Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

26-POS Board 26 Site-Specific Probes for Enteroviruses for Detailed Imaging in Light and Electron Microscopy Varpu Marjomäki 1,2 , Mari Martikainen 1,2 , Kirsi Salorinne 3,2 , Tanja Lahtinen 3,2 , Sami Malola 4,3 , Jaakko Koivisto 4,3 , Mika Pettersson 3,2 , Perttu Permi 1,3,2 , Hannu Häkkinen 3,4,2 . 1 University of Jyväskylä, Jyväskylä, Finland, 3 University of Jyväskylä, Jyväskylä,, Finland, 4 University of Jyväskylä, Jyväskylä, Finland. 2 University of Jyväskylä, Jyväskylä, Finland, We are focused on understanding the mechanisms and cellular factors behind enterovirus infection. Our previous results have suggested that enteroviruses follow a macropinocytic entry to host cells. In order to follow virus uncoating and other details in the infectious pathway, covalently conjugated probes for virus capsid or genome are needed. On the other hand, visualization of virus opening in cellular structures profits from dynamics probes that are released upon virus uncoating. We have developed a protocol for site-specific covalent conjugation of atomically monodisperse gold clusters with 1.5 nm metal core to viral surfaces (PNAS 2014). Water-soluble Au102(para- mercaptobenzoic acid)44 clusters, functionalized by maleimide linkers to target cysteines of viral capsid were conjugated to echovirus 1 and coxsackievirus B3 without compromising the infectivity. Quantitative analysis showed ordering of the bound gold clusters on the viral surface and a clear correlation between the clusters and the cysteine sites close to the viral surface. Another site-specific probe was developed for the hydrophobic pocket of enteroviruses. A derivative of Pleconaril was conjugated to fluorescent labels and Au102. The probe mildly stabilized the virus particle and caused a delay in the virus uncoating, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding was proven by STD and tr-NOESY NMR methods and TEM. The virus-fluorescent probe accumulated in endosomes but was seen to leak from the virus-positive endosomes from the capsid proteins suggesting that, like the physiological hydrophobic content, the probe may be released upon virus uncoating. Our results collectively thus show that the gold and fluorescently labeled probes may be used to track and visualize the studied enteroviruses during early phases of infection opening new avenues to follow virus uncoating in cells.

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