in humans and animals. The infection starts by attachment of the virus to a receptor protein on the host cells surface, followed by its transport into the cell for release ("uncoating") of the genomic ssRNA from the protective capsid into the cytosol for virus progeny production. A detailed understanding of these steps is a prerequisite for the design of novel antiviral agents. To this end, using rhinovirus as a model, this research project originally aimed to characterize the structure of its ssRNA, map possible interaction sites with the capsid, and identify putative uncoating facilitators. However, after several unsuccessful attempts it became foreseeable that achievement of these goals could not be guaranteed within the funding period. Realizing that rhinovirus RNA features several G-quadruplex (G4) motifs predicted by a bioinformatic analysis, we decided to investigate the putative role of these atypical nucleic acid secondary structures instead. Based on extensive biophysical studies, we could clearly confirm G4 formation by these sequences and show that pretreatment with G4 binding substances such as pyridostatin (PDS) substantially curtails ssRNA release from the rhinovirus capsid. In certain buffers, we further found that PDS formed fibers, which, similar to neutrophil extracellular nets, efficiently trapped rhinovirus particles, thereby preventing their host cell attachment. In addition, in collaboration with other researchers, we have devised a monolithic anion exchange chromatography procedure yielding rhinovirus of high purity. This virus has then been used to thoroughly validate nanoDSF as a potent method for future high-throughput identification of novel uncoating inhibitors. Finally, we explored in-depth the role of a fatty acid attached to the capsid protein VP4 in most picornaviruses. We found that inhibiting the human enzymes responsible for this modification by DDD85646, a drug originally developed against Trypanosomes, led to a dramatic decrease in infectivity of most virus family members. This work showed multiple defects as underlying cause, ranging from inefficient virus assembly and maturation to a severely diminished uncoating of the still produced viral particles. Importantly, in a follow-up study in collaboration with a USA based research group, we have also found efficient inhibition of arenaviruses, responsible for Lassa fever and related human diseases by DDD85646. Altogether, these results provide important new insight into basic steps of the picornavirus life cycle and will also serve as a foundation for the development of novel broad-spectrum antiviral agents.