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1) Essential viral enzymes
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+RNA viruses have several key enzymes that are essential for their replication and thus inhibitors of these enzymes are potential drugs to combat viral diseases. Most of current antiviral drugs (some, such as tenofovir, developed at IOCB) target the viral polymerase, protease or integrase. We focus on structural analysis of key viral enzymes of newly emerged +RNA viruses to aid structure-based drug design. |
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The SARS-CoV-2 methyltransferase (Krafcikova et al. 2020). RNA methylation is important for recognition of RNA by the ribosome (mRNA translation) and for RNA stability (non-methylated RNA, such as viral RNA, is quickly degraded in our cells). SARS-CoV-2 possess an RNA methyltrasferase that is composed of two proteins - nsp10 and nsp16. We have crystalized the protein complex and devised a model describing how the viral RNA is recognized and methylated. |
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2) Viral hijacking of human host factors
Viruses are way too small organism (not even considered living by many) to encode all the proteins they require to reproduce in our cells. Therefore, they evolved mechanisms how to hijack our proteins for their selfish benefits. We use protein crystallography to understand molecular principles of viral reproduction in our cells. Our research not only reveals the mysteries from viral life cycle but also provides new targets for drug design. |
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Viral non-structural 3A proteins act as a molecular lasso to hijack the Golgi resident ACBD3 protein (Klima et al. 2017). Molecular dynamics simulation-based model of the ACBD3 GOLD domain in complex with viral 3A protein based on our recent crystal structure (Klima et al. 2017) on the lipid bilayer. The viral 3A protein is colored in aquamarine and shown in cartoon representation except for the myristoylated G1 residue which is shown in spheres representation and colored according to elements - carbons are colored in aquamarine, oxygens in red, nitrogen in blue. The ACBD3 GOLD domain is shown in cartoon representation with semi-transparent surface and colored in gold except for the membrane-binding site composed of R399, L514, W515, and R516, which is colored in green. |
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3) Inhibitors of essential host factors as potential antivirals
An essential host factor is a protein that is hijacked by a virus to help its procreation instead of fulfilling its normal function in our physiology. For many +RNA viruses, such as HepatitisC virus or SARS, the essential host factor is an enzyme called PI4KB (phosphatidylinositol 4-kinase B). The goal of this research is to develop highly specific inhibitors of PI4KB that could be used as antiviral agents. Recently, we have solved the crystal structure of PI4KB with an inhibitor that was synthesized by our collaborators - the Nencka group. The structure explains how the inhibitor works and it helped us to synthesize next generation sub-nanomolar inhibitors (Mejdrova et al. 2017). |
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| PI4K III beta crystallized with an inhibitor synthesized at IOCB by the group of Dr. Nencka | ||
4) Reconstitution of the key events of viral lifecycle
ACBD3 hijacked by viral 3A efficiently recruits and activates PI4KB To reproduce in our cells, picornaviruses need membranes that are rich in the PI4P (phosphatidylinositol 4-phosphate) lipid and serve as viral replication organelles. The lipid is produced by the PI4KB enzyme. We use a biomimetic GUV (giant unilamellar vesicle) system to reveal how viruses build their replication organelles.
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| GUVs decorated with the viral3A-CFP protein were incubated with 250 nM PI4KB labeled by Alexa488 and 100 nM SidC-mCherry (PI4P biosensor). Upper panel - without ACBD3 the PI4KB enzyme is not membrane localized. Lower panel - viral 3A protein recruits PI4KB to the membrane via ACBD3 protein and stimulates its enzymatic activity. | ||
