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

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.

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

Journal of Virology ◽

10.1128/jvi.02447-07 ◽

2008 ◽

Vol 82 (7) ◽

pp. 3342-3352 ◽

Cited By ~ 80

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.

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Immature particles and capsid-free viral RNA produced by Yellow fever virus-infected cells stimulate plasmacytoid dendritic cells to secrete interferons

Scientific Reports ◽

10.1038/s41598-018-29235-7 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 9

Author(s):

Laura Sinigaglia ◽

Ségolène Gracias ◽

Elodie Décembre ◽

Matthieu Fritz ◽

Daniela Bruni ◽

...

Keyword(s):

Dendritic Cells ◽

Yellow Fever ◽

Yellow Fever Virus ◽

Plasmacytoid Dendritic Cells ◽

Fever Virus ◽

Viral Rna ◽

Infected Cells

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Yellow Fever Virus Down-Regulates mRNA Expression of SOCS1 in the Initial Phase of Infection in Human Cell Lines

Viruses ◽

10.3390/v12080802 ◽

2020 ◽

Vol 12 (8) ◽

pp. 802

Author(s):

Michael B. Yakass ◽

David Franco ◽

Osbourne Quaye

Keyword(s):

Mrna Expression ◽

Yellow Fever ◽

Yellow Fever Virus ◽

Expression Patterns ◽

Fever Virus ◽

Effective Vaccine ◽

Interferon Β ◽

Infected Cells ◽

Insight Into

Flaviviruses are constantly evolving diverse immune evasion strategies, and the exploitation of the functions of suppressors of cytokine signalling (SOCS) and protein inhibitors of activated STATs (PIAS) to favour virus replication has been described for Dengue and Japanese encephalitis viruses but not for yellow fever virus (YFV), which is still of global importance despite the existence of an effective vaccine. Some mechanisms that YFV employs to evade host immune defence has been reported, but the expression patterns of SOCS and PIAS in infected cells is yet to be determined. Here, we show that SOCS1 is down-regulated early in YFV-infected HeLa and HEK 293T cells, while SOCS3 and SOCS5 are not significantly altered, and PIAS mRNA expression appears to follow a rise-dip pattern akin to circadian-controlled genes. We also demonstrate that YFV evades interferon-β application to produce comparable viral titres. This report provides initial insight into the in vitro expression dynamics of SOCS and PIAS upon YFV infection and a basis for further investigation into SOCS/PIAS expression and how these modulate the immune response in animal models.

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