scholarly journals Yellow Fever Virus NS3 Plays an Essential Role in Virus Assembly Independent of Its Known Enzymatic Functions

2008 ◽  
Vol 82 (7) ◽  
pp. 3342-3352 ◽  
Author(s):  
Chinmay G. Patkar ◽  
Richard J. Kuhn
Keyword(s):  
Yellow Fever ◽  
Infectious Virus ◽  
Yellow Fever Virus ◽  
Virus Assembly ◽  
Nonstructural Protein ◽  
Fever Virus ◽  
Helicase Domain ◽  
Genome Replication ◽  
Wild Type ◽  
Nonstructural Proteins

ABSTRACT In flaviviruses it has been proposed that there is a coupling between genome replication and virion assembly and that nonstructural proteins are involved in this process. It was previously reported that mutations in yellow fever virus (YFV) nonstructural protein NS2A blocked production of infectious virus and that this block could be released by a suppressor mutation in NS3. Here, based on studies using a YFV replicon-based trans-packaging system as well as full-length YFV cDNA, we report that mutation of a conserved tryptophan at position 349 in the helicase domain of NS3 blocks production of infectious virus particles, revealing an as-yet-unknown role for NS3 in virus assembly. Mutation of tryptophan 349 to alanine (W349A) had no effect on viral replication, as demonstrated by wild-type levels of viral RNA amplification and protein expression in W349A-transfected cells. Although release of infectious virus was not detected, release of capsidless subviral particles was not blocked. The assembly defect in W349A could be trans-complemented inefficiently using BHK-REP cells (a cell line containing persistently replicating YFV replicon RNA). trans-complementation was also demonstrated by supplying wild-type NS2B-3 or NS3 protein alone as well as by supplying inactive NS2B-3 protein, indicating that this function of NS3 in virus assembly was independent of its known enzymatic functions.

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 Identification of envelope protein epitopes that are important in the attenuation process of wild-type yellow fever virus.

1992 ◽  
Vol 66 (7) ◽  
pp. 4265-4270 ◽  
Author(s):  
B K Sil ◽  
L M Dunster ◽  
T N Ledger ◽  
M R Wills ◽  
P D Minor ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Envelope Protein ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Wild Type

Host-cell interaction of attenuated and wild-type strains of yellow fever virus can be differentiated at early stages of hepatocyte infection

2006 ◽  
Vol 8 (6) ◽  
pp. 1530-1538 ◽  
Author(s):  
Anabelle Lefeuvre ◽  
Hugues Contamin ◽  
Thierry Decelle ◽  
Christophe Fournier ◽  
Jean Lang ◽  
...  
Keyword(s):  
Host Cell ◽  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Cell Interaction ◽  
Fever Virus ◽  
Wild Type ◽  
Early Stages ◽  
Type Strains ◽  
Host Cell Interaction

scholarly journals Production of Pseudoinfectious Yellow Fever Virus with a Two-Component Genome

2007 ◽  
Vol 81 (21) ◽  
pp. 11737-11748 ◽  
Author(s):  
Alexandr V. Shustov ◽  
Peter W. Mason ◽  
Ilya Frolov
Keyword(s):  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Structural Proteins ◽  
Multiplicity Of Infection ◽  
Nonstructural Proteins ◽  
Replication Complex ◽  
Packaging Process ◽  
E Protein ◽  
Two Component

ABSTRACT Application of genetically modified, deficient-in-replication flaviviruses that are incapable of developing productive, spreading infection is a promising means of designing safe and effective vaccines. Here we describe a two-component genome yellow fever virus (YFV) replication system in which each of the genomes encodes complete sets of nonstructural proteins that form the replication complex but expresses either only capsid or prM/E instead of the entire structural polyprotein. Upon delivery to the same cell, these genomes produce together all of the viral structural proteins, and cells release a combination of virions with both types of genomes packaged into separate particles. In tissue culture, this modified YFV can be further passaged at an escalating scale by using a high multiplicity of infection (MOI). However, at a low MOI, only one of the genomes is delivered into the cells, and infection cannot spread. The replicating prM/E-encoding genome produces extracellular E protein in the form of secreted subviral particles that are known to be an effective immunogen. The presented strategy of developing viruses defective in replication might be applied to other flaviviruses, and these two-component genome viruses can be useful for diagnostic or vaccine applications, including the delivery and expression of heterologous genes. In addition, the achieved separation of the capsid-coding sequence and the cyclization signal in the YFV genome provides a new means for studying the mechanism of the flavivirus packaging process.

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
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