Research

17 pages, 2276 KiB

Open AccessArticle

Production and Characterisation of Stabilised PV-3 Virus-like Particles Using Pichia pastoris

by Lee Sherry, Keith Grehan, Jessica J. Swanson, Mohammad W. Bahar, Claudine Porta, Elizabeth E. Fry, David I. Stuart, David J. Rowlands and Nicola J. Stonehouse

Viruses 2022, 14(10), 2159; https://0-doi-org.brum.beds.ac.uk/10.3390/v14102159 - 30 Sep 2022

Cited by 5 | Viewed by 2423

Abstract

Following the success of global vaccination programmes using the live-attenuated oral and inactivated poliovirus vaccines (OPV and IPV), wild poliovirus (PV) is now only endemic in Afghanistan and Pakistan. However, the continued use of these vaccines poses potential risks to the eradication of [...] Read more.

Following the success of global vaccination programmes using the live-attenuated oral and inactivated poliovirus vaccines (OPV and IPV), wild poliovirus (PV) is now only endemic in Afghanistan and Pakistan. However, the continued use of these vaccines poses potential risks to the eradication of PV. The production of recombinant PV virus-like particles (VLPs), which lack the viral genome offer great potential as next-generation vaccines for the post-polio world. We have previously reported production of PV VLPs using Pichia pastoris, however, these VLPs were in the non-native conformation (C Ag), which would not produce effective protection against PV. Here, we build on this work and show that it is possible to produce wt PV-3 and thermally stabilised PV-3 (referred to as PV-3 SC8) VLPs in the native conformation (D Ag) using Pichia pastoris. We show that the PV-3 SC8 VLPs provide a much-improved D:C antigen ratio as compared to wt PV-3, whilst exhibiting greater thermostability than the current IPV vaccine. Finally, we determine the cryo-EM structure of the yeast-derived PV-3 SC8 VLPs and compare this to previously published PV-3 D Ag structures, highlighting the similarities between these recombinantly expressed VLPs and the infectious virus, further emphasising their potential as a next-generation vaccine candidate for PV Full article

(This article belongs to the Special Issue Rosalind Franklin's 100th Birthday)

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17 pages, 7053 KiB

Open AccessArticle

Molecular Organisation of Tick-Borne Encephalitis Virus

by Lauri I. A. Pulkkinen, Sarah V. Barrass, Aušra Domanska, Anna K. Överby, Maria Anastasina and Sarah J. Butcher

Viruses 2022, 14(4), 792; https://0-doi-org.brum.beds.ac.uk/10.3390/v14040792 - 11 Apr 2022

Cited by 22 | Viewed by 5844

Abstract

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species, with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a [...] Read more.

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species, with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a 3.3 Å cryo-EM structure of the TBEV virion of the Kuutsalo-14 isolate, confirming the overall organisation of the virus. We observe conformational switching of the peripheral and transmembrane helices of M protein, which can explain the quasi-equivalent packing of the viral proteins and highlights their importance in stabilising membrane protein arrangement in the virion. The residues responsible for M protein interactions are highly conserved in TBEV but not in the structurally studied Hypr strain, nor in mosquito-borne flaviviruses. These interactions may compensate for the lower number of hydrogen bonds between E proteins in TBEV compared to the mosquito-borne flaviviruses. The structure reveals two lipids bound in the E protein which are important for virus assembly. The lipid pockets are comparable to those recently described in mosquito-borne Zika, Spondweni, Dengue, and Usutu viruses. Our results thus advance the understanding of tick-borne flavivirus architecture and virion-stabilising interactions. Full article

(This article belongs to the Special Issue Rosalind Franklin's 100th Birthday)

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16 pages, 26760 KiB

Open AccessArticle

Cryo-EM Structures of Two Bacteriophage Portal Proteins Provide Insights for Antimicrobial Phage Engineering

by Abid Javed, Hugo Villanueva, Shadikejiang Shataer, Sara Vasciaveo, Renos Savva and Elena V. Orlova

Viruses 2021, 13(12), 2532; https://0-doi-org.brum.beds.ac.uk/10.3390/v13122532 - 16 Dec 2021

Cited by 1 | Viewed by 2863

Abstract

Widespread antibiotic resistance has returned attention to bacteriophages as a means of managing bacterial pathogenesis. Synthetic biology approaches to engineer phages have demonstrated genomic editing to broaden natural host ranges, or to optimise microbicidal action. Gram positive pathogens cause serious pastoral animal and [...] Read more.

Widespread antibiotic resistance has returned attention to bacteriophages as a means of managing bacterial pathogenesis. Synthetic biology approaches to engineer phages have demonstrated genomic editing to broaden natural host ranges, or to optimise microbicidal action. Gram positive pathogens cause serious pastoral animal and human infections that are especially lethal in newborns. Such pathogens are targeted by the obligate lytic phages of the Salasmaviridae and Guelinviridae families. These phages have relatively small ~20 kb linear protein-capped genomes and their compact organisation, relatively few structural elements, and broad host range, are appealing from a phage-engineering standpoint. In this study, we focus on portal proteins, which are core elements for the assembly of such tailed phages. The structures of dodecameric portal complexes from Salasmaviridae phage GA1, which targets Bacillus pumilus, and Guelinviridae phage phiCPV4 that infects Clostridium perfringens, were determined at resolutions of 3.3 Å and 2.9 Å, respectively. Both are found to closely resemble the related phi29 portal protein fold. However, the portal protein of phiCPV4 exhibits interesting differences in the clip domain. These structures provide new insights on structural diversity in Caudovirales portal proteins and will be essential for considerations in phage structural engineering. Full article

(This article belongs to the Special Issue Rosalind Franklin's 100th Birthday)

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15 pages, 3576 KiB

Open AccessArticle

Binding of a Pocket Factor to Hepatitis B Virus Capsids Changes the Rotamer Conformation of Phenylalanine 97

by Cihan Makbul, Christian Kraft, Matthias Grießmann, Tim Rasmussen, Kilian Katzenberger, Melina Lappe, Paul Pfarr, Cato Stoffer, Mara Stöhr, Anna-Maria Wandinger and Bettina Böttcher

Viruses 2021, 13(11), 2115; https://0-doi-org.brum.beds.ac.uk/10.3390/v13112115 - 20 Oct 2021

Cited by 4 | Viewed by 2127

Abstract

(1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a [...] Read more.

(1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses. Full article

(This article belongs to the Special Issue Rosalind Franklin's 100th Birthday)

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16 pages, 5445 KiB

Open AccessArticle

Cryo EM Analysis Reveals Inherent Flexibility of Authentic Murine Papillomavirus Capsids

by Samantha R. Hartmann, Daniel J. Goetschius, Jiafen Hu, Joshua J. Graff, Carol M. Bator, Neil D. Christensen and Susan L. Hafenstein

Viruses 2021, 13(10), 2023; https://0-doi-org.brum.beds.ac.uk/10.3390/v13102023 - 07 Oct 2021

Cited by 2 | Viewed by 2531

Abstract

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. However, studies have been hampered due to restricted tropism that makes production and purification of high titer virus problematic. This issue has been overcome by developing alternative HPV production [...] Read more.

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. However, studies have been hampered due to restricted tropism that makes production and purification of high titer virus problematic. This issue has been overcome by developing alternative HPV production methods such as virus-like particles (VLPs), which are devoid of a native viral genome. Structural studies have been limited in resolution due to the heterogeneity, fragility, and stability of the VLP capsids. The mouse papillomavirus (MmuPV1) presented here has provided the opportunity to study a native papillomavirus in the context of a common laboratory animal. Using cryo EM to solve the structure of MmuPV1, we achieved 3.3 Å resolution with a local symmetry refinement method that defined smaller, symmetry related subparticles. The resulting high-resolution structure allowed us to build the MmuPV1 asymmetric unit for the first time and identify putative L2 density. We also used our program ISECC to quantify capsid flexibility, which revealed that capsomers move as rigid bodies connected by flexible linkers. The MmuPV1 flexibility was comparable to that of a HPV VLP previously characterized. The resulting MmuPV1 structure is a promising step forward in the study of papillomavirus and will provide a framework for continuing biochemical, genetic, and biophysical research for papillomaviruses. Full article

(This article belongs to the Special Issue Rosalind Franklin's 100th Birthday)

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Review

Jump to: Research, Other

14 pages, 3637 KiB

Open AccessReview

Structural Biology of Nanobodies against the Spike Protein of SARS-CoV-2

by Qilong Tang, Raymond J. Owens and James H. Naismith

Viruses 2021, 13(11), 2214; https://0-doi-org.brum.beds.ac.uk/10.3390/v13112214 - 03 Nov 2021

Cited by 12 | Viewed by 3483

Abstract

Nanobodies are 130 amino acid single-domain antibodies (V_HH) derived from the unique heavy-chain-only subclass of Camelid immunogloblins. Their small molecular size, facile expression, high affinity and stability have combined to make them unique targeting reagents with numerous applications in the biomedical [...] Read more.

Nanobodies are 130 amino acid single-domain antibodies (V_HH) derived from the unique heavy-chain-only subclass of Camelid immunogloblins. Their small molecular size, facile expression, high affinity and stability have combined to make them unique targeting reagents with numerous applications in the biomedical sciences. The first nanobody agent has now entered the clinic as a treatment against a blood disorder. The spread of the SARS-CoV-2 virus has seen the global scientific endeavour work to accelerate the development of technologies to try to defeat a pandemic that has now killed over four million people. In a remarkably short period of time, multiple studies have reported nanobodies directed against the viral Spike protein. Several agents have been tested in culture and demonstrate potent neutralisation of the virus or pseudovirus. A few agents have completed animal trials with very encouraging results showing their potential for treating infection. Here, we discuss the structural features that guide the nanobody recognition of the receptor binding domain of the Spike protein of SARS-CoV-2. Full article

(This article belongs to the Special Issue Rosalind Franklin's 100th Birthday)

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20 pages, 4997 KiB

Open AccessReview

How Antibodies Recognize Pathogenic Viruses: Structural Correlates of Antibody Neutralization of HIV-1, SARS-CoV-2, and Zika

by Morgan E. Abernathy, Kim-Marie A. Dam, Shannon R. Esswein, Claudia A. Jette and Pamela J. Bjorkman

Viruses 2021, 13(10), 2106; https://0-doi-org.brum.beds.ac.uk/10.3390/v13102106 - 19 Oct 2021

Cited by 7 | Viewed by 5358

Abstract

The H1N1 pandemic of 2009-2010, MERS epidemic of 2012, Ebola epidemics of 2013-2016 and 2018-2020, Zika epidemic of 2015-2016, and COVID-19 pandemic of 2019-2021, are recent examples in the long history of epidemics that demonstrate the enormous global impact of viral infection. The [...] Read more.

The H1N1 pandemic of 2009-2010, MERS epidemic of 2012, Ebola epidemics of 2013-2016 and 2018-2020, Zika epidemic of 2015-2016, and COVID-19 pandemic of 2019-2021, are recent examples in the long history of epidemics that demonstrate the enormous global impact of viral infection. The rapid development of safe and effective vaccines and therapeutics has proven vital to reducing morbidity and mortality from newly emerging viruses. Structural biology methods can be used to determine how antibodies elicited during infection or vaccination target viral proteins and identify viral epitopes that correlate with potent neutralization. Here we review how structural and molecular biology approaches have contributed to our understanding of antibody recognition of pathogenic viruses, specifically HIV-1, SARS-CoV-2, and Zika. Determining structural correlates of neutralization of viruses has guided the design of vaccines, monoclonal antibodies, and small molecule inhibitors in response to the global threat of viral epidemics. Full article

(This article belongs to the Special Issue Rosalind Franklin's 100th Birthday)

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Other

Jump to: Research, Review

8 pages, 24653 KiB

Open AccessBrief Report

Structural Basis for the Immunogenicity of the C-Terminus of VP1 of Echovirus 3 Revealed by the Binding of a Neutralizing Antibody

by Shuai Qi, Wangjun Fu, Jinyan Fan, Li Zhang, Binyang Zheng, Kang Wang, Xiangxi Wang, Ling Zhu, Xinjian Li and Yuxia Zhang

Viruses 2022, 14(11), 2322; https://0-doi-org.brum.beds.ac.uk/10.3390/v14112322 - 22 Oct 2022

Viewed by 1345

Abstract

Echovirus 3 (E3), a serotype of human enterovirus B (HEV-B), causes severe diseases in infants. Here, we determined the structures of E3 with a monoclonal antibody (MAb) 6D10 by cryo-EM to comprehensively understand the specificities and the immunological characteristic of this serotype. The [...] Read more.

Echovirus 3 (E3), a serotype of human enterovirus B (HEV-B), causes severe diseases in infants. Here, we determined the structures of E3 with a monoclonal antibody (MAb) 6D10 by cryo-EM to comprehensively understand the specificities and the immunological characteristic of this serotype. The solved cryo-EM structures of the F-, A-, and E-particles of E3 bound with 6D10 revealed the structural features of the virus–antibody interface. Importantly, the structures of E-particles bound with 6D10 revealed for the first time the nature of the C-terminus of VP1 for HEV-Bs at the structural level. The highly immunogenic nature of this region in the E-particles provides new strategies for vaccine development for HEV-Bs. Full article

(This article belongs to the Special Issue Rosalind Franklin's 100th Birthday)

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