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

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.

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

mBio ◽

10.1128/mbio.00467-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 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.

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

Proceedings ◽

10.3390/proceedings2020050147 ◽

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.

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Yellow fever virus strains Asibi and 17D-204 infect human umbilical cord endothelial cells and induce novel changes in gene expression

Virology ◽

10.1016/j.virol.2005.07.035 ◽

2005 ◽

Vol 342 (2) ◽

pp. 167-176 ◽

Cited By ~ 37

Author(s):

Svetlana F. Khaiboullina ◽

Albert A. Rizvanov ◽

Michael R. Holbrook ◽

Stephen St. Jeor

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Umbilical Cord ◽

Yellow Fever ◽

Yellow Fever Virus ◽

Fever Virus ◽

Human Umbilical Cord ◽

Virus Strains

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

mBio ◽

10.1128/mbio.01956-15 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 32

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.

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Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

Journal of Virology ◽

10.1128/jvi.03077-15 ◽

2016 ◽

Vol 90 (6) ◽

pp. 3212-3228 ◽

Cited By ~ 17

Author(s):

Leonia Bozzacco ◽

Zhigang Yi ◽

Ursula Andreo ◽

Claire R. Conklin ◽

Melody M. H. Li ◽

...

Keyword(s):

Protein Folding ◽

Yellow Fever ◽

Virus Replication ◽

Yellow Fever Virus ◽

Rna Replication ◽

Fever Virus ◽

Viral Rna ◽

Wild Type ◽

Polyprotein Processing ◽

Wide Range

ABSTRACTDNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication.IMPORTANCEFlaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development.

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Fine Mapping of a cis-Acting Sequence Element in Yellow Fever Virus RNA That Is Required for RNA Replication and Cyclization

Journal of Virology ◽

10.1128/jvi.77.3.2265-2270.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 2265-2270 ◽

Cited By ~ 92

Author(s):

Jeroen Corver ◽

Edith Lenches ◽

Kayla Smith ◽

R. Aaron Robison ◽

Trisha Sando ◽

...

Keyword(s):

Amino Acids ◽

Capsid Protein ◽

Yellow Fever ◽

Fine Mapping ◽

Yellow Fever Virus ◽

Rna Replication ◽

Fever Virus ◽

Sequence Element ◽

Complementary Sequence ◽

Virus Rna

ABSTRACT We present fine mapping of a cis-acting nucleotide sequence found in the 5′ region of yellow fever virus genomic RNA that is required for RNA replication. There is evidence that this sequence interacts with a complementary sequence in the 3′ region of the genome to cyclize the RNA. Replicons were constructed that had various deletions in the 5′ region encoding the capsid protein and were tested for their ability to replicate. We found that a sequence of 18 nucleotides (residues 146 to 163 of the yellow fever virus genome, which encode amino acids 9 to 14 of the capsid protein) is essential for replication of the yellow fever virus replicon and that a slightly longer sequence of 21 nucleotides (residues 146 to 166, encoding amino acids 9 to 15) is required for full replication. This region is larger than the core sequence of 8 nucleotides conserved among all mosquito-borne flaviviruses and contains instead the entire sequence previously proposed to be involved in cyclization of yellow fever virus RNA.

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A histone-like motif in yellow fever virus contributes to viral replication

10.1101/2020.05.05.078782 ◽

2020 ◽

Author(s):

Diego Mourão ◽

Shoudeng Chen ◽

Uwe Schaefer ◽

Leonia Bozzacco ◽

Leticia A. Carneiro ◽

...

Keyword(s):

Gene Expression ◽

Yellow Fever ◽

Yellow Fever Virus ◽

Rna Virus ◽

Fever Virus ◽

Host Protein ◽

Histone H4 ◽

Host Proteins ◽

Control Of Gene Expression ◽

Lysine Residues

The mimicry of host proteins by viruses contributes to their ability to suppress antiviral immunity and hijack host biosynthetic machinery1. Host adaptation to evade this exploitation depends on host protein functional redundancy2. Non-redundant, essential host proteins have limited potential to adapt without severe consequences3. Histones, which are essential for genome architecture and control of gene expression, are among the most evolutionary conserved proteins4. Here we show that the capsid protein of the flavivirus yellow fever virus (YFV), mimics histone H4 and interferes with chromatin gene regulation by BRD4, a bromodomain and extraterminal domain (BET) protein. Two acetyl-lysine residues of YFV capsid are embedded in a histone-like motif that interacts with the BRD4 bromodomain, affecting gene expression and influencing YFV replication. These findings reveal histone mimicry as a strategy employed by an RNA virus that replicates in the cytosol5 and define convergent and distinct molecular determinants for motif recognition of the viral mimic versus histone H4.

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