scholarly journals A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Harish N. Ramanathan ◽  
Shuo Zhang ◽  
Florian Douam ◽  
Katrina B. Mar ◽  
Jinhong Chang ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Mammalian Cells ◽  
Yellow Fever Virus ◽  
Virus Entry ◽  
Critical Role ◽  
Cellular Protein ◽  
Fever Virus ◽  
Mosquito Cells ◽  
Rna Transfection ◽  
Endosomal Acidification

ABSTRACT While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry. IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

scholarly journals A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

2019 ◽  
Author(s):  
Harish N. Ramanathan ◽  
Shuo Zhang ◽  
Florian Douam ◽  
Jinhong Chang ◽  
Priscilla L. Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Virus Entry ◽  
Critical Role ◽  
Rna Replication ◽  
Cellular Protein ◽  
Fever Virus ◽  
Rna Transfection ◽  
Endosomal Acidification

ABSTRACTWhile the basic mechanisms of flavivirus entry and fusion are understood, little is known about the post-fusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E expression; however, as expected, gene expression from the defective reporter virus was insensitive to a small molecule inhibitor of YFV RNA replication. YFVΔSK/Nluc gene expression was also shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur, as well as valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large macromolecular complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a post-fusion, pre-translation step in YFV entry. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a post-fusion step in virus entry.IMPORTANCEFlaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here we show that the yellow fever virus nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

scholarly journals A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 147
Author(s):  
Shuo Zhang ◽  
Harish N. Ramanathan ◽  
Florian Douam ◽  
Katrina B. Mar ◽  
Jinhong Chang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Virus Entry ◽  
Critical Role ◽  
Viral Gene Expression ◽  
Rna Replication ◽  
Fever Virus ◽  
Viral Gene ◽  
Positive Strand Rna

Flaviviruses are enveloped, arthropod-borne, positive-strand RNA viruses that cause significant human disease. While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We recently developed a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter to quantitively monitor the translation of incoming virus particle-delivered genomes. We validated that viral gene expression can be neutralized by YFV-specific antisera and requires known pathways of flavivirus entry; however, as expected, gene expression from the defective reporter virus was insensitive to a small molecule inhibitor of YFV RNA replication. The initial round of viral gene expression was also shown to require: (i) cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur, and (ii) valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large macromolecular complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.

scholarly journals A Yellow Fever Virus 17D Infection and Disease Mouse Model Used to Evaluate a Chimeric Binjari-Yellow Fever Virus Vaccine

Vaccines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 368 ◽  
Author(s):  
Kexin Yan ◽  
Laura J. Vet ◽  
Bing Tang ◽  
Jody Hobson-Peters ◽  
Daniel J. Rawle ◽  
...  
Keyword(s):  
Weight Loss ◽  
Mouse Model ◽  
Yellow Fever ◽  
Virus Vaccine ◽  
Neutralizing Antibodies ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Liver Pathology ◽  
Vaccine Production ◽  
Mosquito Cells

Despite the availability of an effective, live attenuated yellow fever virus (YFV) vaccine (YFV 17D), this flavivirus still causes up to ≈60,000 deaths annually. A number of new approaches are seeking to address vaccine supply issues and improve safety for the immunocompromised vaccine recipients. Herein we describe an adult female IFNAR-/- mouse model of YFV 17D infection and disease that recapitulates many features of infection and disease in humans. We used this model to evaluate a new YFV vaccine that is based on a recently described chimeric Binjari virus (BinJV) vaccine technology. BinJV is an insect-specific flavivirus and the chimeric YFV vaccine (BinJ/YFV-prME) was generated by replacing the prME genes of BinJV with the prME genes of YFV 17D. Such BinJV chimeras retain their ability to replicate to high titers in C6/36 mosquito cells (allowing vaccine production), but are unable to replicate in vertebrate cells. Vaccination with adjuvanted BinJ/YFV-prME induced neutralizing antibodies and protected mice against infection, weight loss and liver pathology after YFV 17D challenge.

scholarly journals Vaccine and Wild-Type Strains of Yellow Fever Virus Engage Distinct Entry Mechanisms and Differentially Stimulate Antiviral Immune Responses

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Maria Dolores Fernandez-Garcia ◽  
Laurent Meertens ◽  
Maxime Chazal ◽  
Mohamed Lamine Hafirassou ◽  
Ophélie Dejarnac ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Virus Entry ◽  
Fever Virus ◽  
Host Cells ◽  
Target Cells ◽  
Wild Type ◽  
E Protein ◽  
Virulence Attenuation ◽  
Molecular Determinants

ABSTRACTThe live attenuated yellow fever virus (YFV) vaccine 17D stands as a “gold standard” for a successful vaccine. 17D was developed empirically by passaging the wild-type Asibi strain in mouse and chicken embryo tissues. Despite its immense success, the molecular determinants for virulence attenuation and immunogenicity of the 17D vaccine are poorly understood. 17D evolved several mutations in its genome, most of which lie within the envelope (E) protein. Given the major role played by the YFV E protein during virus entry, it has been hypothesized that the residues that diverge between the Asibi and 17D E proteins may be key determinants of attenuation. In this study, we define the process of YFV entry into target cells and investigate its implication in the activation of the antiviral cytokine response. We found that Asibi infects host cells exclusively via the classical clathrin-mediated endocytosis, while 17D exploits a clathrin-independent pathway for infectious entry. We demonstrate that the mutations in the 17D E protein acquired during the attenuation process are sufficient to explain the differential entry of Asibi versus 17D. Interestingly, we show that 17D binds to and infects host cells more efficiently than Asibi, which culminates in increased delivery of viral RNA into the cytosol and robust activation of the cytokine-mediated antiviral response. Overall, our study reveals that 17D vaccine and Asibi enter target cells through distinct mechanisms and highlights a link between 17D attenuation, virus entry, and immune activation.IMPORTANCEThe yellow fever virus (YFV) vaccine 17D is one of the safest and most effective live virus vaccines ever developed. The molecular determinants for virulence attenuation and immunogenicity of 17D are poorly understood. 17D was generated by serially passaging the virulent Asibi strain in vertebrate tissues. Here we examined the entry mechanisms engaged by YFV Asibi and the 17D vaccine. We found the two viruses use different entry pathways. We show that the mutations differentiating the Asibi envelope (E) protein from the 17D E protein, which arose during attenuation, are key determinants for the use of these distinct entry routes. Finally, we demonstrate that 17D binds and enters host cells more efficiently than Asibi. This results in a higher uptake of viral RNA into the cytoplasm and consequently a greater cytokine-mediated antiviral response. Overall, our data provide new insights into the biology of YFV infection and the mechanisms of viral attenuation.

scholarly journals An RNA Pseudoknot Is Required for Production of Yellow Fever Virus Subgenomic RNA by the Host Nuclease XRN1

2010 ◽  
Vol 84 (21) ◽  
pp. 11395-11406 ◽  
Author(s):  
Patrícia A. G. C. Silva ◽  
Carina F. Pereira ◽  
Tim J. Dalebout ◽  
Willy J. M. Spaan ◽  
Peter J. Bredenbeek
Keyword(s):  
Yellow Fever ◽  
Rna Structure ◽  
Mammalian Cells ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Infected Mosquito ◽  
Molecular Characteristics ◽  
Subgenomic Rna ◽  
Rna Pseudoknot

ABSTRACT Cells and mice infected with arthropod-borne flaviviruses produce a small subgenomic RNA that is colinear with the distal part of the viral 3′-untranslated region (UTR). This small subgenomic flavivirus RNA (sfRNA) results from the incomplete degradation of the viral genome by the host 5′-3′ exonuclease XRN1. Production of the sfRNA is important for the pathogenicity of the virus. This study not only presents a detailed description of the yellow fever virus (YFV) sfRNA but, more importantly, describes for the first time the molecular characteristics of the stalling site for XRN1 in the flavivirus genome. Similar to the case for West Nile virus, the YFV sfRNA was produced by XRN1. However, in contrast to the case for other arthropod-borne flaviviruses, not one but two sfRNAs were detected in YFV-infected mammalian cells. The smaller of these two sfRNAs was not observed in infected mosquito cells. The larger sfRNA could also be produced in vitro by incubation with purified XRN1. These two YFV sfRNAs formed a 5′-nested set. The 5′ ends of the YFV sfRNAs were found to be just upstream of the previously predicted RNA pseudoknot PSK3. RNA structure probing and mutagenesis studies provided strong evidence that this pseudoknot structure was formed and served as the molecular signal to stall XRN1. The sequence involved in PSK3 formation was cloned into the Sinrep5 expression vector and shown to direct the production of an sfRNA-like RNA. These results underscore the importance of the RNA pseudoknot in stalling XRN1 and also demonstrate that it is the sole viral requirement for sfRNA production.

scholarly journals Yellow fever virus outbreak in Brazil under current and future climate

2021 ◽  
Vol 6 ◽  
pp. 664-677
Author(s):  
Tara Sadeghieh ◽  
Jan M. Sargeant ◽  
Amy L. Greer ◽  
Olaf Berke ◽  
Guillaume Dueymes ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Future Climate

scholarly journals Yellow Fever Outbreak in Eastern Senegal, 2020–2021

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1475
Author(s):  
Moussa Moïse Diagne ◽  
Marie Henriette Dior Ndione ◽  
Alioune Gaye ◽  
Mamadou Aliou Barry ◽  
Diawo Diallo ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Mosquito Species ◽  
Virus Transmission ◽  
Genomic Analysis ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
World Health ◽  
Effective Vaccine ◽  
Ministry Of Health

Yellow fever virus remains a major threat in low resource countries in South America and Africa despite the existence of an effective vaccine. In Senegal and particularly in the eastern part of the country, periodic sylvatic circulation has been demonstrated with varying degrees of impact on populations in perpetual renewal. We report an outbreak that occurred from October 2020 to February 2021 in eastern Senegal, notified and managed through the synergistic effort yellow fever national surveillance implemented by the Senegalese Ministry of Health in collaboration with the World Health Organization, the countrywide 4S network set up by the Ministry of Health, the Institut Pasteur de Dakar, and the surveillance of arboviruses and hemorrhagic fever viruses in human and vector populations implemented since mid 2020 in eastern Senegal. Virological analyses highlighted the implication of sylvatic mosquito species in virus transmission. Genomic analysis showed a close relationship between the circulating strain in eastern Senegal, 2020, and another one from the West African lineage previously detected and sequenced two years ago from an unvaccinated Dutch traveler who visited the Gambia and Senegal before developing signs after returning to Europe. Moreover, genome analysis identified a 6-nucleotide deletion in the variable domain of the 3′UTR with potential impact on the biology of the viral strain that merits further investigations. Integrated surveillance of yellow fever virus but also of other arboviruses of public health interest is crucial in an ecosystem such as eastern Senegal.

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