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Characterization of Scaffolding Proteins Altered in the Ability to Perform a Critical Conformational SwitchCherwa, Jr., James Edward January 2009 (has links)
Throughout recent history scientists have struggled to elucidate the biochemical and biophysical mechanisms that guide the assembly of macromolecular structures. The early models of "sub-assembly" or "self assembly" attempted to explain how individual components could interact in a precisely regulated manner to form higher-ordered complex biological structures. Subsequent studies, using viral systems as assembly models, demonstrated how protein-protein and protein-nucleic acid interactions assist in lowering the thermodynamic barriers that typically disfavor assembly.Due to their simplicity, viruses provide an ideal system to investigate the biophysical mechanisms that drive the assembly of complex biological structures. Proper virion assembly requires numerous macromolecular interactions that proceed along an ordered morphogenetic pathway. While structural proteins are incorporated into the final product, morphogenesis is equally dependent upon scaffolding proteins, which are not included in the mature virion. Since the identification of scaffolding proteins in the bacteriophage P22, homologues have been discovered in many systems. Scaffolding proteins play multiple roles during morphogenesis by inducing protein conformational switches and lowering the thermodynamic barriers to promote virion assembly, while ensuring the efficiency and fidelity of the final product.
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Structure-Function Relationships in Microviridae External Scaffolding ProteinsUchiyama, Asako January 2007 (has links)
Microviruses (canonical members: øX174, G4, and alpha3) are T=1 icosahedral virions with a two scaffolding protein-mediated assembly pathway. The external scaffolding protein D mainly mediates the assembly of coat protein pentamers into procapsids. The results of previous genetic studies suggest that helix 1 of D protein may act as a substrate specificity domain, mediating the initial coat-scaffolding protein recognition in a species-specific manner. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were initially constructed in a plasmid, which over-expresses the protein, between the closely related phages G4 and øX174. The results of biochemical and genetic analyses identify coat-scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.However, protein concentrations higher than those found in typical infections could drive reactions that may not occur under physiological conditions. In order to elucidate a more detailed mechanistic model, the same chimeric external scaffolding gene was placed directly in the øX174 genome, and the chimeric virus was characterized. The results of the genetic and biochemical analyses indicate that helix 1 most likely mediates the nucleation reaction for the formation of the first assembly intermediate containing the external scaffolding protein. Mutants that can more efficiently use the chimeric scaffolding protein were isolated. These second-site mutations appear to act on a kinetic level, shortening the lag phase before virion production.Finally, by using improved protocols, two novel early assembly intermediates, the 9S* and 12S* particles, have been isolated and characterized. The 9S* particle consists of a coat protein pentamer associated with the internal scaffolding protein. The 12S* intermediate is a complex of a 9S* particle with the major spike protein, and the DNA pilot protein. The existence of internal scaffolding and DNA pilot proteins that were absent in previously characterized intermediates suggest that 9S* and 12S* particles are biologically active intermediates. Moreover, preliminary in vitro assembly experiments performed with the 12S* particle and exogenous external scaffolding protein yield empty capsids-like particle, strongly suggesting that these are the physiologically relevant intermediates.
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The mechanisms of foamy virus capsid assembly /Eastman, Scott Walton. January 2002 (has links)
Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 104-125).
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Protein adaptability involved in self-assembled icosahedral capsids /Nilsson, Josefina, January 2006 (has links)
Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.
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Characterization of a minimal avian leukosis-sarcoma virus packaging signal /Banks, Jennifer Dawn. January 1999 (has links)
Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 89-108).
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Modeling and simulations of single stranded rna virusesBoz, Mustafa Burak 21 June 2012 (has links)
The presented work is the application of recent methodologies on modeling and
simulation of single stranded RNA viruses. We first present the methods of modeling
RNA molecules using the coarse-grained modeling package, YUP. Coarse-grained
models simplify complex structures such as viruses and let us study general behavior of
the complex biological systems that otherwise cannot be studied with all-atom details.
Second, we modeled the first all-atom T=3, icosahedral, single stranded RNA
virus, Pariacoto virus (PaV). The x-ray structure of PaV shows only 35% of the total
RNA genome and 88% of the capsid. We modeled both missing portions of RNA and
protein. The final model of the PaV demonstrated that the positively charged protein N-
terminus was located deep inside the RNA. We propose that the positively charged N-
terminal tails make contact with the RNA genome and neutralize the negative charges in
RNA and subsequently collapse the RNA/protein complex into an icosahedral virus.
Third, we simulated T=1 empty capsids using a coarse-grained model of three
capsid proteins as a wedge-shaped triangular capsid unit. We varied the edge angle and
the potentials of the capsid units to perform empty capsid assembly simulations. The final
model and the potential are further improved for the whole virus assembly simulations.
Finally, we performed stability and assembly simulations of the whole virus using
coarse-grained models. We tested various strengths of RNA-protein tail and capsid
protein-capsid protein attractions in our stability simulations and narrowed our search for
optimal potentials for assembly. The assembly simulations were carried out with two
different protocols: co-transcriptional and post-transcriptional. The co-transcriptional
assembly protocol mimics the assembly occurring during the replication of the new RNA.
Proteins bind the partly transcribed RNA in this protocol. The post-transcriptional
assembly protocol assumes that the RNA is completely transcribed in the absence of
proteins. Proteins later bind to the fully transcribed RNA. We found that both protocols
can assemble viruses, when the RNA structure is compact enough to yield a successful
virus particle. The post-transcriptional protocol depends more on the compactness of the
RNA structure compared to the co-transcriptional assembly protocol. Viruses can exploit
both assembly protocols based on the location of RNA replication and the compactness
of the final structure of the RNA.
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Retroviral recombination during reverse transcription an analysis of the mechanism, frequency, and effect of the viral packaging signal [psi] /Anderson, Jeffrey A. January 2001 (has links)
Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains viii, 174 p. : ill. Vita. Includes abstract. Includes bibliographical references.
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Evolutionary Recovery and the Thermodynamic Aftermath of Horizontal Gene Transfer in MicrovirusesDoore, Sarah Marie January 2015 (has links)
Experimental evolution has been used to investigate both general and specific evolutionary processes. More recently, it has also been used to resolve protein-protein interactions. Viruses assemble through a series of protein-protein interactions which must remain more favorable than any competing off-pathway reaction. By constructing chimeric viruses with genes or segments of genes from another species, foreign elements are introduced into this system of assembly. Characterization of the resulting chimeras provides information about which proteins interact, the protein-protein interacting interface, the role of particular domains, and the importance of specific residues. Chimeric viruses often exhibit a reduction in fitness, as the foreign element is unable to interact as efficiently in the system as the native element. Through experimental evolution, mutations accumulate that affect interacting partners in the system, leading to a more optimal assembly pathway. The microviruses are well-characterized single-stranded (ss) DNA bacteriophages. They are divided into three clades, represented by φX174, G4, and α3. Incidences of horizontal gene transfer between microvirus clades are unusually rare and may be due to a complex assembly pathway with multiple stages: a foreign element has the potential to disrupt a multitude of morphogenetic steps. In this study, we exchanged major spike genes between the two microvirus species φX174 and G4, then monitored the evolutionary recovery. Results can be interpreted within this thermodynamic paradigm. Although the G4-φXG chimera could only form plaques at low temperature and exhibited reduced fitness, its evolutionary recovery was relatively straightforward. The other chimera, φX-G4G, could only form plaques when complemented with two wild-type φX174 genes. Isolating a complementation-independent chimera required the passaging of mutants through a series of different environments. The first selection yielded mutations of the largest effects. First, the truncation of a protein involved in DNA synthesis was recovered, resulting in a global decrease in gene expression. Next, a recombination event at the 3' end of the foreign gene resulted in a modification of the protein’s C-terminus. These mutations were subjected to further analysis to determine why they were so critical at this early stage of experimental evolution. Subsequent passages of the φX-G4G chimera eventually yielded viable strains, with additional mutations affecting stages of late assembly. Overall, results indicate how gene exchange can drastically affect flux through the pathway. When the system is initially perturbed, the process of experimental evolution allows the pathway to return to a normalized state. The mutations isolated during this recovery stage indicates how the flux was initially altered, and how it can be restored.
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The requirement of the DEAD-box protein DDX24 for the packaging of human immunodeficiency virus type 1 RNA /Ma, Jing, 1978- January 2008 (has links)
Human immunodeficiency virus (HIV) is the causing agent of the acquired immune deficiency syndrome (AIDS). Like all retroviruses, HIV carries two copies of viral genomic RNA in each virion. HIV genome encodes three structural genes, including gag, pol and env, as well as two regulatory genes (rev and tat) and four accessory genes (vif, vpr, vpu and nef). It is noted that none of these nine viral proteins bears the helicase activity. Helicases are able to unwind RNA duplex and remodel the structure of RNA-protein (RNP) complexes using energy derived from hydrolysis of nucleotide triphosphates (NTPs). They are involved in every step of cellular RNA metabolisms. It is conceivable that HIV needs to exploit cellular RNA helicases to promote the replication of its RNA at various steps such as transcription, folding and transport. / In this study, we found that a DEAD-box protein named DDX24 associates with HIV-1 Gag in an RNA-dependent manner but is not found within virus particles. Knockdown of DDX24 inhibits the packaging of HIV-1 RNA and thus diminishes viral infectivity. The decreased viral RNA packaging as a result of DDX24-knockdown is observed only in the context of the Rev/RRE (Rev response element)-dependent but not the CTE (constitutive transport element)-mediated nuclear export of viral RNA, which is explained by the specific interaction of DDX24 with the Rev protein. We propose that DDX24 acts at the early phase of HIV-1 RNA metabolism prior to nuclear export and the consequence of this action extends to the viral RNA packaging stage during virus assembly.
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Unraveling the Role of Cellular Factors in Viral Capsid FormationSmith, Gregory Robert 01 March 2015 (has links)
Understanding the mechanisms of virus capsid assembly has been an important research objective over the past few decades. Determining critical points along the pathways by which virus capsids form could prove extremely beneficial in producing more stable DNA vectors or pinpointing targets for antiviral therapy. The inability of current experimental technology to address this objective has resulted in a need for alternative approaches. Theoretical and computational studies offer an unprecedented opportunity for detailed examination of capsid assembly. The Schwartz Lab has previously developed a discrete event stochastic simulator to model virus assembly based upon local rules detailing the geometry and interaction kinetics of individual capsid subunits. Applying numerical optimization methods to learn kinetic rate parameters that fit simulation output to in vitro static light scattering data has been a successful avenue to understand the details of virus assembly systems; however, information describing in vitro assembly processes does not necessarily translate to real virus assembly pathways in vivo. There are a number of important distinctions between experimental and realistic assembly environments that must be addressed to produce an accurate model. This thesis will describe work expanding upon previous parameter estimation algorithms for more complex data over three model icosahedral virus systems: human papillomavirus (HPV), hepatitis B virus (HBV) and cowpea chlorotic mottle virus (CCMV). Then it will consider two important modifications to assembly environment to more accurately reflect in vivo conditions: macromolecular crowding and the presence of nucleic acid about which viruses may assemble. The results of this work led to a number of surprising revelations about the variability in potential assembly rates and mechanisms discovered and insight into how assembly mechanisms are affected by changes in concentration, fluctuations in kinetic rates and adjustments to the assembly environment.
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