AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15(E15) using genetic and biochemical methodology METHODS: Hydroxylamine was used to create nonsense mutants of bacteriop...AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15(E15) using genetic and biochemical methodology METHODS: Hydroxylamine was used to create nonsense mutants of bacteriophage E15. The mutants were then screened for defects in their adsorption apparatus proteins, initially by measuring the concentrations of free tail spike proteins in lysates of cells that had been infected by the phage mutants under nonpermissive growth conditions. Phage strains whose infected cell lysates contained above-average levels of free tail spike protein under non-permissive growth conditions were assumed to contain nonsense mutations in genes coding for adsorption apparatus proteins.These mutants were characterized by classical genetic mapping methods as well as automated sequencing of several of their genes. Finally, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography were used to examine the protein compositions of the radioactive particles produced when the various mutants were grown on a non-permissive host cell in the presence of 35S-methionine and co-purified along with E15 wt phage on Cs Cl block gradients.RESULTS: Our results are consistent with gp4 forming the portal ring structure of E15. In addition, they show that proteins gp15 and gp17 likely comprise the central tube portion of the E15 adsorption apparatus, with gp17 being more distally positioned than gp15 and dependent upon both gp15 and gp16 for its attachment. Finally, our data indicates that tail spike proteins comprised of gp20 can assemble onto nascent virions that contain gp7, gp10, gp4 and packaged DNA, but which lack both gp15 and gp17, thereby forming particles that are of sufficient stability to survive Cs Cl buoyant density centrifugation.CONCLUSION: The portal ring(gp4) of E15 is bound to tail spikes(gp20) and the tail tube(gp15 and gp17); gp17's attachment requires both gp15 and gp16.展开更多
Dicistroviruses comprise a newly characterized and rapidly expanding family of small RNA viruses of invertebrates. Several features of this virus group have attracted considerable research interest in recent years. In...Dicistroviruses comprise a newly characterized and rapidly expanding family of small RNA viruses of invertebrates. Several features of this virus group have attracted considerable research interest in recent years. In this review I provide an overview of the Dicistroviridae and describe progress made toward the understanding and practical application of dicistroviruses, including (i) construction of the first infectious clone of a dicistrovirus, (ii) use of the baculovirus expression system for production of an infectious dicistrovirus, (iii) the use of Drosophila C virus for analysis of host response to virus infection, and (iv) correlation of the presence of Israeli acute paralysis virus with honey bee colony collapse disorder. The potential use of dicistroviruses for insect pest management is also discussed. The structure, mechanism and practical use of the internal ribosome entry site (IRES) elements has recently been reviewed elsewhere.展开更多
While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus,these characteristics have been largely ne...While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus,these characteristics have been largely neglected in studies on rabies virus(RABV).Here,we purified the RABV virions with good purity and integrity,and analyzed their proteome by nano LC–MS/MS,followed by the confirmation with immunoblot and immuno-electronic microscopy.In addition to the 5 viral proteins,49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions.Function annotation suggested that 24 of them were likely involved in virus replication.Furthermore,cryo-EM was employed to observe the purified RABV virions,generating high-resolution pictures of the bullet-shaped virion structure of RABV.This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection,as well as the discovery of new anti-RABV therapeutics.展开更多
In the current SARS-CoV-2 disease(COVID-19)pandemic,the structural understanding of new emerging viruses in relation to developing effective treatment and interventions are very necessary.Viruses present remarkable di...In the current SARS-CoV-2 disease(COVID-19)pandemic,the structural understanding of new emerging viruses in relation to developing effective treatment and interventions are very necessary.Viruses present remarkable differences in geometric shapes,sizes,molecular compositions and organizations.A detailed structural knowledge of a virion is essential for understanding the mechanisms of capsid assembly/disassembly,antigenicity,cell-receptor interaction,and designing therapeutic strategies.X-ray crystallography,cryoelectron microscopy and molecular simulations have elucidated atomic-level structure of several viruses.In view of this,a recently determined crystal structure of SARS-CoV-2 nucleocapsid has revealed its architecture and self-assembly very similar to that of the SARS-CoV-1 and the Middle-East respiratory syndrome virus(MERS-CoV).In structure determination,capsid symmetry is an important factor greatly contributing to its stability and balance between the packaged genome and envelope.Since the capsid protein subunits are asymmetrical,the maximum number of inter-subunit interactions can be established only when they are arranged symmetrically.Therefore,a stable capsid must be in a perfect symmetry and lowest possible free-energy.Isometric virions are spherical but geometrically icosahedrons as compared to complex virions that are both isometric and helical.Enveloped icosahedral or helical viruses are very common in animals but rare in plants and bacteria.Icosahedral capsids are defined by triangulation number(T=1,3,4,13,etc.),i.e.,the identical equilateral-triangles formed of subunits.Biologically significant defective capsids with or without nucleic acids are common in enveloped alpha-,flavi-and hepadnaviruses.The self-assembling,stable and non-infectious virus-like particles have been widely exploited as vaccine candidates and therapeutic molecules delivery vehicles.展开更多
基金Supported by The NIH-AREA Grant,No.1R15GM52696-01the NSF-RUI Grant,No.DMB-8608480+2 种基金the Howard Hughes Medical Research Institute(two grants to the PLNU Biology Department)Research Associates of PLNU(a 300 member alumni support group)the PLNU Administration
文摘AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15(E15) using genetic and biochemical methodology METHODS: Hydroxylamine was used to create nonsense mutants of bacteriophage E15. The mutants were then screened for defects in their adsorption apparatus proteins, initially by measuring the concentrations of free tail spike proteins in lysates of cells that had been infected by the phage mutants under nonpermissive growth conditions. Phage strains whose infected cell lysates contained above-average levels of free tail spike protein under non-permissive growth conditions were assumed to contain nonsense mutations in genes coding for adsorption apparatus proteins.These mutants were characterized by classical genetic mapping methods as well as automated sequencing of several of their genes. Finally, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography were used to examine the protein compositions of the radioactive particles produced when the various mutants were grown on a non-permissive host cell in the presence of 35S-methionine and co-purified along with E15 wt phage on Cs Cl block gradients.RESULTS: Our results are consistent with gp4 forming the portal ring structure of E15. In addition, they show that proteins gp15 and gp17 likely comprise the central tube portion of the E15 adsorption apparatus, with gp17 being more distally positioned than gp15 and dependent upon both gp15 and gp16 for its attachment. Finally, our data indicates that tail spike proteins comprised of gp20 can assemble onto nascent virions that contain gp7, gp10, gp4 and packaged DNA, but which lack both gp15 and gp17, thereby forming particles that are of sufficient stability to survive Cs Cl buoyant density centrifugation.CONCLUSION: The portal ring(gp4) of E15 is bound to tail spikes(gp20) and the tail tube(gp15 and gp17); gp17's attachment requires both gp15 and gp16.
基金This journal paper of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa,Project No. 6673, was supported by the Iowa State University Plant Sciences Institute, and the Consortium for Plant Biotechnology Research
文摘Dicistroviruses comprise a newly characterized and rapidly expanding family of small RNA viruses of invertebrates. Several features of this virus group have attracted considerable research interest in recent years. In this review I provide an overview of the Dicistroviridae and describe progress made toward the understanding and practical application of dicistroviruses, including (i) construction of the first infectious clone of a dicistrovirus, (ii) use of the baculovirus expression system for production of an infectious dicistrovirus, (iii) the use of Drosophila C virus for analysis of host response to virus infection, and (iv) correlation of the presence of Israeli acute paralysis virus with honey bee colony collapse disorder. The potential use of dicistroviruses for insect pest management is also discussed. The structure, mechanism and practical use of the internal ribosome entry site (IRES) elements has recently been reviewed elsewhere.
基金This research was funded by the National Key Research and Development Plan(Grant No.2016YFD0500401)National Natural Science Foundation of China(Grant No.31402214)China Postdoctoral Science Foundation(Grant No.2014M552638).
文摘While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus,these characteristics have been largely neglected in studies on rabies virus(RABV).Here,we purified the RABV virions with good purity and integrity,and analyzed their proteome by nano LC–MS/MS,followed by the confirmation with immunoblot and immuno-electronic microscopy.In addition to the 5 viral proteins,49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions.Function annotation suggested that 24 of them were likely involved in virus replication.Furthermore,cryo-EM was employed to observe the purified RABV virions,generating high-resolution pictures of the bullet-shaped virion structure of RABV.This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection,as well as the discovery of new anti-RABV therapeutics.
文摘In the current SARS-CoV-2 disease(COVID-19)pandemic,the structural understanding of new emerging viruses in relation to developing effective treatment and interventions are very necessary.Viruses present remarkable differences in geometric shapes,sizes,molecular compositions and organizations.A detailed structural knowledge of a virion is essential for understanding the mechanisms of capsid assembly/disassembly,antigenicity,cell-receptor interaction,and designing therapeutic strategies.X-ray crystallography,cryoelectron microscopy and molecular simulations have elucidated atomic-level structure of several viruses.In view of this,a recently determined crystal structure of SARS-CoV-2 nucleocapsid has revealed its architecture and self-assembly very similar to that of the SARS-CoV-1 and the Middle-East respiratory syndrome virus(MERS-CoV).In structure determination,capsid symmetry is an important factor greatly contributing to its stability and balance between the packaged genome and envelope.Since the capsid protein subunits are asymmetrical,the maximum number of inter-subunit interactions can be established only when they are arranged symmetrically.Therefore,a stable capsid must be in a perfect symmetry and lowest possible free-energy.Isometric virions are spherical but geometrically icosahedrons as compared to complex virions that are both isometric and helical.Enveloped icosahedral or helical viruses are very common in animals but rare in plants and bacteria.Icosahedral capsids are defined by triangulation number(T=1,3,4,13,etc.),i.e.,the identical equilateral-triangles formed of subunits.Biologically significant defective capsids with or without nucleic acids are common in enveloped alpha-,flavi-and hepadnaviruses.The self-assembling,stable and non-infectious virus-like particles have been widely exploited as vaccine candidates and therapeutic molecules delivery vehicles.
