scholarly journals High resolution cryo EM analysis of HPV16 identifies minor structural protein L2 and describes capsid flexibility

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniel J. Goetschius ◽  
Samantha R. Hartmann ◽  
Suriyasri Subramanian ◽  
Carol M. Bator ◽  
Neil D. Christensen ◽  
...  
Keyword(s):  
High Resolution ◽  
High Titer ◽  
Rigid Bodies ◽  
Keratinocyte Differentiation ◽  
Structural Studies ◽  
Structural Protein ◽  
Cottontail Rabbit ◽  
Particle Level ◽  
Minor Structural Protein ◽  
Local Movement

AbstractHuman papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. HPV is epitheliotropic and its replication is tightly associated with terminal keratinocyte differentiation making production and purification of high titer virus preparations for research problematic, therefore alternative HPV production methods have been developed for virological and structural studies. In this study we use HPV16 quasivirus, composed of HPV16 L1/L2 capsid proteins with a packaged cottontail rabbit papillomavirus genome. We have achieved the first high resolution, 3.1 Å, structure of HPV16 by using a local subvolume refinement approach. The high resolution enabled us to build L1 unambiguously and identify L2 protein strands. The L2 density is incorporated adjacent to conserved L1 residues on the interior of the capsid. Further interpretation with our own software for Icosahedral Subvolume Extraction and Correlated Classification revealed flexibility, on the whole-particle level through diameter analysis and local movement with inter-capsomer analysis. Inter-capsomer expansion or contraction, governed by the connecting arms, showed no bias in the magnitude or direction of capsomer movement. We propose that papillomavirus capsids are dynamic and capsomers move as rigid bodies connected by flexible linkers. The resulting virus structure will provide a framework for continuing biochemical, genetic and biophysical research for papillomaviruses. Furthermore, our approach has allowed insight into the resolution barrier that has previously been a limitation in papillomavirus structural studies.

scholarly journals High resolution cryo EM analysis of HPV16 identifies minor structural protein L2 and describes capsid flexibility

2020 ◽  
Author(s):  
Daniel J. Goetschius ◽  
Samantha R. Hartmann ◽  
Suriyasri Subramanian ◽  
Carol Bator ◽  
Neil D. Christensen ◽  
...  
Keyword(s):  
High Resolution ◽  
High Titer ◽  
Rigid Bodies ◽  
Keratinocyte Differentiation ◽  
Structural Studies ◽  
Structural Protein ◽  
Cottontail Rabbit ◽  
Particle Level ◽  
Minor Structural Protein ◽  
Local Movement

AbstractHuman papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. HPV is epitheliotropic and its replication is tightly associated with terminal keratinocyte differentiation making production and purification of high titer virus preparations for research problematic, therefore alternative HPV production methods have been developed for virological and structural studies. In this study we use HPV16 quasivirus, composed of HPV16 L1/L2 capsid proteins with a packaged cottontail rabbit papillomavirus genome. We have achieved the first high resolution, 3.1Å, structure of HPV16 by using a local subvolume refinement approach. The high resolution enabled us to build L1 unambiguously and identify L2 protein strands. The L2 density is incorporated adjacent to conserved L1 residues on the interior of the capsid. Further interpretation with our own software for Icosahedral Subvolume Extraction and Correlated Classification (ISECC) revealed flexibility, on the whole-particle level through diameter analysis and local movement with inter-capsomer analysis. Inter-capsomer expansion or contraction, governed by the connecting arms, showed no bias in the magnitude or direction of capsomer movement. We propose that papillomavirus capsids are dynamic and capsomers move as rigid bodies connected by flexible linkers. The resulting virus structure will provide a framework for continuing biochemical, genetic and biophysical research for papillomaviruses. Furthermore, our approach has allowed insight into the resolution barrier that has previously been a limitation in papillomavirus structural studies.

scholarly journals RG1-VLP and Other L2-Based, Broad-Spectrum HPV Vaccine Candidates

2021 ◽  
Vol 10 (5) ◽  
pp. 1044
Author(s):  
Bettina Huber ◽  
Joshua Weiyuan Wang ◽  
Richard B. S. Roden ◽  
Reinhard Kirnbauer
Keyword(s):  
Small Animal ◽  
Good Manufacturing Practice ◽  
Special Focus ◽  
T Cell Epitopes ◽  
Hpv Vaccines ◽  
Structural Protein ◽  
Vaccine Candidates ◽  
Protective Antibodies ◽  
Minor Structural Protein ◽  
Hpv Types

Licensed human papillomavirus (HPV) vaccines contain virus-like particles (VLPs) self-assembled from L1 major-capsid proteins that are remarkably effective prophylactic immunogens. However, the induced type-restricted immune response limits coverage to the included vaccine types, and costly multiplex formulations, restrictive storage and distribution conditions drive the need for next generation HPV vaccines. Vaccine candidates based upon the minor structural protein L2 are particularly promising because conserved N-terminal epitopes induce broadly cross-type neutralizing and protective antibodies. Several strategies to increase the immunological potency of such epitopes are being investigated, including concatemeric multimers, fusion to toll-like receptors ligands or T cell epitopes, as well as immunodominant presentation by different nanoparticle or VLP structures. Several promising L2-based vaccine candidates have reached or will soon enter first-in-man clinical studies. RG1-VLP present the HPV16L2 amino-acid 17–36 conserved neutralization epitope “RG1” repetitively and closely spaced on an immunodominant surface loop of HPV16 L1-VLP and small animal immunizations provide cross-protection against challenge with all medically-significant high-risk and several low-risk HPV types. With a successful current good manufacturing practice (cGMP) campaign and this promising breadth of activity, even encompassing cross-neutralization of several cutaneous HPV types, RG1-VLP are ready for a first-in-human clinical study. This review aims to provide a general overview of these candidates with a special focus on the RG1-VLP vaccine and its road to the clinic.

scholarly journals Синтез, структура и магнитные свойства многослойных наногетероструктур Fe|MgO|Cr|MgO|Fe

2021 ◽  
Vol 63 (9) ◽  
pp. 1306
Author(s):  
Е.М. Якунина ◽  
Е.А. Кравцов ◽  
Yu.N. Khaydukov ◽  
Н.О. Антропов ◽  
В.В. Проглядо
Keyword(s):  
Magnetic Properties ◽  
High Resolution ◽  
Exchange Interaction ◽  
Magnetic Moments ◽  
Ferromagnetic Material ◽  
Structural Studies ◽  
X Ray ◽  
Structural And Magnetic Properties ◽  
Metal Layers

Layered nanoheterostructures Fe | MgO | Cr | MgO | Fe is an artificial ferromagnetic material in which the exchange interaction of the magnetic moments of Fe layers through intermediate dielectric and metal layers can lead to magnetic configurations that are not realized in the well-studied Fe | MgO | Fe and Fe | Cr | Fe. In this work, we investigated the correlations of the structural and magnetic properties of layered nanoheterostructures Fe (10 nm) | MgO (1.5 nm) | Cr (t nm) | MgO (1.5 nm) | Fe (7 nm) (thickness t = 0.9 and 1.8 nm). Structural studies performed using X-ray diffractometry and high-resolution reflectometry confirmed the formation of a textured structure and revealed its well-defined layered character with sharp interlayer boundaries.

scholarly journals Minor structural protein VP2 in rabbit hemorrhagic disease virus downregulates the expression of the viral capsid protein VP60

2009 ◽  
Vol 90 (12) ◽  
pp. 2952-2955 ◽  
Author(s):  
Liu Chen ◽  
Guangqing Liu ◽  
Zheng Ni ◽  
Bin Yu ◽  
Tao Yun ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Disease Virus ◽  
Luciferase Assay ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Hemorrhagic Disease ◽  
Mrna Levels ◽  
Structural Protein ◽  
Rabbit Hemorrhagic Disease ◽  
Minor Structural Protein ◽  
Capsid Protein Vp60

Rabbit hemorrhagic disease virus (RHDV) has two structural proteins: the major capsid protein VP60 and the minor capsid protein VP2. VP2 is speculated to play an important role in the virus life cycle. To investigate the effect of VP2 on VP60 expression, three types of experiment (baculovirus–insect cell system, mammalian–luciferase assay system and in vitro coupled transcription/translation system) were used to express VP60 alone or co-expressed with VP2. Both forms of VP60 were able to form virus-like particles in insect cells. Western blot analysis and dual-luciferase assays demonstrated that the presence of VP2 results in downregulation of the expression of VP60 in vivo. Real-time RT-PCR of mRNA levels showed that downregulation of VP60 occurs at the transcriptional level. The ability of the viral minor structural protein VP2 to regulate capsid protein levels may contribute to effective virus infection.

scholarly journals In situ proteolysis of an N-terminal His tag with thrombin improves the diffraction quality of human aldo-keto reductase 1C3 crystals

2018 ◽  
Vol 74 (5) ◽  
pp. 300-306 ◽  
Author(s):  
Jovana J. Plavša ◽  
Pavlína Řezáčová ◽  
Michael Kugler ◽  
Petr Pachl ◽  
Jiří Brynda ◽  
...  
Keyword(s):  
High Resolution ◽  
Enzyme Inhibitor ◽  
Structural Studies ◽  
Potential Therapeutic Target ◽  
Thrombin Cleavage ◽  
His Tag ◽  
Structure Solution ◽  
Biotransformation Of Xenobiotics

Human aldo-keto reductase 1C3 (AKR1C3) stereospecifically reduces steroids and prostaglandins and is involved in the biotransformation of xenobiotics. Its role in various cancers makes it a potential therapeutic target for the development of inhibitors. Recombinant AKR1C3 with a thrombin-cleavable N-terminal His6 tag was expressed from a pET-28(+) vector for structural studies of enzyme–inhibitor complexes. A modified in situ proteolysis approach was applied to specifically remove the His tag by thrombin cleavage during crystallization screening trials. This improved the morphology and diffraction quality of the crystals and allowed the acquisition of high-resolution diffraction data and structure solution. This approach may be generally applicable to other proteins expressed using the pET-28(+) vector.

scholarly journals Detergent-free extraction, reconstitution and characterization of membrane-anchored cytochrome-b5 in native lipids

2020 ◽  
Vol 56 (48) ◽  
pp. 6511-6514 ◽  
Author(s):  
Bankala Krishnarjuna ◽  
Thirupathi Ravula ◽  
Ayyalusamy Ramamoorthy
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Cytochrome B5 ◽  
Target Protein ◽  
Structural Studies ◽  
Net Charge

Directly extracted membrane proteins are stable and suitable for functional and high-resolution structural studies; however, the efficacy is modulated by the net charge of the target protein and the polymer used at a given pH.

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