Establishment of Stably Transfected Cells Constitutively Expressing the Full-Length and Truncated Antigenic Proteins of Two Genetically Distinct Mink Astroviruses

Bidokhti, Mehdi R. M.; Ullman, Karin; Jensen, Trine Hammer; Chriél, Mariann; Mottahedin, Amin; Munir, Muhammad; Andersson, Anna Maria; Detournay, Olivier; Hammer, Anne Sofie; Baule, Claudia

doi:10.1371/journal.pone.0082978

Cited by 6 publications

(15 citation statements)

References 53 publications

(76 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Divergent strains of HAstV have been associated with encephalitis ( 13 – 15 ). The Astroviridae family also includes many nonhuman astroviruses (AstVs) that cause infections in mammals and birds, causing a range of symptoms, including gastroenteritis, fatal hepatitis, and neurological disease ( 16 , 17 ).…”

Section: Introductionmentioning

confidence: 99%

Structural, Mechanistic, and Antigenic Characterization of the Human Astrovirus Capsid

York

Yousefi

Bogdanoff

et al. 2016

J Virol

View full text Add to dashboard Cite

Human astroviruses (HAstVs) are nonenveloped, positive-sense, single-stranded RNA viruses that are a leading cause of viral gastroenteritis. HAstV particles display T=3 icosahedral symmetry formed by 180 copies of the capsid protein (CP), which undergoes proteolytic maturation to generate infectious HAstV particles. Little is known about the molecular features that govern HAstV particle assembly, maturation, infectivity, and immunogenicity. Here we report the crystal structures of the two main structural domains of the HAstV CP: the core domain at 2.60-Å resolution and the spike domain at 0.95-Å resolution. Fitting of these structures into the previously determined 25-Å-resolution electron cryomicroscopy density maps of HAstV allowed us to characterize the molecular features on the surfaces of immature and mature T=3 HAstV particles. The highly electropositive inner surface of HAstV supports a model in which interaction of the HAstV CP core with viral RNA is a driving force in T=3 HAstV particle formation. Additionally, mapping of conserved residues onto the HAstV CP core and spike domains in the context of the immature and mature HAstV particles revealed dramatic changes to the exposure of conserved residues during virus maturation. Indeed, we show that antibodies raised against mature HAstV have reactivity to both the HAstV CP core and spike domains, revealing for the first time that the CP core domain is antigenic. Together, these data provide new molecular insights into HAstV that have practical applications for the development of vaccines and antiviral therapies.IMPORTANCE Astroviruses are a leading cause of viral diarrhea in young children, immunocompromised individuals, and the elderly. Despite the prevalence of astroviruses, little is known at the molecular level about how the astrovirus particle assembles and is converted into an infectious, mature virus. In this paper, we describe the high-resolution structures of the two main astrovirus capsid proteins. Fitting these structures into previously determined low-resolution maps of astrovirus allowed us to characterize the molecular surfaces of immature and mature astroviruses. Our studies provide the first evidence that astroviruses undergo viral RNA-dependent assembly. We also provide new insight into the molecular mechanisms that lead to astrovirus maturation and infectivity. Finally, we show that both capsid proteins contribute to the adaptive immune response against astrovirus. Together, these studies will help to guide the development of vaccines and antiviral drugs targeting astrovirus.

show abstract

Section: Introductionmentioning

confidence: 99%

Structural, Mechanistic, and Antigenic Characterization of the Human Astrovirus Capsid

York

Yousefi

Bogdanoff

et al. 2016

J Virol

View full text Add to dashboard Cite

show abstract

“…For this, stable clonal host cells would also be an alternative, as it has been shown for other viruses. 38. , 39.…”

Section: Discussionmentioning

confidence: 99%

Investigating Tick-borne Flaviviral-like Particles as a Delivery System for Gene Therapy

Neddermeyer

Hultenby

Paidikondala

et al. 2018

Current Therapeutic Research

View full text Add to dashboard Cite

BackgroundResearch on the biogenesis of tick-borne encephalitis virus (TBEV) would benefit gene therapy. Due to specific arrangements of genes along the TBEV genome, its viral-like particles (VLPs) could be exploited as shuttles to deliver their replicon, which carries therapeutic genes, to immune system cells.ObjectiveTo develop a flaviviral vector for gene delivery as a part of gene therapy research that can be expressed in secretable VLP suicidal shuttles and provide abundant unique molecular and structural data supporting this gene therapy concept.MethodTBEV structural gene constructs of a Swedish Torö strain were cloned into plasmids driven by the promoters CAG and CMV and then transfected into various cell lines, including COS-1 and BHK-21. Time-course sampling of the cells, culture fluid, cell lysate supernatant, and pellet specimens were performed. Western blotting and electron microscopy analyses of collected specimens were used to investigate molecular and structural processing of TBEV structural proteins.ResultsWestern blotting analysis showed differences between promoters in directing the gene expression of the VLPs constructs. The premature flaviviral polypeptides as well as mature VLPs could be traced. Using electron microscopy, the premature and mature VLP accumulation in cellular compartments—and also endoplasmic reticulum proliferation as a virus factory platform—were observed in addition to secreted VLPs.ConclusionsThe abundant virologic and cellular findings in this study show the natural processing and safety of inserting flaviviral structural genes into suicidal VLP shuttles. Thus, we propose that these VLPs are a suitable gene delivering system model in gene therapy.

show abstract

“…The construction and expression of the full-length and truncated MAstV CP of strain DK5790 using pDual-GC vector (Agilent Technologies, Santa Clara, CA, USA) have been described [36]. CP refers to the full-length capsid protein (spanning amino acids (aa) 1–775 of CP); CPΔN refers to an N-terminal truncated protein (spanning aa 161–775 of CP); CPΔC is a C-terminal truncated protein (spanning aa 1–621 of CP).…”

Section: Methodsmentioning

confidence: 99%

“…CP refers to the full-length capsid protein (spanning amino acids (aa) 1–775 of CP); CPΔN refers to an N-terminal truncated protein (spanning aa 161–775 of CP); CPΔC is a C-terminal truncated protein (spanning aa 1–621 of CP). The MAstV CPs were expressed in stably transfected fetal mink cells and were purified by affinity in a nickel resin, as described in details in our previous study [36].…”

Section: Methodsmentioning

confidence: 99%

“…The HAstV CP that contains the immunogenic domains has been expressed mostly for functional studies [16,34,35]. Our previous work with a full-length CP of MAstV expressed in bacteria has shown partial protection against the manifestation of clinical symptoms of pre-weaning diarrhea syndrome in mink [36]. In an immunization trial with a full-length CP of the chicken AstV expressed in baculovirus, partial protection was also reported [37].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Immunogenicity and Efficacy Evaluation of Subunit Astrovirus Vaccines

et al. 2019

Self Cite

View full text Add to dashboard Cite

A full understanding of the immune response to astrovirus (AstV) infection is required to treat and control AstV-induced gastroenteritis. Relative contributions of each arm of the immune system in restricting AstV infection remain unknown. In this study, two novel subunit AstV vaccines derived from capsid protein (CP) of mink AstV (MAstV) such as CPΔN (spanning amino acids 161–775) and CPΔC (spanning amino acids 1–621) were evaluated. Their immunogenicity and cytokine production in mice, as well as protective efficacy in mink litters via maternal immunization, were studied. Truncated CPs induced higher levels of serum anti-CP antibodies than CP, with the highest level for CPΔN. No seronegativity was detected after booster immunization with either AstV CP truncates in both mice and mink. All mink moms stayed seropositive during the entire 104-day study. Furthermore, lymphoproliferation responses and Th1/Th2 cytokine induction of mice splenocytes ex vivo re-stimulated by truncated CPs were significantly higher than those by CP, with the highest level for CPΔN. Immunization of mink moms with truncated CPs could suppress virus shedding and clinical signs in their litters during a 51-day study after challenge with a heterogeneous MAstV strain. Collectively, AstV truncated CPs exhibit better parameters for protection than full-length CP.

show abstract

Establishment of Stably Transfected Cells Constitutively Expressing the Full-Length and Truncated Antigenic Proteins of Two Genetically Distinct Mink Astroviruses

Cited by 6 publications

References 53 publications

Structural, Mechanistic, and Antigenic Characterization of the Human Astrovirus Capsid

Structural, Mechanistic, and Antigenic Characterization of the Human Astrovirus Capsid

Investigating Tick-borne Flaviviral-like Particles as a Delivery System for Gene Therapy

Immunogenicity and Efficacy Evaluation of Subunit Astrovirus Vaccines

Contact Info

Product

Resources

About