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Self-assembly of Rous Sarcoma Virus capsid protein, probed by Solid-state NMR and TEM
Title
Self-assembly of Rous Sarcoma Virus capsid protein, probed by Solid-state NMR and TEM.
Creator
Jeon, Jaekyun, Chen, Bo, Tatulian, Suren, Schulte, Alfons, Cole, Alexander, University of Central Florida
Abstract / Description
The retroviral capsid protein (CA) is derived from the cleavage of Gag polyprotein during the maturation process, and self-assembles into a polymorphic fullerene-like shell encasing the viral genome materials. The orthoretroviral CAs, for instance, Human Immunodeficiency Virus (HIV) and Rous Sarcoma Virus (RSV) CA, has little similarity in their sequence composition but a common 3D structure. They form distinct capsid assembly in vivo and a range of similar assemblies in vitro. Due to the...
Show moreThe retroviral capsid protein (CA) is derived from the cleavage of Gag polyprotein during the maturation process, and self-assembles into a polymorphic fullerene-like shell encasing the viral genome materials. The orthoretroviral CAs, for instance, Human Immunodeficiency Virus (HIV) and Rous Sarcoma Virus (RSV) CA, has little similarity in their sequence composition but a common 3D structure. They form distinct capsid assembly in vivo and a range of similar assemblies in vitro. Due to the substantial polymorphism, such assemblies are not amenable for conventional structural biology techniques such as X-ray diffraction crystallography and cryo-electron microscopy (cryo-EM). Solid-state NMR spectroscopy is the optimal platform to study these CA assemblies to attain site-specific structural and dynamic information. However, it is challenging to make signal assignments for such non-crystalline and large biomolecules as retroviral CA assemblies. In this study, we were to elucidate the assembly mechanism of retroviral capsids by applying the state-of-art solid-state NMR techniques on the RSV CA assembly system and establishing an atomistic resolution structural model. The RSV CA is the second most studied protein among the retroviral family after HIV that causes AIDS (acquired immune deficiency syndrome), but there is no atomistic model for RSV CA assemblies available.In this study, we showed that highly uniform tubular RSV CA assembly can be prepared. Screened by TEM, our tubular assembly showed sharp 6-fold symmetry under diffraction, illustrating the quasi-crystalline character. Subsequently we acquired a series of solid-state NMRivspectra for tubular RSV CA assembly, and completed chemical shift signal assignments with samples of various isotope labeling. Then, combining cryo-EM electron density map of tubular assembly of RSV CA with the secondary structures derived from solid-state NMR, we established an atomistic resolution structure model. In this model, we identified the residue-specific assembly interfaces. Interestingly, our model revealed the structural re-arrangements upon the assembly and suggested that the tubular assembly of RSV CA may take a different assembly pathway from that of HIV capsid.
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Date Issued
2016
Identifier
CFE0006332, ucf:51572
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006332
The cytopathic activity of cholera toxin requires a threshold quantity of cytosolic toxin.
Title
The cytopathic activity of cholera toxin requires a threshold quantity of cytosolic toxin.
Creator
Bader, Carly, Teter, Kenneth, Zervos, Antonis, Jewett, Travis, Tatulian, Suren, University of Central Florida
Abstract / Description
Cholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much...
Show moreCholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much of the toxin is transported to the lysosomes for degradation, but a secondary pool of toxin is diverted to the Golgi apparatus and then to the ER. Here the A1 subunit detaches from the rest of the toxin and enters the cytosol. The disordered conformation of free CTA1 facilitates toxin export to the cytosol by activating a quality control mechanism known as ER-associated degradation. The return to a folded structure in the cytosol allows CTA1 to attain an active conformation for modification of its Gs? target through ADP-ribosylation. This modification locks the protein in an active state which stimulates adenylate cyclase and leads to elevated levels of cAMP. A chloride channel located in the apical enterocyte membrane opens in response to signaling events induced by these elevated cAMP levels. The osmotic movement of water into the intestinal lumen that results from the chloride efflux produces the characteristic profuse watery diarrhea that is seen in intoxicated individuals.The current model of intoxication proposes only one molecule of cytosolic toxin is required to affect host cells, making therapeutic treatment nearly impossible. However, based on emerging evidence, we hypothesize a threshold quantity of toxin must be present within the cytosol of the target cell in order to elicit a cytopathic effect. Using the method of surface plasmon resonance along with toxicity assays, I have, for the first time, directly measured the efficiency of toxin delivery to the cytosol and correlated the levels of cytosolic toxin to toxin activity. I have shown CTA1 delivery from the cell surface to the cytosol is an inefficient process with only 2.3 % of the surface bound CTA1 appearing in the cytosol after 2 hours of intoxication. I have also determined and a cytosolic quantity of more than approximately .05ng of cytosolic CTA1 must be reached in order to elicit a cytopathic effect. Furthermore, CTA1 must be continually delivered from the cell surface to the cytosol in order to overcome the constant proteasome-mediated clearance of cytosolic toxin. When toxin delivery to the cytosol was blocked, this allowed the host cell to de-activate Gs?, lower cAMP levels, and recover from intoxication. Our work thus indicates it is possible to treat cholera even after the onset of disease. These findings challenge the idea of irreversible cellular toxicity and open the possibility of post-intoxication treatment options.
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Date Issued
2013
Identifier
CFE0004810, ucf:49759
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004810

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