Biophysical Society Thematic Meeting

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

University of Jyväskylä, Jyväskylä, Finland,

University of Jyväskylä, Jyväskylä,,

Finland,

University of Jyväskylä, Jyväskylä, Finland.

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.