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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 role of a highly conserved eubacterial ribosomal protein in translation quality control
Title
The role of a highly conserved eubacterial ribosomal protein in translation quality control.
Creator
Naganathan, Anusha, Moore, Sean, Cole, Alexander, Teter, Kenneth, Roy, Herve, Koculi, Eda, University of Central Florida
Abstract / Description
The process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of...
Show moreThe process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of decoding, making it difficult to envision how it might affect decoding and reading frame maintenance. Twenty years after the identification of L9's putative function, there is no mechanism for how a remotely located L9 improves translation fidelity. This mystery makes our picture of translation incomplete. Despite the high conservation of L9 in eubacteria, E.coli lacking L9 does not exhibit any obvious growth defects. Thus, the evolutionary advantage conferred by L9 in bacteria is masked under laboratory conditions. In order to uncover unique L9-dependent conditions, a library of E. coli mutants was screened to isolate those that rely on L9 for fitness. Interestingly, factors found to be synergistic with L9 had no known role in fidelity. Six independent mutants were isolated, each exhibiting a severe growth defect that is partially suppressed in the presence of L9. One class of L9-dependent mutations was present in an essential ribosome biogenesis factor, Der. Der's established function is in the maturation of the large ribosomal subunit. The identified mutations severely impaired the GTPase activity of Der. Interestingly, L9 did not directly compensate for the defective GTPase activity of mutant Der. The second class of L9-dependent mutations was present in EpmA and EpmB, factors required to post-translationally modify elongation factor, EF-P. EF-P's established function is in the translation of poly-proline containing proteins. EF-P deficient cells were nearly inviable in the absence of L9; however, L9 did not directly influence poly-proline translation. Therefore, in each case, L9 improved cell health without altering the activity of either Der or EF-P. Remarkably, the der mutants required only the N domain of L9, whereas the absence of active EF-P required full-length, wild-type L9 for growth complementation. Thus, each mutant class needed a different aspect of L9's unique architecture. In cells lacking either active EF-P or Der, there was a severe deficiency of 70S ribosomes and the indication of small subunit maturation defects, both of which worsened upon L9 depletion. These results strongly suggest that L9 plays a role in improving ribosome quality and abundance under certain conditions.Overall, the genetic screen lead to the discovery that bacteria need L9 when either of two important translation factors (Der or EF-P) is inactivated. This work has characterized the physiological requirement for L9 in each case and offers a new insight into L9's assigned role in translation fidelity.
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Date Issued
2015
Identifier
CFE0005674, ucf:50169
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005674

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