Region Required for Protein Expression from the Stop-Start Pentanucleotide in the M Gene of Influenza B Virus

ABSTRACT Segment 7 of influenza B virus encodes two proteins, M1 and BM2. BM2 is expressed from a stop-start pentanucleotide, in which the BM2 initiation codon overlaps with the M1 stop codon. Here, we demonstrate that 45 nucleotides of the 3′ end of the M1 coding region, but not the 5′ end of the BM2 coding region, are sufficient for the efficient expression of the downstream protein. Placing these 45 nucleotides and the stop-start pentanucleotide in between the coding sequences induced the expression of at least three noninfluenza proteins, suggesting the utility of this system for expressing multiple proteins from one mRNA.

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Influenza B Virus Requires BM2 Protein for Replication

Journal of Virology ◽

10.1128/jvi.78.11.5576-5583.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5576-5583 ◽

Cited By ~ 25

Author(s):

Masato Hatta ◽

Hideo Goto ◽

Yoshihiro Kawaoka

Keyword(s):

Reverse Genetics ◽

Mdck Cells ◽

Open Reading Frame ◽

Initiation Codon ◽

Influenza B Virus ◽

Influenza B ◽

M Gene ◽

Reading Frame ◽

Sialidase Activity ◽

B Virus

ABSTRACT The BM2 protein of influenza B virus functions as an ion channel, which is suggested to be important for virus uncoating in endosomes of virus-infected cells. Because direct support for this function is lacking, whether BM2 plays an essential role in the viral life cycle remains unknown. We therefore attempted to generate BM2 knockout viruses by reverse genetics. Mutant viruses possessing M segments with the mutated initiation codon of BM2 protein at the stop-start pentanucleotide were viable and still expressed BM2. The introduction of multiple stop codons and a one-nucleotide deletion downstream of the stop-start pentanucleotide, in addition to disablement of the BM2 initiation codon, failed to generate viable mutant viruses, but the mutant M segments still expressed proteins that reacted with the BM2 peptide antiserum. To completely abolish BM2 expression, we generated a mutant M gene whose BM2 open reading frame was deleted. Although this mutant was not able to replicate in normal MDCK cells, it did replicate in a cell line that we established which constitutively expresses BM2. Furthermore, a virus possessing the mutant M gene lacking the BM2 open reading frame and a mutant NA gene containing the BM2 open reading frame instead of the NA open reading frame underwent multiple cycles of replication in MDCK cells, with exogenous sialidase used to supplement the deleted viral sialidase activity. These findings demonstrate that the BM2 protein is essential for influenza B virus replication.

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The N-Terminal Extension of the Influenza B Virus Nucleoprotein Is Not Required for Nuclear Accumulation or the Expression and Replication of a Model RNA

Journal of Virology ◽

10.1128/jvi.72.6.5307-5312.1998 ◽

1998 ◽

Vol 72 (6) ◽

pp. 5307-5312 ◽

Cited By ~ 25

Author(s):

Mark P. Stevens ◽

Wendy S. Barclay

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Viral Rna ◽

Nuclear Accumulation ◽

Influenza B Virus ◽

Virus Protein ◽

Influenza B ◽

Coding Region ◽

B Virus ◽

N Terminus

ABSTRACT The nucleoprotein (NP) of influenza B virus is 50 amino acids longer at the N-terminus than influenza A virus NP and lacks homology to the A virus protein over the first 69 residues. We have deleted the N-terminal 51 and 69 residues of the influenza B/Ann Arbor/1/66 virus NP and show that nuclear accumulation of the protein is unaffected. This indicates that the nuclear localization signal is not located at the extreme N terminus, as in influenza A virus NP. To determine if the N-terminal mutants could support the expression and replication of a model influenza B virus RNA, the genes encoding the subunits of the viral RNA-dependent RNA polymerase (PA, PB1, and PB2) were cloned. Coexpression of NP and the P proteins in 293 cells was found to permit the expression and replication of a transfected model RNA based on segment 4 of B/Maryland/59, in which the hemagglutinin-coding region was replaced by a chloramphenicol acetyltransferase gene. The expression and replication of the synthetic RNA were not affected by the replacement of NP with NP mutants lacking the N-terminal 51 or 69 residues, indicating that the N-terminal extension is not required for transcription or replication of the viral RNA. In addition, we report that the influenza B virus NP cannot be functionally replaced by type A virus NP in this system.

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Isolation of a novel type of interfering influenza B virus defective in the function of M gene

Archives of Virology ◽

10.1007/bf01317372 ◽

1986 ◽

Vol 90 (3-4) ◽

pp. 223-236 ◽

Cited By ~ 5

Author(s):

K. Tobita ◽

T. Odagiri ◽

T. Tanaka

Keyword(s):

Influenza B Virus ◽

Influenza B ◽

M Gene ◽

B Virus

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Recurring Influenza B Virus Infections in Seals

Emerging Infectious Diseases ◽

10.3201/eid1903.120965 ◽

2013 ◽

Vol 19 (3) ◽

pp. 511-512 ◽

Cited By ~ 53

Author(s):

Rogier Bodewes ◽

Danny Morick ◽

Gerrie de Mutsert ◽

Nynke Osinga ◽

Theo Bestebroer ◽

...

Keyword(s):

Influenza B Virus ◽

Influenza B ◽

Virus Infections ◽

B Virus

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Molecular Epidemiology and Phylogenetic Analyses of Influenza B Virus in Thailand during 2010 to 2014

PLoS ONE ◽

10.1371/journal.pone.0116302 ◽

2015 ◽

Vol 10 (1) ◽

pp. e0116302 ◽

Cited By ~ 31

Author(s):

Nipaporn Tewawong ◽

Kamol Suwannakarn ◽

Slinporn Prachayangprecha ◽

Sumeth Korkong ◽

Preeyaporn Vichiwattana ◽

...

Keyword(s):

Molecular Epidemiology ◽

Phylogenetic Analyses ◽

Influenza B Virus ◽

Influenza B ◽

B Virus

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Mutation E48K in PB1 Polymerase Subunit Improves Stability of a Candidate Live Attenuated Influenza B Virus Vaccine

Vaccines ◽

10.3390/vaccines9070800 ◽

2021 ◽

Vol 9 (7) ◽

pp. 800

Author(s):

Jongsuk Mo ◽

Stivalis Cardenas-Garcia ◽

Jefferson J. S. Santos ◽

Lucas M. Ferreri ◽

C. Joaquín Cáceres ◽

...

Keyword(s):

Vaccine Candidate ◽

The Elderly ◽

Potential Interaction ◽

Respiratory Pathogen ◽

Influenza B Virus ◽

Influenza B ◽

Epitope Tag ◽

Live Attenuated Vaccine ◽

B Virus ◽

Homologous Challenge

Influenza B virus (IBV) is a major respiratory pathogen of humans, particularly in the elderly and children, and vaccines are the most effective way to control it. In previous work, incorporation of two mutations (E580G, S660A) along with the addition of an HA epitope tag in the PB1 segment of B/Brisbane/60/2008 (B/Bris) resulted in an attenuated strain that was safe and effective as a live attenuated vaccine. A third attempted mutation (K391E) in PB1 was not always stable. Interestingly, viruses that maintained the K391E mutation were associated with the mutation E48K. To explore the contribution of the E48K mutation to stability of the K391E mutation, a vaccine candidate was generated by inserting both mutations, along with attenuating mutations E580G and S660A, in PB1 of B/Bris (B/Bris PB1att 4M). Serial passages of the B/Bris PB1att 4M vaccine candidate in eggs and MDCK indicated high stability. In silico structural analysis revealed a potential interaction between amino acids at positions 48 and 391. In mice, B/Bris PB1att 4M was safe and provided complete protection against homologous challenge. These results confirm the compensatory effect of mutation E48K to stabilize the K391E mutation, resulting in a safer, yet still protective, IBV LAIV vaccine.

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