Browsing by Subject "cryo-EM"
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(2024)Coxsackievirus A9 (CVA9) is an enterovirus in the family Picornaviridae. While most infections are asymptomatic, it can cause diseases ranging from mild to serious, especially in neonates where it is known to cause aseptic meningitis and sepsis. There are currently no available vaccines or specific antiviral therapies against coxsackievirus infections. CVA9 has a 30 nm icosahedrally-symmetric capsid comprised of four viral proteins, VP1-4. These four proteins associate together to form a single protomer which is repeated 60 times to make the complete capsid. VP1 forms the five-fold vertex, at the base of which is located a hydrophobic pocket. This pocket is typically occupied by the pocket factor, palmitate. Upon internalization in an endosome, the pocket factor disassociates from the pocket, leading to pocket collapse and subsequently capsid expansion and loss of VP4 and the viral genome. Antiviral therapeutics against CVA9 have focused on stabilizing the hydrophobic pocket to ultimately prevent genome release, but there are currently no approved treatments. Ongoing research has expanded the search to the N-phenyl benzamides class of compounds, which have demonstrated antiviral activity against CVA9. An in-situ docking analysis proposed the hydrophobic pocket as one of several potential binding sites. Here, I used cryogenic electron microscopy and single particle processing to determine the binding site of one such compound, CL301, validating one proposed binding site and the antiviral mechanism of action. A dataset of 24,208 particles was reconstructed resulting in a 2.6 Å resolution electron density map. Atomic modelling showed that an averaged density contributed by both CL301 and palmitate occupied the VP1 hydrophobic pocket. There is not sufficient space for both molecules to occupy the pocket at once, so there is a mixed population, which could be individual virions having all 60 pockets occupied with one or the other molecule, or only a fraction of pockets per virion being occupied. As there is little conformational difference between the two, the statistical analysis used to remove heterogeneity in the dataset was not sensitive enough. In conclusion, CL301 is effective through capsid stabilization preventing expansion and release of the viral genome. Future work should concentrate on improving the affinity of this class of compounds.
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(2019)OBJECTIVES and RESEARCH QUESTION. Human parechovirus 3 (HPeV3) is a (+)ssRNA icosahaedrally symmetric virus which causes meningoencephalitis and sepsis in children and neonates. As it causes the most severe symptoms among parechoviruses it is attracting more attention (4). Currently there are no approved broad treatment strategies against parechoviruses, however recent research by Rhoden et al., 2017, reported the antiviral activity of posaconazole (PSZ) against HPeV3 in cell culture. Posaconazole is an antifungal drug approved for use against Candida and Aspergillus infections. It targets lanosterol-14alpha-demethylase and prevents the production of ergosterol, a lipid vital for fungal membranes not present in mammalian cells (24). In mammalian cells PSZ accumulates at the endoplasmic reticulum (ER) and binds to the oxysterol-binding protein (OSBP) and Niemann-pick type C1 (NPC1) (59, 28, 30). The drug may affect cellular components and thusly block parechoviral infection or could bind to the viral capsid. METHODS. To test viral capsid-binding hypothesis PSZ activity was tested in a range of concentrations against two HPeV3 isolates and HPeV1 Harris in Vero and HT29 cell lines. HPeV3 isolate 152037 was purified on a CsCl step gradient and imaged by cryo electron microscopy (cryo-EM). Single particle analysis was done in Scipion (40) and acquired density maps visualized in UCSF Chimera (49). Atomic model of a different isolate of HPeV3 (PDB ID: 6GV4, 16) was changed at 6 sites and fitted to density maps from this work in Coot (52). Maps were subtracted in search of density that would represent PSZ. RESULTS. PSZ was effective against both HPeV3 isolates at 1 μM in Vero cells when added to the virus prior to infection, however not in HT29 cells. At higher concentrations (>10 μM) PSZ formed crystals which limited the concentration that can be used for cryo-EM. In order to test the hypothesis of PSZ being a capsid binder 3 datasets were collected, HPeV3 control, HPeV3+DMSO and HPeV3+PSZ (4 μM) with final resolutions after single particle analysis of 3.3 Å, 3.9 Å and 3.4 Å respectively. Subtraction of maps yielded no difference that would represent PSZ. DISCUSSION and CONCLUSION. PSZ does not appear to be a capsid binder although it appears to work early in the infection. Absence of PSZ density in HPeV3+PSZ density map could be due to low saturation and images containing PSZ were filtered out in image processing. Another possibility is low affinity of PSZ for the capsid. As PSZ binds various membranes it is possible that it blocks HPeV3 infection by targeting cell components. Additional experiments could be performed in the future in order to provide insight into which stages of infection PSZ affects.
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