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

2003 ◽  
Vol 77 (3) ◽  
pp. 2265-2270 ◽  
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.

scholarly journals Mutations in the Yellow Fever Virus Nonstructural Protein NS2A Selectively Block Production of Infectious Particles

2002 ◽  
Vol 76 (10) ◽  
pp. 4773-4784 ◽  
Author(s):  
Beate M. Kümmerer ◽  
Charles M. Rice
Keyword(s):  
Amino Acids ◽  
Yellow Fever ◽  
Cleavage Site ◽  
Infectious Virus ◽  
Yellow Fever Virus ◽  
Nonstructural Protein ◽  
Virus Production ◽  
Fever Virus ◽  
Helicase Domain ◽  
Infected Cells

ABSTRACT Little is known about the function of flavivirus nonstructural protein NS2A. Two forms of NS2A are found in yellow fever virus-infected cells. Full-length NS2A (224 amino acids) is the product of cleavage at the NS1/2A and NS2A/2B sites. NS2Aα, a C-terminally truncated form of 190 amino acids, results from partial cleavage by the viral NS2B-3 serine protease at the sequence QK↓T within NS2A. Exchange of serine for lysine at this site (QKT→QST) blocks the production of both NS2Aα and infectious virus. The present study reveals that this defect is not at the level of RNA replication. Despite normal structural region processing, infectious particles containing genome RNA and capsid protein were not released from cells transfected with the mutant RNA. Nevertheless, production of subviral prM/M- and E-containing particles was unimpaired. The NS2A defect could be complemented in trans by providing NS1-2A or NS1-2Aα. However, trans complementation was not observed when the C-terminal lysine of NS1-2Aα was replaced with serine. In addition to true reversions, NS2Aα cleavage site mutations could be suppressed by two classes of second-site changes. The first class consisted of insertions at the NS2Aα cleavage site that restored its basic character and cleavability. A second class of suppressors occurred in the NS3 helicase domain, in which NS3 aspartate 343 was replaced with an uncharged residue (either valine, alanine, or glycine). These mutations in NS3 restored infectious-virus production in the absence of cleavage at the mutant NS2Aα site. Taken together, our results reveal an unexpected role for NS2A and NS3 in the assembly and/or release of infectious flavivirus particles.

scholarly journals High Fidelity of Yellow Fever Virus RNA Polymerase

2004 ◽  
Vol 78 (2) ◽  
pp. 1032-1038 ◽  
Author(s):  
Konstantin V. Pugachev ◽  
Farshad Guirakhoo ◽  
Simeon W. Ocran ◽  
Fred Mitchell ◽  
Megan Parsons ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
Yellow Fever ◽  
Error Rate ◽  
Yellow Fever Virus ◽  
Protein A ◽  
Fever Virus ◽  
Genome Replication ◽  
Virus Rna

ABSTRACT Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 × 10−4 per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 × 10−7 to 2.3 × 10−7. Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.

scholarly journals Wild-Type Yellow Fever Virus RNA in Cerebrospinal Fluid of Child

2019 ◽  
Vol 25 (8) ◽  
pp. 1567-1570 ◽  
Author(s):  
Paula E.S. Marinho ◽  
Pedro P.M. Alvarenga ◽  
Ana P.C. Crispim ◽  
Talitah M.S. Candiani ◽  
Alice M. Alvarenga ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Wild Type ◽  
Virus Rna

scholarly journals Isolation of yellow fever virus (YFV) from naturally infectied Haemagogus (Conopostegus) leucocelaenus (diptera, cukicudae) in São Paulo State, Brazil, 2009

2011 ◽  
Vol 53 (3) ◽  
pp. 133-139 ◽  
Author(s):  
Renato Pereira de Souza ◽  
Selma Petrella ◽  
Terezinha Lisieux Moraes Coimbra ◽  
Adriana Yurika Maeda ◽  
Iray Maria Rocco ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Cultured Cells ◽  
Yellow Fever Virus ◽  
Sao Paulo ◽  
Fever Virus ◽  
São Paulo ◽  
Paulo State ◽  
Virus Rna ◽  
São Paulo State ◽  
Entomological Research

After detecting the death of Howlers monkeys (genus Alouatta) and isolation of yellow fever virus (YFV) in Buri county, São Paulo, Brazil, an entomological research study in the field was started. A YFV strain was isolated from newborn Swiss mice and cultured cells of Aedes albopictus - C6/36, from a pool of six Haemagogus (Conopostegus) leucocelaenus (Hg. leucocelaenus) mosquitoes (Dyar & Shannon) collected at the study site. Virus RNA fragment was amplified by RT-PCR and sequenced. The MCC Tree generated showed that the isolated strain is related to the South American I genotype, in a monophyletic clade containing isolates from recent 2008-2010 epidemics and epizootics in Brazil. Statistical analysis commonly used were calculated to characterize the sample in relation to diversity and dominance and indicated a pattern of dominance of one or a few species. Hg. leucocelaenus was found infected in Rio Grande do Sul State as well. In São Paulo State, this is the first detection of YFV in Hg. leucocelaenus.

scholarly journals Functional Requirements of the Yellow Fever Virus Capsid Protein

2007 ◽  
Vol 81 (24) ◽  
pp. 13944-13944 ◽  
Author(s):  
Chinmay G. Patkar ◽  
Christopher T. Jones ◽  
Yu-hsuan Chang ◽  
Ranjit Warrier ◽  
Richard J. Kuhn
Keyword(s):  
Capsid Protein ◽  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Functional Requirements ◽  
Virus Capsid ◽  
Virus Capsid Protein

scholarly journals Functional Requirements of the Yellow Fever Virus Capsid Protein

2007 ◽  
Vol 81 (12) ◽  
pp. 6471-6481 ◽  
Author(s):  
Chinmay G. Patkar ◽  
Christopher T. Jones ◽  
Yu-hsuan Chang ◽  
Ranjit Warrier ◽  
Richard J. Kuhn
Keyword(s):  
Capsid Protein ◽  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Virus Assembly ◽  
Fever Virus ◽  
Site Directed Mutagenesis ◽  
Packaging System ◽  
Virus Capsid ◽  
Double Mutation ◽  
Virus Capsid Protein

ABSTRACT Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning.

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 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.

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