Adaptive mutation in influenza A virus non-structural gene is linked to host switching and induces a novel protein by alternative splicing

The NS1 protein encoded by influenza A virus antagonizes the interferon response through various mechanisms, including blocking cellular mRNA maturation by binding the cellular CPSF30 3= end processing factor and/or suppressing the activation of interferon regulatory factor 3 (IRF3). In the present study, we identified two truncated NS1 proteins that are translated from internal AUGs at positions 235 and 241 of the NS1 open reading frame. We analyzed the cellular localization and function of the N-truncated NS1 proteins encoded by two influenza A virus strains, Udorn/72/H3N2 (Ud) and Puerto Rico/8/34/H1N1 (PR8). The NS1 protein of PR8, but not Ud, inhibits the activation of IRF3, whereas the NS1 protein of Ud, but not PR8, binds CPSF30. The truncated PR8 NS1 proteins are localized in the cytoplasm, whereas the full-length PR8 NS1 protein is localized in the nucleus. The infection of cells with a PR8 virus expressing an NS1 protein containing mutations of the two in-frame AUGs results in both the absence of truncated NS1 proteins and the reduced inhibition of activation of IRF3 and beta interferon (IFN-␤) transcription. The expression of the truncated PR8 NS1 protein by itself enhances the inhibition of the activation of IRF3 and IFN-␤ transcription in Ud virus-infected cells. These results demonstrate that truncated PR8 NS1 proteins contribute to the inhibition of activation of this innate immune response. In contrast, the N-truncated NS1 proteins of the Ud strain, like the full-length NS1 protein, are localized in the nucleus, and mutation of the two in-frame AUGs has no effect on the activation of IRF3 and IFN-␤ transcription. IMPORTANCEInfluenza A virus causes pandemics and annual epidemics in the human population. The viral NS1 protein plays a critical role in suppressing type I interferon expression. In the present study, we identified two novel truncated NS1 proteins that are translated from the second and third in-frame AUG codons in the NS1 open reading frame. The N-terminally truncated NS1 encoded by the H1N1 PR8 strain of influenza virus that suppresses IRF3 activation is localized primarily in the cytoplasm. We demonstrate that this truncated NS1 protein by itself enhances this suppression, demonstrating that some strains of influenza A virus express truncated forms of the NS1 protein that function in the inhibition of cytoplasmic antiviral events.

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confidence: 99%

Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation

Kuo

Lin

et al. 2016

“…For example, M42 and NS3 are translated from spliced mRNAs transcribed from the M and NS segments, respectively (15,16). M42 functions in place of M2 as a proton channel (15), and NS3 is associated with the adaptation of avian influenza A virus to new mammalian hosts (16). Approximately 30 of Ͼ18,000 isolates likely express M42 and NS3, based on nucleotide sequence analyses (15,16).…”

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confidence: 99%

“…M42 functions in place of M2 as a proton channel (15), and NS3 is associated with the adaptation of avian influenza A virus to new mammalian hosts (16). Approximately 30 of Ͼ18,000 isolates likely express M42 and NS3, based on nucleotide sequence analyses (15,16). PB1-F2, PB1-N40, PA-N155, and PA-N182 are expressed from alternative translation initiation sites in the PB1 and PA segments (17)(18)(19)(20)(21).…”

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confidence: 99%

“…Moreover, several other novel viral proteins have been shown to be expressed by splicing, alternative initiation, or ribosomal frameshifts. For example, M42 and NS3 are translated from spliced mRNAs transcribed from the M and NS segments, respectively (15,16). M42 functions in place of M2 as a proton channel (15), and NS3 is associated with the adaptation of avian influenza A virus to new mammalian hosts (16).…”

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confidence: 99%

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Identification of a Novel Viral Protein Expressed from the PB2 Segment of Influenza A Virus

Yamayoshi

Watanabe

Goto

et al. 2016

Over the past 2 decades, several novel influenza virus proteins have been identified that modulate viral infections in vitro and/or in vivo. The PB2 segment, which is one of the longest influenza A virus segments, is known to encode only one viral protein, PB2. In the present study, we used reverse transcription-PCR (RT-PCR) targeting viral mRNAs transcribed from the PB2 segment to look for novel viral proteins encoded by spliced mRNAs. We identified a new viral protein, PB2-S1, encoded by a novel spliced mRNA in which the region corresponding to nucleotides 1513 to 1894 of the PB2 mRNA is deleted. PB2-S1 was detected in virusinfected cells and in cells transfected with a protein expression plasmid encoding PB2. PB2-S1 localized to mitochondria, inhibited the RIG-I-dependent interferon signaling pathway, and interfered with viral polymerase activity (dependent on its PB1-binding capability). The nucleotide sequences around the splicing donor and acceptor sites for PB2-S1 were highly conserved among pre-2009 human H1N1 viruses but not among human H1N1pdm and H3N2 viruses. PB2-S1-deficient viruses, however, showed growth kinetics in MDCK cells and virulence in mice similar to those of wild-type virus. The biological significance of PB2-S1 to the replication and pathogenicity of seasonal H1N1 influenza A viruses warrants further investigation. IMPORTANCE Transcriptome analysis of cells infected with influenza

“…The genome of IAV contains eight RNA segments that encode at least 17 viral proteins, including 8 initially identified proteins (PB2, PB1, PA, HA, NP, NA, M1, and NS1), 2 splicing variants of the M and NS genes (M2 and NS2) (1)(2)(3), and the recently identified proteins PB1-N40 (4), PB1-F2 (5), PA-X (6), M42 (7), NS3 (8), PA-N155, and PA-N182 (9). PB1-N40 is an N-terminally truncated version of the PB1 protein that lacks the transcriptase function but can still interact with other polymerase complex subunits and regulate virus replication in a specific genetic background (4).…”

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confidence: 99%

PA-X Decreases the Pathogenicity of Highly Pathogenic H5N1 Influenza A Virus in Avian Species by Inhibiting Virus Replication and Host Response

Wang

et al. 2015

128

PA-X is a newly discovered protein that decreases the virulence of the 1918 H1N1 virus in a mouse model. However, the role of PA-X in the pathogenesis of highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype in avian species is totally unknown. By generating two PA-X-deficient viruses and evaluating their virulence in different animal models, we show here that PA-X diminishes the virulence of the HPAIV H5N1 strain A/Chicken/Jiangsu/k0402/2010 (CK10) in mice, chickens, and ducks. Expression of PA-X dampens polymerase activity and virus replication both in vitro and in vivo. Using microarray analysis, we found that PA-X blunts the global host response in chicken lungs, markedly downregulating genes associated with the inflammatory and cell death responses. Correspondingly, a decreased cytokine response was recapitulated in multiple organs of chickens and ducks infected with the wild-type virus relative to those infected with the PA-X-deficient virus. In addition, the PA-X protein exhibits antiapoptotic activity in chicken and duck embryo fibroblasts. Thus, our results demonstrated that PA-X acts as a negative virulence regulator and decreases virulence by inhibiting viral replication and the host innate immune response. Therefore, we here define the role of PA-X in the pathogenicity of H5N1 HPAIV, furthering our understanding of the intricate pathogenesis of influenza A virus. Influenza A virus (IAV) can infect diverse host species, from wild and domestic birds to mammalian species, and the pathogenesis of IAV is complex due to its remarkable genetic variability. The genome of IAV contains eight RNA segments that encode at least 17 viral proteins, including 8 initially identified proteins (PB2, PB1, PA, HA, NP, NA, M1, and NS1), 2 splicing variants of the M and NS genes (M2 and NS2) (1-3), and the recently identified proteins PB1-N40 (4), PB1-F2 (5), PA-X (6), M42 (7), NS3 (8), PA-N155, and PA-N182 (9). PB1-N40 is an N-terminally truncated version of the PB1 protein that lacks the transcriptase function but can still interact with other polymerase complex subunits and regulate virus replication in a specific genetic background (4). PB1-F2, encoded by an alternative open reading frame (ORF) of PB1, has multiple functions, including the induction of apoptosis (10), aggravation of inflammation (11,12), and secondary bacterial infection (13). PA-X is a frameshift product of the ribosome and acts to decrease the virulence of the 1918 H1N1 virus in mice (6). PA-N155 and PA-N182 are N-terminally truncated forms of PA, and the mutant viruses lacking these two proteins exhibit attenuated in vitro replication and pathogenicity in mice relative to the wild-type (wt) virus (9). M42 is the M2 isoform with an alternative ectodomain that can functionally replace M2 and support efficient viral replication (7). Selman et al. have identified NS3 as the isoform of NS1 and speculated that the codon providing NS3 expression could be associated with host adaptation and the overcoming of the species barrier (8)....