Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The energetic model for DNA delivery is based on two opposing forces, an inward pressure-based force, created by the highly condensed packaged genome, and a counter frictional force, mediated by the DNA conduit's amide and guanidinium groups. Thus far, we have altered the balance of these forces by increasing and decreasing the frictional force. To the best of our knowledge, our study represents the first such study that explore the use of potential energy and how it is mediated. Moreover the hypothesis that single-stranded viral genomes may be evolutionarily honed for packaging and delivery is novel. It is likely that the target audience is broad, including structural biologists, biochemists, geneticist, and biophysicists. The genetic studies incorporate several high school teacher-student teams, thus adding a strong educational outreach component to the work. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has directly provided scientific training to two young scientists: Dr. Aaron Roznowski, who received his degree in the PI's laboratory in 2019, and an undergraduate student, Sam Love, who started as a high school student but is now a freshman at the University of Arizona. Computational studies also indicate that actual genome sequence, its chemical makeup, may be evolutionary honed for packaging and transport (8, 9). We are investigating this phenomenon, which is providing scientific training to a graduate, Elizabeth Ogunbunmi, and two high school students at the Berkshire School. 8. Chechetkin VR, Lobzin VV. 2017. Large-scale chromosome folding versus genomic DNA sequences: A discrete double Fourier transform technique. J Theor Biol 426:162-179. 9. Chechetkin VR, Lobzin VV. 2018. Genome packaging within icosahedral capsids and large-scale segmentation in viral genomic sequences. J Biomol Struct Dyn doi:10.1080/07391102.2018.1479660:1-50. How have the results been disseminated to communities of interest?The results have been published in the Journal "Viruses"(6). 6. Roznowski AP, Fisher JM, Fane BA. 2020. Mutagenic Analysis of a DNA Translocating Tube's Interior Surface. Viruses 12:10.3390/v12060670. What do you plan to do during the next reporting period to accomplish the goals?The results of computational analyses indicate the single-stranded DNA genomes may be evolutionarily honed for packaging and delivery (8,9). Briefly, microvirus genomes appear to be divided into 60 segments, each containing a consensus sequence, which facilitate the genome's arrangement within the 60 icosahedrally ordered capsid proteins. We are currently de-optimizing the genome to study the effects of mutated consensus sequences on genome packaging and transport. 8. Chechetkin VR, Lobzin VV. 2017. Large-scale chromosome folding versus genomic DNA sequences: A discrete double Fourier transform technique. J Theor Biol 426:162-179. 9. Chechetkin VR, Lobzin VV. 2018. Genome packaging within icosahedral capsids and large-scale segmentation in viral genomic sequences. J Biomol Struct Dyn doi:10.1080/07391102.2018.1479660:1-50.
Impacts
What was accomplished under these goals?
Elucidating the energy requirements of DNA transport: In microviruses, the single-stranded DNA genome enters the cell through a narrow tube called the H-tube. The H-tube is not a permanent feature of the mature viruses. It emerges from the particles interior when on the surface of the host cell (1-3). The entire inner passage of the H-tube is lined with amide and guanidinium side chains, which are known to interact with purines. Only one other high-resolution single-stranded nucleic acid conduit structure have been determined (4). This conduit, which emerges from the caliciviruses, RNA animal viruses, is also lined with amide and guanidium groups, indicating that this may be a common feature, perhaps mediating energy use during penetration. Virions are high-energy metastable complexes. Potential energy is stored as internal capsid pressure in the form of a highly compacted, volumetrically constrained genome (5). However, potential energy more efficiently performs work if released in small usable increments. The amide side chains may act like the air gauges that regulate compressed gas driven machines. These gauges prevent energy from overwhelming the system. In this analogy, the compressed gas represents the volumetrically constrained genome and its potential energy, whereas the gas cylinder represents the capsid. The H-tube is the conduit through which the energy is released. If this model is correct, adding additional amide and guanidinium groups, via site directed mutagenesis, may slow the rate of DNA delivery. Indeed, X->Q mutations results in genomes becoming kinetically trapped with the H tube, whereas Q->X mutations result in tubes that appear to collapse during transport (6). Defining the molecular basis of host range: As described in last year's progress report, the results of our genetic and biochemical studies had broader ecological implications. In particular, virions attached and eclipsed to both native and unsusceptible hosts; however, they breached only the native host's cell wall. This suggests that unsusceptible host-phage interactions promote off-pathway reactions that can inactivate viruses without penetration (7). This may be advantageous to potential new hosts; culling the viral population from which an expanded host range mutant could emerge, a novel mechanism in the host-virus arms race, and implies that the virus can be experimentally evolved to avoid unsusceptible hosts. To investigate this further, we devised an experimental evolution protocol that requires viruses to "run the gauntlet." They must survive passage through a layer of unsusceptible cells, which can deactivate them, to reach the permissive cells in which they can replicate. After 20 runs, two mutations emerged in the four experimental populations that did not emerge in the control populations. These two mutations are located adjacent to previously identified host range mutations. They have now been placed within an otherwise wild-type background. This project was being conducted with high-school students from Tucson Magnet High School. This work has obviously been impacted by the COVID-19 pandemic. 1. Sun L, Rossmann MG, Fane BA. 2014. High-resolution structure of a virally encoded DNA-translocating conduit and the mechanism of DNA penetration. J Virol 88:10276-9. 2. Sun L, Young LN, Zhang X, Boudko SP, Fokine A, Zbornik E, Roznowski AP, Molineux IJ, Rossmann MG, Fane BA. 2014. Icosahedral bacteriophage PhiX174 forms a tail for DNA transport during infection. Nature 505:432-5. 3. Sun Y, Roznowski AP, Tokuda JM, Klose T, Mauney A, Pollack L, Fane BA, Rossmann MG. 2017. Structural changes of tailless bacteriophage PhiX174 during penetration of bacterial cell walls. Proc Natl Acad Sci U S A 114:13708-13713. 4. Conley MJ, McElwee M, Azmi L, Gabrielsen M, Byron O, Goodfellow IG, Bhella D. 2019. Calicivirus VP2 forms a portal-like assembly following receptor engagement. Nature doi:10.1038/s41586-018-0852-1. 5. Evilevitch A. 2013. Physical evolution of pressure-driven viral infection. Biophys J 104:2113-4. 6. Roznowski AP, Fisher JM, Fane BA. 2020. Mutagenic Analysis of a DNA Translocating Tube's Interior Surface. Viruses 12:10.3390/v12060670. 7. Roznowski AP, Young RJ, Love SD, Andromita AA, Guzman VA, Wilch MH, Block A, McGill A, Lavelle M, Romanova A, Sekiguchi A, Wang M, Burch AD, Fane BA. 2019. Recessive Host Range Mutants and Unsusceptible Cells That Inactivate Virions without Genome Penetration: Ecological and Technical Implications. J Virol 93.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Roznowski AP, Fisher JM, Fane BA. 2020. Mutagenic Analysis of a DNA Translocating Tube's Interior Surface. Viruses 12:10.3390/v12060670.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:While tailed double-stranded DNA viruses predominate the viruses that infect free-living bacteria in medically relevant microbiomes, single-stranded DNA microviruses are the dominant species infecting obligate intracellular, pathogenic bacteria, such as chlamydia. As most prokaryotic virus research focus on the double-stranded DNA viruses, the molecular basis of single-stranded DNA transport and single-stranded viral tropism has not been thoroughly elucidated. The results of a structural study (1) and a genetic/biochemical study (2) have illustrated the unique mechanisms utilized by the single-stranded DNA viruses. The genetic study, published in 2019, had broad impact, which was described in last year's report. Briefly, unsusceptible host cells can inactivate viruses. The genetic studies incorporated several high school teachers-students teams. 1. Sun Y, Roznowski AP, Tokuda JM, Klose T, Mauney A, Pollack L, Fane BA, Rossmann MG. 2017. Structural changes of tailless bacteriophage PhiX174 during penetration of bacterial cell walls. Proc Natl Acad Sci U S A 114:13708-13713 2. Roznowski AP, Young RJ, Love SD, Andromita AA, Guzman VA, Wilch MH, Block A, McGill A, Lavelle M, Romanova A, Sekiguchi A, Wang M, Burch AD, Fane BA. 2019. Recessive host range mutants and unsusceptible cells that inactivate virions without genome penetration: ecological and technical implications. J Virol doi:10.1128/JVI.01767-18. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has directly provided scientific training to two young scientists: Dr. Aaron Roznowski, who received his degree in the PI's laboratory in 2019, and an undergraduate student, Robert Young, who started in the lab as a participant in the STEP 2STEM program for under-represented groups in science, and Samuel Love, who started as a high school student but is now a freshman at the University of Arizona. In addition, a high school student-teacher team from Tucson Magnet High participated in the research. At the Southern Arizona Science Fair, the team or its members won three awards:a first prize in Cellular and Molecular Biology, The Critical Thinking Award from the Wallace Foundation, and Samuel Love (2019) earned the Top Young Scientist Award (Roche). How have the results been disseminated to communities of interest?The results have been presented at the International Virus Structure and Assembly meeting, and published in the Journal of Virology. What do you plan to do during the next reporting period to accomplish the goals?During the next year, we anticipate finishing the analysis of H-tube mutants and determining the effects of the mutations identified during the experimnetal evolution on host cell attachment, penetration, and the ability to survive incubation with unsusceptible cells.
Impacts
What was accomplished under these goals?
Defining the molecular basis of host range: As described in last year's progress report, the results of our genetic and biochemical studies had broader ecological implications. In particular, virions attached and eclipsed to both native and unsusceptible hosts; however, they breached only the native host's cell wall. This suggests that unsusceptible host-phage interactions promote off-pathway reactions that can inactivate viruses without penetration. This may be advantageous to potential new hosts; culling the viral population from which an expanded host range mutant could emerge, a novel mechanism in the host-virus arms race. This implies that the virus can be experimentally evolved to avoid unsusceptible hosts. To investigate this further, we devised an experimental evolution protocol that requires viruses to "run the gauntlet." They must survive passage through a layer of unsusceptible cells, which can deactivate them, to reach the permissive cells in which they can replicate. After 20 runs, two mutations emerged in the four experimental populations that did not emerge in the control populations. These two mutations are located adjacent to previously identified host range mutations. These mutations will be placed in an otherwise wild-type background and the resulting mutants will be characterized more thoroughly. Elucidating the energy requirements of DNA transport: The entire inner passage of the H-tube is lined with amide and guanidinium side chains, which are known to interact with purines. Although other high-resolution DNA conduit structures have yet to be determined, their sequences contain many glutamine, arginine and asparagine residues. Virions represent a high-energy metastable complex. Potential energy is stored as internal capsid pressure in the form of a highly compacted, volumetrically constrained genome. Potential energy more efficiently performs work if released in small usable increments. The amide side chains may act like the air gauges that regulate compressed gas driven machines. These gauges prevent energy from overwhelming the system. In this analogy, the compressed gas represents the volumetrically constrained genome and its potential energy, whereas the gas cylinder represents the capsid. The H-tube is the conduit through which the energy is released. If this model is correct, adding additional amide and guanidinium groups, via site directed mutagenesis, may slow the rate of DNA delivery. X->Q mutations were made and thoroughly characterized. As described in last year's progress report, genomic DNA becomes kinetically trapped during transport.However, this is a two-part analysis, the effects of amide and guanidinium group removal also need to be analyzed. The H protein easily tolerated X->Q substitutions. The mutations neither affected the ability of the H protein to be incorporated during virus assembly, nor, the ability of protein to oligomerize into a tube, as evinced by robust K+ efflux. However, identifying suitable Q->X proteins were more problematic vis-à-vis their effects on DNA transport. folding and incorporation. However, four suitable sites have now been identified: Q191, Q195, Q241, and Q247.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:While tailed double-stranded DNA viruses predominate the viruses that infect free-living bacteria in medically relevant microbiomes, single-stranded DNA microviruses are the dominant species infecting obligate intracellular, pathogenic bacteria, such as chlamydia. In 2014, the atomic structure of the DNA pilot protein was solved. The structure was paradigm shifting creating a large target audience, including structural biologists and biochemists, representing basic science disciplines, and translational scientists, who may endeavor to develop biotechnology applications. In the last year, we published further structural studies utilizing an in vitro reconstituted the DNA delivery reaction. The results provided insights into viral host range mechanisms, which have been further explored. An "in press" manuscript will be spotlighted by the Journal of Virology in an upcoming volume. Three key findings led the editors to choose our article. 1) Unsusceptible host cells can inactivate viruses without genome penetration. This may be advantageous to potential new hosts; culling the viral population from which an expanded host range mutant could emerge. 2) When identified, altered host range mutations were recessive. Accordingly, isolation required populations generated in low MOI environments. However, in laboratory settings, viral propagation includes high MOI conditions. This has likely impacted similar studies with many other viruses. Lastly, structural and genetic data could be used to predict site-directed mutant phenotypes, which may broaden the classic antireceptor definition to include interfaces between capsid complexes. Thus, it is likely that the target audience will likely continue to be broad. Vis-a-vis education, for the studies described in this upcoming manuscript, we devised ways to incorporate teams of high school teachers and students into the research program. This augments the impact beyond science. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has directly provided scientific training to two young scientists: Mr. Aaron Roznowski, a current graduate student in the PI's laboratory, and an undergraduate student, Robert Young, who started in the lab as a participant in the STEP 2STEM program for under-represented groups in science. In addition, the host range studies incorporated two high school teacher-student teams, a total of 11 participants. The team from Tucson Magnet High School won the grand prize in the local science fair. The members of both teams are on the byline of an "in press" manuscript. How have the results been disseminated to communities of interest?The results have been presented at the FASEB Meeting for Virus Structure and Assembly, published in PNAS and awaiting publication in the Journal of Virology. What do you plan to do during the next reporting period to accomplish the goals?1) Extend the host range studies. Extracellular host range is determined by two distinct reactions: reversible attachment and eclipse, which is an irreversible reaction. Our studies thus far have focused on eclipse. Using molecular modeling and structure, we hypothesize that a 6-carbon sugar binding site found on the viral coat protein may regulate reversible attachment. We will begin these studies with mutational analyses. 2) The energetic model for DNA delivery is based on two opposing forces, an inward pressure-based force, created by the highly condensed packaged genome, and a counter frictional force, mediated by the DNA conduit's amide and guanidinium groups. Thus far, we have altered the balance of these forces by increasing the frictional force. We will explore whether genome deletions and insertions can be used to alter the pressure-based force.
Impacts
What was accomplished under these goals?
Defining the molecular basis of host range: Although microviruses do not possess a visible tail structure, one vertex rearranges after interacting with host lipopolysaccharides. Most examinations of host range, eclipse, and penetration were conducted before this "host-induced" unique vertex was discovered and before DNA sequencing became routine. Consequently, structure-function relationships dictating host range remain undefined. Biochemical and genetic analyses were conducted with two closely related microviruses, alpha3 and ST-1. Despite ~90% amino acid identity, the natural host of alpha3 is E. coli C; whereas ST-1 is a K12-specific phage. Virions attached and eclipsed to both native and unsusceptible hosts; however, they breached only the native host's cell wall. This suggests that unsusceptible host-phage interactions promote off-pathway reactions that can inactivate viruses without penetration. This phenomenon may have broader ecological implications. To determine which structural proteins conferred host range specificity, chimeric virions were generated by individually interchanging the coat, spike, or DNA pilot proteins. Interchanging the coat protein switched host range. However, host range expansion could be conferred by single point mutations in the coat protein. The expansion phenotype was recessive: genetically mutant progeny from co-infected cells did not display the phenotype. Thus, mutant isolation required populations generated in low MOI environments: a phenomenon that may have impacted past host range studies in both prokaryotic and eukaryotic systems. The resulting genetic and structural data were consistent enough that host range expansion could be predicted, broadening the classical definition of antireceptors to include interfaces between protein complexes within the capsid. Elucidating the energy requirements of DNA transport: The entire inner passage of the H-tube is lined with amide and guanidinium side chains, which are known to interact with purines. Although other high-resolution DNA conduit structures have yet to be determined, their sequences contain many glutamine, arginine and asparagine residues. Virions represent a high-energy metastable complex. Potential energy is stored as internal capsid pressure in the form of a highly compacted, volumetrically constrained genome. Potential energy more efficiently performs work if released in small usable increments. The amide side chains may act like the air gauges that regulate compressed gas driven machines. These gauges prevent energy from overwhelming the system. In this analogy, the compressed gas represents the volumetrically constrained genome and its potential energy, whereas the gas cylinder represents the capsid. The H-tube is the conduit through which the energy is released. If this model is correct, adding additional amide and guanidinium groups, via site directed mutagenesis, may slow the rate of DNA delivery. Thus, adding additional amide groups would lead to DNA becoming kinetically trapped during transport. As described in the project's initial submission, additional amide groups were added. All of our many observations were consistent with the above model. However, no one experiment provided direct evidence. During the last year, we established a direct assay. It is possible to follow infecting genomes through the host cell wall. Initially, they are found associated with the outer membrane but then move to the host's inner membrane. If the too many amide groups cause a significant portion of the infecting genomes to stall mid-transit, then a higher proportion of them may remain associated with the outer membrane, which can be detected using qPCR after separating the cell wall into inner and outer membrane fractions. After viral eclipse, infected cells were collected, washed twice to remove unattached and uneclipsed particles, converted to spheroplasts, and sonicated. The resulting cytoplasmic and outer membrane vesicles were pelletized and then separated in percoll gradients. Separation efficiency was determined by analyzing gradient fractions for succinate dehydrogenase activity and Keto-deoxy-d-manno-8-octanoic acid (KDO) content, respective markers of the inner and outer membrane fractions (49). Fractions containing the most KDO had a density of 1.03 g/mL. Those having the highest succinate dehydrogenase activity had a density of 1.01 g/mL. These values are consistent with those typically obtained in membrane separation protocols. The identified fractions were analyzed by qPCR for viral DNA. Wild-type H proteins delivered approximately 10-fold more genomic DNA to the inner membrane than the mutant proteins.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2019
Citation:
Roznowski AP, Young RJ, Love SD, Andromita AA, Guzman VA, Wilch MH, Block A, McGill A, Lavelle M, Romanova A, Sekiguchi A, Wang M, Burch AD, Fane BA. 2019. Recessive host range mutants and unsusceptible cells that inactivate virions without genome penetration: ecological and technical implications. J Virol doi:10.1128/JVI.01767-18.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Sun Y, Roznowski AP, Tokuda JM, Klose T, Mauney A, Pollack L, Fane BA, Rossmann MG. 2017. Structural changes of tailless bacteriophage PhiX174 during penetration of bacterial cell walls. Proc Natl Acad Sci U S A 114:13708-13713.
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