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 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

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 New insights into internal ribosome entry site elements relevant for viral gene expression

2008 ◽  
Vol 89 (3) ◽  
pp. 611-626 ◽  
Author(s):  
Encarnación Martínez-Salas ◽  
Almudena Pacheco ◽  
Paula Serrano ◽  
Noemi Fernandez
Keyword(s):  
Gene Expression ◽  
Rna Binding ◽  
Viral Gene Expression ◽  
Rna Replication ◽  
Host Factors ◽  
Viral Gene ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Positive Strand ◽  
Positive Strand Rna

A distinctive feature of positive-strand RNA viruses is the presence of high-order structural elements at the untranslated regions (UTR) of the genome that are essential for viral RNA replication. The RNA of all members of the family Picornaviridae initiate translation internally, via an internal ribosome entry site (IRES) element present in the 5′ UTR. IRES elements consist of cis-acting RNA structures that usually require specific RNA-binding proteins for translational machinery recruitment. This specialized mechanism of translation initiation is shared with other viral RNAs, e.g. from hepatitis C virus and pestivirus, and represents an alternative to the cap-dependent mechanism. In cells infected with many picornaviruses, proteolysis or changes in phosphorylation of key host factors induces shut off of cellular protein synthesis. This event occurs simultaneously with the synthesis of viral gene products since IRES activity is resistant to the modifications of the host factors. Viral gene expression and RNA replication in positive-strand viruses is further stimulated by viral RNA circularization, involving direct RNA–RNA contacts between the 5′ and 3′ ends as well as RNA-binding protein bridges. In this review, we discuss novel insights into the mechanisms that control picornavirus gene expression and compare them to those operating in other positive-strand RNA viruses.

Epigenetic reprogramming of host and viral genes by Human Cytomegalovirus infection in Kasumi-3 myeloid progenitor cells at early times post-infection

2021 ◽  
Author(s):  
Eleonora Forte ◽  
Fatma Ayaloglu Butun ◽  
Christian Marinaccio ◽  
Matthew J. Schipma ◽  
Andrea Piunti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Post Infection ◽  
De Novo ◽  
Critical Role ◽  
Myeloid Cells ◽  
Viral Gene Expression ◽  
Chromatin Accessibility ◽  
Viral Gene ◽  
Dynamic Changes ◽  
Pol Ii

HCMV establishes latency in myeloid cells. Using the Kasumi-3 latency model, we previously showed that lytic gene expression is activated prior to establishment of latency in these cells. The early events in infection may have a critical role in shaping establishment of latency. Here, we have used an integrative multi-omics approach to investigate dynamic changes in host and HCMV gene expression and epigenomes at early times post infection. Our results show dynamic changes in viral gene expression and viral chromatin. Analyses of Pol II, H3K27Ac and H3K27me3 occupancy of the viral genome showed that 1) Pol II occupancy was highest at the MIEP at 4 hours post infection. However, it was observed throughout the genome; 2) At 24 hours, H3K27Ac was localized to the major immediate early promoter/enhancer and to a possible second enhancer in the origin of replication OriLyt; 3) viral chromatin was broadly accessible at 24 hpi. In addition, although HCMV infection activated expression of some host genes, we observed an overall loss of de novo transcription. This was associated with loss of promoter-proximal Pol II and H3K27Ac, but not with changes in chromatin accessibility or a switch in modification of H3K27. Importance. HCMV is an important human pathogen in immunocompromised hosts and developing fetuses. Current anti-viral therapies are limited by toxicity and emergence of resistant strains. Our studies highlight emerging concepts that challenge current paradigms of regulation of HCMV gene expression in myeloid cells. In addition, our studies show that HCMV has a profound effect on de novo transcription and the cellular epigenome. These results may have implications for mechanisms of viral pathogenesis.

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

Virology ◽  
2005 ◽  
Vol 342 (2) ◽  
pp. 167-176 ◽  
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

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

2016 ◽  
Vol 90 (6) ◽  
pp. 3212-3228 ◽  
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.

scholarly journals Activation of p90 Ribosomal S6 Kinases by ORF45 of Kaposi's Sarcoma-Associated Herpesvirus Is Critical for Optimal Production of Infectious Viruses

2014 ◽  
Vol 89 (1) ◽  
pp. 195-207 ◽  
Author(s):  
Bishi Fu ◽  
Ersheng Kuang ◽  
Wenwei Li ◽  
Denis Avey ◽  
Xiaojuan Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacterial Artificial Chromosome ◽  
Critical Role ◽  
Viral Gene Expression ◽  
Artificial Chromosome ◽  
Lytic Replication ◽  
Viral Gene ◽  
Wild Type ◽  
Lytic Reactivation ◽  
Infected Cells

ABSTRACTWe have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma-associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and extracellular regulated kinase (ERK) (E. Kuang, Q. Tang, G. G. Maul, and F. Zhu, J Virol 82:1838–1850, 2008,http://dx.doi.org/10.1128/JVI.02119-07). We now have identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK and, consequently, its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant, BAC16-45A66F. The reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion also was accompanied by a noticeable decreased in viral gene expression during lytic reactivation. Consequently, the ORF45-F66A mutant produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication.IMPORTANCEKSHV is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment and to facilitate efficient production of progeny viral particles. We previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replication.

scholarly journals HCMV miRNA Targets Reveal Important Cellular Pathways for Viral Replication, Latency, and Reactivation

2018 ◽  
Vol 4 (4) ◽  
pp. 29 ◽  
Author(s):  
Nicole Diggins ◽  
Meaghan Hancock
Keyword(s):  
Gene Expression ◽  
Latent Infection ◽  
Current Knowledge ◽  
Critical Role ◽  
Viral Gene Expression ◽  
Lytic Replication ◽  
Progeny Virus ◽  
Viral Gene ◽  
Cellular Targets ◽  
Differentiated Cells

It is now well appreciated that microRNAs (miRNAs) play a critical role in the lifecycles of many herpes viruses. The human cytomegalovirus (HCMV) replication cycle varies significantly depending on the cell type infected, with lytic replication occurring in fully-differentiated cells such as fibroblasts, endothelial cells, or macrophages, and latent infection occurring in less-differentiated CD14+ monocytes and CD34+ hematopoietic progenitor cells where viral gene expression is severely diminished and progeny virus is not produced. Given their non-immunogenic nature and their capacity to target numerous cellular and viral transcripts, miRNAs represent a particularly advantageous means for HCMV to manipulate viral gene expression and cellular signaling pathways during lytic and latent infection. This review will focus on our current knowledge of HCMV miRNA viral and cellular targets, and discuss their importance in lytic and latent infection, highlight the challenges of studying HCMV miRNAs, and describe how viral miRNAs can help us to better understand the cellular processes involved in HCMV latency.

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