IDENTIFICATION and CHARACTERIZATION of the HRV-C RECEPTOR(S)

Using modern RT/PCR diagnostic tools, a number of so far unknown human rhinovirus (HRV) types have been identified in clinical samples. Sequences of their RNA genomes revealed that some of these viruses were only distantly related to the established HRV species A and B. The novel isolates were classified as HRV-C. The new serotypes had escaped detection because of their inability to replicate in tissue culture. They cause influenza-like symptoms, febrile obstructive bronchitis, and asthma and appear to be more aggressive than the "classical" HRVs. Recently it was shown that at least HRV-C102 could be grown in nasal tissue explants. When bypassing the requirement for the receptor via transfection with viral genomic RNA transcribed in vitro from a synthetic cDNA clone, it also replicated in HeLa cells. This suggests that HRV-Cs do not infect via the so far known and ubiquitously expressed receptors, intercellular adhesion molecule 1 and members of the low-density lipoprotein receptor family. Rather, they most probably recognize a cell surface structure only present in a subset of cells found in the nasal mucosa, the inner ear, and the lower respiratory tract. This view is supported by sequence similarity-based 3D-molecular modelling of the viral capsid; HRV-Cs lack the signature sequences and loops recognized by the known HRV receptors. Therefore, the hunt is open for this enigmatic receptor(s). We thus propose to identify and characterize the HRV-C receptor(s) and eventually solve the 3D-structure of a virus- receptor complex to understand the molecular mechanism of recognition.

Rhinoviruses (RVs) are the leading cause of upper respiratory tract infections but are also exacerbating lower airway diseases causing substantial morbidity in the patients. They are classified as species A, B, and C consisting of over 150 different strains. Several epidemiologic studies concluded that RV-A and RV-C are more virulent than RV-B and more frequently linked with childhood asthma. In order to infect a host cell, rhinoviruses must attach to a receptor, be transported into the cells interior by endocytosis and release (uncoat) their single-stranded RNA genome from the protective capsid into the cytosol for reproduction. To get insight into these early events for the difficult-to-grow RV-Cs, we wanted to identify the elusive RV-C receptor(s). Initially, we put much effort into producing RV-C15 at quantities sufficient for receptor detection aiding its isolation. However, despite using a number of complementary approaches, this goal could not be accomplished due to the extremely low yields of C-species rhinoviruses. In April 2015, by using a bioinformatics approach, a RV-C receptor was reported by an American research team. We thus shifted our focus on comparing two rhinovirus types of the evolutionary separate species A and B but binding the same receptor (ICAM-1) with respect to their infectious pathway. The unexepected result was that they are shuttled along different endocytic routes towards distinct endosomal compartments for productive uncoating. This finding is of relevance for the reportedly different pathogenic potential of RV-A and -B. Furthermore, we developed a novel assay for quantification of experimentally triggered rhinovirus RNA uncoating, based on measuring the fluorescence emitted by a special probe (a molecular beacon) paired with CHIP electrophoresis. This shall, in the future, aid in determining details on the still elusive mechanism of viral RNA release. Finally, we have investigated the role of a fatty acid modification normally present in VP4, the smallest capsid protein of picornaviruses. We found that inhibition of the enzymes responsible for attachment of this myristic acid chain with a novel inhibitor led to a drastic drop in infectivity of rhino-, coxsackie- and other picornaviruses tested. As underlying cause we identified a less efficient virus assembly, and, even more importantly, a severe uncoating defect of the still produced viral particles. Altogether these data provide a basis for the development of an entirely novel antiviral therapy across a wide spectrum of picornaviruses including A-, B-, and C-type rhinoviruses.