Progress 10/01/12 to 09/30/17
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. To date, it remains the only viral DNA translocating conduit solved to atomic resolution. The structure was paradigm shifting and published in the journal Nature. The target audience was broad, including structural biologists and biochemists, representing basic science disciplines, and translational scientists, who may endeavor to develop biotechnology applications. Toward this end, we demonstrated that the length of the conduit could be altered without affecting function, a feature that demonstrates the system's nano-technological potential. We have reconstituted the DNA delivery reaction in vitro. The DNA delivery reaction could be followed in real time and has led to further structural studies. Lastly, the structure also suggests an energetic model for DNA delivery; we are currently testing this model. Thus, it is likely that the target audience will continue to be broad. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Over the course of its entire duration, the project has directly provided scientific training to three young scientists: Mr. Aaron Roznowski, a current graduate student: Ms. Lindsey Young, who started as an undergraduate student in the PI's laboratory, worked as a technician and then matriculated in the graduate program at the University of California Berkeley, in the PI's laboratory, and Mr. Brody Blackburn, an undergraduate student, who became a technician in the lab before accepting a position in industry. He is now applying to graduate programs. How have the results been disseminated to communities of interest?Over the course of the entire project, the results have been presented annually at either the International Virus and Phage Assembly meetings or the FASEB Virus Structure and Assembly Meeting. Moreover, the PI has given several invited seminars related to this research at Michigan State University (2013) the University of Missouri, Kansas City (2014), Washington State University (2015), and a plenary talk at the National ASM Meeting (2015). In addition, there have been four published peer review manuscripts: in Nature (2014), Virology (2014), Journal of Virology (2016), and PNAS (2017). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The atomic structure of the H-tube, a virally encoded DNA translocating conduit: The atomic structure of the øX174 DNA pilot protein H was solved in collaboration with Dr. M. G. Rossmann's group (Purdue University). The central domain consists of 10 alpha helices arrayed into a 170-angstrom long coiled-coil structure, or helical barrel. The external diameter is 48 angstroms, whereas the internal diameter ranges from 22 - 24 angstroms. To the best of our knowledge, this is the first description of a decameric coiled coil structure, and to date, the only viral DNA translocating conduit solved to atomic resolution. The length of the barrel is long enough to transverse the periplasmic space. The 22-angstrom internal diameter can easily accommodate two separate un-paired ssDNA strands with intercalated bases. Moreover, there was no evidence of H-barrels in cryo-electron microscopic images of more than five hundred mature virions. Thus, the packaged H proteins most likely form these barrels sometime during attachment and/or penetration, which may represent a novel DNA delivery mechanism. Indeed, the results of further studies, see below support this model. Upon cell contact, H proteins interact and a fully formed tube emerges from the capsid. After DNA delivery, the conduit dissociates within the cell wall. Thus, the newly discovered H-tube is ephemeral. The biological relevance of this paradigm-shifting structure was tested genetically in vivo, biochemically in vitro, and tomographically in situ. Using the atomic structure as a guide, mutations were designed to eliminate inter-helix contacts without affecting the helical fold. Particles sedimenting at 114S, the S value of the virion, were isolated from mutant-infected cell extracts. Compared to the wild-type virions, there were no apparent differences in DNA and protein content. However, the specific infectivity (plaque forming units /OD280) of the mutant particles was approximately three orders of magnitude lower than the wild-type control. The properties of the mutant H proteins were also investigated in vitro. As evinced by similar circular dichroism spectra, the mutant proteins maintained a wild-type secondary structural content. However, by size exclusion chromatography, the mutant proteins migrate as lower order oligomers. In vitro reconstitution of viral DNA delivery: To further elucidate the molecular mechanisms involved in DNA penetration, an DNA delivery system was reconstituted in vitro. The reaction conditions must favor an asymmetric process. For example, only one site of the virus contacts the host cell receptor. Thus, the receptor must be spatially fixed, unable to interact with the virus in more than one place. Lipopolysaccharides (LPS) from sensitive strains serve as microvirus receptors. Two viruses, phiX174 and ST1, with different host specificities were used. Purified LPS was examined by electron microscopy. Under our reaction conditions, it formed lipid bi-layers, which would spatially fix the receptor vis-à-vis the virion. Incubation of viruses with LPS from a susceptible host results in a greater than 90% loss of infectivity, presumably the viral DNA is leaving the capsid. LPS isolated from the unsusceptible hosts had no effect on virus viability. Thus, the in vitro reaction is species-specific and mimics naturally occurring host cell tropism. Small Angle X-ray scattering (SAXS) was used to study real-time conformational. The intensity of X-rays scattered from a mixture of homogeneous spherical objects such as viruses is a function of their gyration radii and the scattering angle. DNA release results in a loss of mass. Thus, the gyration radius of an empty virus increases. The SAXS data demonstrates that as particles lose their DNA they become less spherical, indicating that reaction induced asymmetry. To characterize the asymmetry, the reactions have been examined by cryo-EM microscopy for image reconstruction. The results demonstrate that the viral vertex embedded in the LPS bilayers undergoes a dramatic reorganization. The G protein spike pentamer is jettisoned. The spikes remain intact at the other 11 vertices. A cylindrical cavity, consisting of low density protein surrounded by high density DNA, appears. This likely represents the formation of the H-tube. Lastly, the underlying coat protein pentamer is rotationally reorganized, creating a pore through which the H-tube can descend. These results were recently published in PNAS. Accessing the bioengineering potential of the system: If a protein can be modified and remain functional, biotechnology applications are broadened. For example, a nanotube's length may need to be altered. To determine if H-tubes could be lengthened, its repetitive primary structure was exploited. The H-tube alpha-helices run parallel to each other. The tube has two motifs, a hendecad (11) motif: three helical turns per 11 amino acids, and a heptad (7) motif; two helical turns per 7 amino acids. The helices are in register i.e. each hendecad (11) or heptad motif contacts itself within neighboring helices. Thus, deletions and insertions (± 11 or 7) should alter tube length without affecting oligomerization. We demonstrated that tubes could be both shortened and lengthened and remain functional. These results were published in the Journal of Virology. The energetics of DNA transport: The H-tube structure demonstrates that the entire inner passage is lined with amide and guanidinium side chains, which are known to interact with purines. As exemplified by the catabolic oxidation of carbon and the electron transport systems, 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. The amide side chains perform the function of a regulatory gauge. If too much air pressure (energy) is released at one time, it can rupture the tube. It may be possible to demonstrate this experimentally by removing amide and guanidinium side chains from the interior lining. Similarly, adding amide and guanidinium side chains may kinetically trap DNA during transport. The requisite mutants needed to test this model have been generated. The addition amide and guanidinium side chains results in cold-sensitive phenotypes. By contrast, removing amide and guanidinium side chains to a defined location in the structure results in temperature-sensitive phenotypes. Protein H participates in several phage-related functions besides DNA transport, such as host cell recognition and particle assembly. Accordingly, our efforts have focused on developing strategies to uncouple these various processes. For example, the mutant genes are plasmid-based and H nonsense mutant is used. To investigate assembly under restrictive conditions, the am(H) phage is first grown in cells with a cloned wild-type gene. This results in particles with a wild-type H protein content that can deliver DNA at the restrictive temperature to cells carry a plasmid with the mutant H gene. Thus, assembly can be characterized in the absence of possible entry defects conferred by the mutant proteins. Similarly, to investigate entry defects, particles with mutant H proteins are synthesized at permissive temperatures, and assayed for entry defects at the restrictive temperature. Preliminary results suggest that the mutations neither affect assembly nor host cell attachment. We are currently developing protocols that specifically assay DNA transport. These include K+ efflux studies to measure phage-mediated breeches to host cells and monitoring the kinetics of phage protein synthesis.
Publications
- Type:
Journal Articles
Status:
Submitted
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, submitted.
This work temporally spans two NIFA projects,
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Raoznowski, A. P. and B. A. Fane Mutagenic analysis of a DNA translocating tube's interior surface
|
Progress 10/01/15 to 09/30/16
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 and published in the journal Nature. The target audience was broad, 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 a manuscript that directly demonstrated the system's biotechnology potential. The structure also suggests an energetic model for DNA delivery; we are currently testing this model. Lastly, we have reconstituted the DNA delivery reaction in vitro. The reaction can be followed in real time and has led to further structural studies. Thus, it is likely that the target audience will likely continue to be broad. 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, Brody Blackburn. Mr Blackburn is currently employed as technician in the laboratory How have the results been disseminated to communities of interest?The results have been presented at the FASEB Meeting for Virus Structure and Assembly and published in the Journal of Virology. What do you plan to do during the next reporting period to accomplish the goals?1) Finish the analyses involving mutant proteins that lack inward facing amide side chains. 2) Complete the structural studies of the in vitro reconstituted DNA delivery reaction.
Impacts
What was accomplished under these goals?
Length alteration: accessing the bioengineering potential of the system: In the atomic structure, H-tube alpha helices run parallel to each other. The tube has two motifs, a hendecad (11) motif: three helical turns per 11 amino acids, and a heptad (7) motif; two helical turns per 7 amino acids. The helices with and in register i.e. each hendecad (11) or heptad motif contacts itself within neighboring helices. Thus, deletions and insertions (± 11 or 7) should alter tube length without affecting oligomerization. As discussed in last year's report, we are able to lengthen the tube, demonstrating that the tube is amenable to bioengineering. In the last year, we also demonstrated that in some region the tube can be shortened and retain function. Results also identified a domain required for H protein incorporation during virion morphogenesis. This analysis has been completed and the manuscript was published in the fall of 2016. 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. As described in last year's progress report, a mutant with additional amide side chains on the inner surface has been generated. It exhibits a severe cold-sensitivity phenotype, failing to form plaques below 24°C. The cold sensitive phenotype is consistent with the above model, as pressure is a function of temperature. Further analyses indicated that DNA has become kinetically trapped within the DNA translocating tube. During the last year, we have been examining the converse situation, the removal of amide and guanidinium side chains from amino acids lining the tube's inner passage. Most of these mutants exhibit a temperature-sensitive phenotype. As pressure is a function of temperature, the heat sensitivity is consistent with the aforementioned model. If the tube's integrity is compromised, it may rupture during DNA transport. The mutations conferring defective phenotypes are not evenly distributed throughout the protein. The mutations conferring the most severe phenotype are found at the transition point between the 11,3 and 7,2 coiled coil motifs. Although the temperature sensitive phenotypes are consistent with the model, protein-folding mutations often exhibit this phenotype. The failure to fold at elevated temperatures will lead to incorporation defects during particle morphogenesis. Thus, we are systematically characterizing the stoichiometry of the mutant particles. Reconstituting DNA delivery in vitro and structural studies: To meaningfully reconstitute viral receptor recognition and DNA delivery in vitro, the reaction conditions must favor an asymmetric process. For example, only one site of the virus contacts the host cell receptor. Thus, the receptor must be spatially fixed, unable to interact with the virus in more than one place. Lipopolysaccharides (LPS) from sensitive strains serve as microvirus receptors. Two viruses, phiX174 and ST1, with different host specificities are being used in these studies. We have purified LPS from both the phiX174 and ST1 hosts. Purified LPS was examined by electron microscopy. Under our reaction conditions, it formed lipid bi-layers, which would spatially fix the receptor vis-à-vis the virion. Incubation of viruses with LPS from a susceptible host results in a greater than 90% loss of infectivity, presumably the viral DNA is leaving the capsid. LPS isolated from the unsusceptible hosts had no effect on virus viability. Thus, the in vitro reaction is species-specific and mimics naturally occurring host cell tropism. Small Angle X-ray scattering (SAXS) is being used to study real-time conformational changes in phiX174 while reacting with LPS in solution. The intensity of X-rays scattered from a mixture of homogeneous spherical objects such as viruses is a function of their gyration radii and the scattering angle. DNA release results in a loss of mass. Thus, the gyration radius of an empty virus increases. The SAXS data demonstrates that particles are losing DNA and as they lose their DNA they become less spherical, indicating that reaction induced asymmetry. To characterize the asymmetry, the reactions have been examined by cryo electron microscopy for image reconstruction. Preliminary results demonstrate that the viral vertex embedded in the LPS bilayers has undergone a dramatic reorganization.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Roznowski AP, Fane BA. 2016. Structure-Function Analysis of the varphiX174 DNA-Piloting Protein Using Length-Altering Mutations. J Virol 90:7956-7966.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Structure-Function Analysis of the phiX174 DNA Piloting Protein using Length Altering Mutations
|
Progress 10/01/14 to 09/30/15
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. Last year, the atomic structure of the DNA pilot protein was solved. The structure was paradigm shifting and published in the journal Nature. The target audience was broad, including structural biologists and biochemists, representing basic science disciplines, and translational scientists, who may endeavor to develop biotechnology applications. The structure suggests several mechanistic hypotheses to be tested. As this is the first, and currently only viral DNA conduit solved at atomic resolution, it is likely that the target audience will likely continue to be broad. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has directly provided scientific training to three young scientists: Mr. Aaron Roznowski, a current graduate student in the PI's laboratory, and two undergraduate students, Brody Blackburn and Nathaniel Yang. How have the results been disseminated to communities of interest?The results have been presented at the International Virus and Phage Assembly meetings. A manuscript describing the length altered mutants is in preparation for submission to the Journal of Virology. What do you plan to do during the next reporting period to accomplish the goals?1) Publish the results of the length-altered analyses. 2) Generate mutants with shorter than the wild-type tubes for functional analyses. 3) To further explore the energy model, mutants that lack the inward facing amide side chains, and to alter the virion's intrinsic potential energy, strains with larger and smaller genomes will be generated.
Impacts
What was accomplished under these goals?
Length alteration: accessing the bioengineering potential of the system: In the atomic structure, H-tube alpha helices run parallel to each other. The tube has two motifs, a hendecad (11) motif: three helical turns per 11 amino acids, and a heptad (7) motif; two helical turns per 7 amino acids. The helices with and in register i.e. each hendecad (11) or heptad motif contacts itself within neighboring helices. Thus, deletions and insertions (± 11 or 7) should alter tube length without affecting oligomerization. As described in last year's report, an addition 14 amino acids were engineered into the protein, creating two additional heptads. This extends the tube by approximately 15 angstroms. The virus (H+14) with the length-altered tube is viable above 28C. Thus, the tube must form after cell contact. Moreover, these results indicate that the tube is amenable to bioengineering. This mutant has now been more thoroughly characterized. The resulting H+14 mutant was viable but exhibited a cold sensitive (cs) phenotype at 24°C. Cold sensitivity was rescued by several single point mutations in the inserted sequence. To determine if the mutant virions were as infectious as wild-type, permissively synthesized particles were purified by rate zonal sedimentation. The mutant virus sediments like wild-type and the OD peak coincides with the specific infectivity peak. OD peak protein content was analyzed via SDS-PAGE and elongated H protein was observed in H+14 particles. The amount of H+14 H protein was assessed via densitometry and it appears to be present at wild-type levels. The specific infectivity of the H+14 mutant was calculated [plaque forming units (pfu)/A260] and was found to be reduced when compared to wild-type by approximately 50%. However, reversion of the cs phenotype (H+14 R→L) also restored specific infectivity to wild-type levels. A similar mutant was recovered with an addition of 11 amino acids (H+11) within the hendecad domain. Its phenotype is indistinguishable from wild-type in all assays described above. Thus, the elongated proteins can be fully tolerated as evinced by the wild-type phenotype of the H+14 R→L and H+11 mutants. The length altered H+14 R→L mutant was used to further validate the biological significance of the rather unexpected x-ray structure. Monomers composing the piloting protein tube are bound together through a set of specific inter-helical contacts. As seen in other coiled coil structures, the contact keeps the monomers parallel and in register, aligning equivalent heptad and hendecad motifs. Having two H proteins of differing lengths would disrupt this arrangement. Since H proteins are incorporated as monomers during assembly, particles containing heterogeneous H proteins can be assembled. These particles should exhibit reduced infectivity. To test this hypothesis, lysis-resistant cells were co-infected with wild-type and the equally infectious H+14 R→L mutant. Particles were purified and assayed for protein content and specific infectivity. Both the wild-type and H+14 R→L H proteins were found in particles at approximately equal levels. The specific infectivity of the heterogeneous particles was reduced almost two orders of magnitude when compared to the parental particles. The coiled-coil domain spans the periplasmic space or a membrane adhesion zone during genome piloting. A minimal length is likely required so that an assembled H tube can bridge the gap between the external environment and the cytoplasm. Although characterizing the phenotypes associated with length-shortened tubes may directly address this question, internal deletions could also affect the proteins' ability to be incorporated during assembly or prevent oligomerization for DNA transport. To explore these various structure-function relationships, nine cloned H genes with internal deletions were constructed. Mutant genes lacked DNA encoding a single hendecad (del11), a single heptad (del7), or two sequential heptads (del14). Cloned gene expression was assayed for the ability to complement an am(H) mutant. Unlike the cloned wild-type gene, none of the cloned mutant genes were able to complement an am(H) mutant on the level of plaque formation. To determine whether the ΔH proteins failed to function in DNA transport or failed to incorporate during assembly, lysis resistant cells expressing the deletion constructs were infected with an am(H) mutant. The resulting infection products were isolated by rate zonal sedimentation and their protein content was determined by SDS-PAGE. The mutations conferred two different molecular phenotypes. The proteins containing deletions at both ends of coiled coil domains were incorporated into uninfectious virus-like particles. This result suggests that these proteins are defective in tube formation and/or the tubes are too short to span the cell wall. However, secondary mutations in gene H restore viability. Thus the primary defect is likely tube formation. By contrast, the proteins with internal deletions were not incorporated into viruses, suggesting that this region may contain an assembly incorporation domain. 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. As described in last year's progress report, a mutant with additional amide side chains on the inner surface has been generated. It exhibits a severe cold-sensitivity phenotype, failing to form plaques below 28C. The cold sensitive phenotype is consistent with the above model, as pressure is a function of temperature. This mutant has now been more thoroughly characterized. The primary defect is DNA transport. In these assays, lysis resistant cells are infected at low and high temperature. The virus has a single-stranded DNA genome of positive polarity. Therefore, the minus strand must be synthesized before viral protein synthesis. This allows the kinetics of viral protein synthesis to be used as a surrogate for DNA transport. Compared to the wild-type control, protein synthesis in mutant infected cells is delayed by 20 minutes and never reaches wild-type levels. These data suggest that DNA has become kinetically trapped within the DNA translocating tube.
Publications
|
Progress 10/01/13 to 09/30/14
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. Last year, the atomic structure of the DNA pilot protein was solved. The structure was paradigm shifting and published in the journal Nature. The target audience was broad, including structural biologists and biochemists, representing basic science disciplines, and translational scientists, who may endeavor to develop biotechnology applications. The structure suggests several mechanistic hypotheses to be tested. As this is the first, and currently only viral DNA conduit solved at atomic resolution, it is likely that the target audience will likely continue to be broad. 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 Mr Brody Blackburn, an undergraduate student. How have the results been disseminated to communities of interest? All microbiology majors are required to take MIC421b, a lab course with an enrollment of approximately 60 students. Although Dr. Fane is not the instructor for this course, he devised a module on mutations and second site genetic analyses. Students conducted a second-site genetic analysis with the mutants that form length-altered H-tubes at 24C. In addition to demonstrating how genetic analyses can be used in the study of protein structure, this portion of the module illustrated the principles of Luria and Delbruck's fluctuation test. What do you plan to do during the next reporting period to accomplish the goals? 1) Finish the functional analyses of the longer length-altered tubes. 2) Generate mutants with shorter than the wild-type tubes for functional analyses. 3) Characterize the low temperature functional defect conferred by additional, inward facing, amide side chains within the tube. 4) Generate mutants that lack the inward facing amide side chains.
Impacts
What was accomplished under these goals?
Length alteration: accessing the bioengineering potential of the system. In the atomic structure, H-tube alpha-helices run parallel to each other. The tube has two motifs, a hendecad (11) motif: three helical turns per 11 amino acids, and a heptad (7) motif; two helical turns per 7 amino acids. The helices with and in register i.e. each hendecad (11) or heptad motif contacts itself within neighboring helices. Thus, deletions and insertions (± 11 or 7) should alter tube length without affecting oligomerization. If unable to span the cell wall, a shorter tube will not deliver DNA. By contrast, phages with longer tubes may retain viability if the capsidcan tolerate the size altered subunits. When the structure was solved, it was unknown whether the tube pre-existed within the capsid as a fully assembled structure or if it formed after cell contact. The capsid has an internal diameter of 172 angstroms. Although the tube, which is 170 angstroms in length, could be accommodated, it would be a rather tight fit. An addition 14 amino acids were engineered into the protein, creating two additional heptads. This extends the tube by approximately 15 angstroms. The virus with the length-altered tube is viable above 28C. Thus, the tube must form after cell contact. Moreover, these results indicate that the tube is amenable to bioengineering. 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. There is enough homology between the fX174 H protein and T7 gp16, a likely component of the tail tube extension in T7, to generate a reasonable alignment in which the positioning of the glutamine residues are similar. 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. Conversely, eliminating these moieties may produce conduits to fragile to transport the DNA. There are only two places in the tube where additional amide groups can be added. This mutant has been generated and exhibits a severe cold-sensitivity phenotype, failing to form plaques below 28°C.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Sun, L., M. G. Rossmann, and B. A. Fane. 2014. High-resolution structure of a virally encoded DNA-translocating conduit and the mechanism of DNA penetration. J Virol 88:10276-10279.
|
Progress 01/01/13 to 09/30/13
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. The microvirus DNA translocation mechanism has been obscure since their discovery in the mid 1920’s. For double-stranded DNA phages, the transport mechanism is often associated with the emblematic phage tail, whereas tail-less phages typically utilize host cell pores. Tail-less and requiring no host cell proteins to penetrate, microvirus DNA delivery cannot be interpreted within these well-defined paradigms. The DNA pilot protein, a minor structural protein, mediates DNA delivery. To gain insights into the DNA delivery mechanism, the atomic structure of the phiX174 DNA pilot protein was solved. As the structure was paradigm-shifting, the target audience was broad, including structural biologists and biochemists, who representing basic science disciplines, and translational scientists, who may endeavor to develop biotechnology applications. It is likely that both audiences were reached via publication in Nature, a higher impact, general scientific journal that spans many disciplines. The phiX174 DNA pilot protein structure represents the first viral DNA conduit solved at atomic resolution. 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. Ms. Lindsey Young, who started as an undergraduate student in the PI’s laboratory. She is currently a graduate student at the University of California Berkeley, and Mr. Aaron Roznowski, a current graduate student in the PI’s laboratory How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? The first viral DNA conduit solved at atomic resolution, it suggests evolutionary and mechanistic hypotheses that would be difficult to test in other systems. For example, the protein is composed of regularly repeating motifs, suggesting length plasticity and evolution via successive genetic duplications. The biological activity of length-modified conduits will test this model. The tube’s interior surface is entirely lined with DNA-interacting amide and guanidinium groups, which are likely critical for DNA piloting: a testable hypothesis.Moreover, attempts will be made to isolate and structurally characterize putative tube forming proteins form other viruses.
Impacts
What was accomplished under these goals?
The atomic structure of the øX174 DNA pilot protein H was solved in collaboration with Dr. M. G. Rossmann’s group (Purdue University). The structure has partially elucidated this historically obscure process of microvirus DNA delivery. Ten H proteins form an a-helical tube (H-tube) with dimensions and physical properties ideal for genome translocation. Although the x-ray model is an oligomeric tube, the protein is monomeric during procapsid assembly. Upon cell contact, H proteins interact and a fully formed tube emerges from the capsid. After DNA delivery, the conduit appears to dissociate within the cell wall. Thus, the newly discovered øX174 phage tail is ephemeral. The biological relevance of this paradigm-shifting structure was tested genetically in vivo, biochemically in vitro, and tomographically in situ. Using the atomic structure as a guide, mutations were designed to eliminate inter-helix contacts without affecting the helical fold. Particles sedimenting at 114S, the S value of the virion, were isolated from mutant-infected cell extracts. Compared to the wild-type virions, there were no apparent differences in DNA and protein content. However, the specific infectivity (plaque forming units /OD280) of the mutant particles was approximately three orders of magnitude lower than the wild-type control. In vitro, mutant proteins maintained a primarily a-helical fold, as assayed by circular dichroism (data not shown). However, the proteins migrated as monomers or lower order oligomers vis-à-vis the wild-type protein by size exclusion chromatography. Trypsin digestion produced small pieces, whereas the wild-type protein generated a stable fragment. This indicates that lysine and arginine residues buried in the wild-type structure were accessible, implying the failure to form tubes. The tube extending from the virion and passing through the entire cell wall was directly visualized in situ by cryo-EM tomography. Moreover, the hypothesized host cell attachment functions of the protein's N-terminus were also tested. These results were also published.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Sun L, Young LN, Zhang X, Boudko SP, Fokine A, Zbornik E, Roznowski AP, Moulineux I, Rossmann MG, Fane BA. 2014. Icosahedral ?X174 forms a tail for DNA transport. Nature 505: 432-435.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Young LN, Hockenberry, Fane BA. 2014. Mutations in the N-terminus of the �X174 DNA pilot protein H confer both assembly and host cell attachment defects. Journal of Virology 88: 1787-1794.
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Efficient and consistent laboratory-based DNA transformation protocols, a critical component for genetic analyses, are sparse within the Bdellovibrionaceae, Spiroplasmatales and Chlamydia: microbial classifications that include animal, human, and plant pathogens. The recently isolated gokushoviruses, which are related to bacteriophage phiX174, could be developed into powerful genetic tools. Unlike the double-stranded (ds) bacteriophages, gokusho- and microviruses lack complex, multi-protein tail organelles utilized for DNA delivery. Instead, a single DNA pilot protein H delivers the viral genome from the outer membrane to the cytoplasm. During the last year, efforts were dedicated to solving the structure of the soluble central domain of the phiX174 protein in collaboration with the M. G. Rossmann (Purdue University). The structure has been solved to 2.4-angstrom resolution. Due to its unusual features, which likely identify a novel mechanism of DNA delivery, extensive biochemical and genetic analyses were conducted to ensure the x-ray structure's biological significance. PARTICIPANTS: University of Arizona: Bentley A Fane, Lindsey N Young. Collaborators at Ourdue University: M.G. Rossmann, Lei Sun. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The central domain consists of 10 alpha helices arrayed into a 170-angstrom long coiled-coil structure, or helical barrel. The external diameter is 48 angstroms, whereas the internal diameter ranges from 22 - 24 angstroms. To the best of our knowledge, this is the first description of a decameric coiled coil structure. The length of the barrel is long enough to transverse the periplasmic space. The N- and C-termini of the protein, which were not crystallized, are hydrophobic and most likely anchor the barrel in the inner and outer membranes. The packaged, circular, ssDNA genome contains mostly unpaired bases. The 22-angstrom internal diameter can easily accommodate two separate un-paired ssDNA strands with intercalated bases. The circular, ssDNA genomes of the filamentous bacterial Inoviruses are packaged into a cylindrical capsid with similar inner dimensions. The formation of an oligomeric barrel was unexpected as the H protein is incorporated into particles as individual monomers. Moreover, there was no evidence of H-barrels in cryo electron microscopic images of more than five hundred mature virions. Thus, the packaged H proteins most likely form these barrels sometime during attachment and/or penetration, which may represent a novel DNA delivery mechanism. If this hypothesis is correct, mutations that inhibit the requisite barrel forming helix-helix interactions should not affect virus assembly. However, if barrel formation is essential for penetration, the resulting particles should lack infectivity. To determine whether mutant H proteins affected particle formation and/or the infectivity of assembled particles, particles were purified from mutant and wild-type infected cells. Although the mutant particles lacked infectivity, they were biophysically indistinguishable from wild-type particles in protein and DNA content. The properties of the mutant H proteins were also investigated in vitro. As evinced by similar circular dichroism spectra, the mutant proteins maintained a wild-type secondary structural content. However by size exclusion chromatography, the mutant proteins migrate as lower order oligomers. Due to the unique nature and evolutionary implications of the crystal structure we are currently preparing a manuscript for submission to Nature.
Publications
- No publications reported this period
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