2016 Theses Doctoral

Characterization of Extracellular Zinc-mediated Inhibition of Human HRVs

Bennett, Ashlee Nicole

As the predominant etiological agent of the “common cold,” human rhinoviruses (HRVs) have a substantial economic impact and contribute to severe respiratory complications in immune compromised and asthmatic individuals. While zinc (Zn) ions have been previously shown to have an inhibitory effect upon HRVs, clinical trials using Zn products have produced conflicting results, and the lack of a known mechanism of Zn inhibition has stymied therapeutic development. Previous research on the potential anti-rhinoviral mechanism of Zn compounds focused upon intracellular processes. My research has demonstrated that extracellular exposure of both major and minor group HRVs to Zn chloride (ZnCl) and Zn gluconate (ZnG) is sufficient to profoundly decrease the infectivity of the viral population. The infectivity of other representatives of the picornavirus family is not decreased in the presence of Zn compounds, suggesting that Zn-mediated virus inhibition is HRV specific. Other metal cations similar to Zn have not demonstrated HRV inhibition. Zn-based inhibition of HRVs is independent of pH, is effected within minutes and is dampened at lower temperatures. Furthermore, whereas EDTA can chelate Zn to prevent inhibition of HRVs, it cannot reverse the Zn-based inhibition after it has occurred. In addition, infectious center plaque assay (ICPA) and competition assay data suggest that this mechanism is not related to the virus-cellular receptor interaction, and that Zn-treated viral capsids are still able to interact with receptor binding sites. Moreover, cultivation and analysis of Zn-resistant HRV1A isolates suggests that genomic discrepancies in the VP1 capsid protein play a role in the mechanism of Zn inhibition. 3 distinct point mutations that conferred amino acid substitutions were found in multiple Zn-resistant isolates. Located in the exterior B-C loop and the interior viral-genome neighboring region of VP1, 2 out of the 3 mutations resulted in a dramatic amino-acid polarity change which likely has the chemical consequence of effectively repelling 2+ cations such as Zn. Furthermore, northern blot analysis reveals that Zn-treated HRVs exhibit an increased susceptibility to genomic RNA degradation, a phenomena that may be facilitated by a Zn-mediated cleavage of viral RNA within the viral capsid. Zn complexed to chelating compounds Hinokital (HK), Pyrithione (PT) and Pyrrolidine Dithiocarbamate (PDTC) also demonstrated extracellular HRV inhibition, with the sequence and ratio of exposure, or precipitate formation, modulating the outcome. It is not clear if these compounds augment the antiviral mechanism of Zn alone, or initiate a distinct antiviral mechanism unrelated to that performed by Zn in isolation. Based upon the data presented here, Zn mediated inhibition of HRVs can occur in a cell-independent, extracellular manner to degrade viral RNA and thereby abrogate viral infectivity. This mechanism may provide novel insight into further therapeutic development of these compounds or template the design of future small molecule therapeutics against these and similar viruses.