Molecular mechanisms enhancing the proteome of influenza A viruses: An overview of recently discovered proteins

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.

Section: Discussionmentioning

confidence: 64%

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

Kuo

Lin

et al. 2016

“…PA-X is a fusion protein containing a common N-terminal endonuclease domain of 191 amino acids (aa) with the polymerase PA open reading frame (ORF) fused to a unique C-terminal region of 41 or 61 aa (depending on the strain) produced by ribosomal frameshifting of the ϩ1 reading frame of PA (Fig. 1A) (12)(13)(14). Although most influenza virus isolates have 61 C-terminal aa residues, some strains of influenza virus, such as 2009 pandemic H1N1 (pH1N1) viruses, possess a stop codon at position 42 of the C terminus that results in a truncated 41-aa C terminus (12)(13)(14).…”

Section: Virus-induced Host Protein Shutoff Is Considered To Be a Majmentioning

confidence: 99%

“…1A) (12)(13)(14). Although most influenza virus isolates have 61 C-terminal aa residues, some strains of influenza virus, such as 2009 pandemic H1N1 (pH1N1) viruses, possess a stop codon at position 42 of the C terminus that results in a truncated 41-aa C terminus (12)(13)(14). We and Jagger et al demonstrated that transfection of a PA-X expression plasmid inhibited reporter gene expression to a greater extent than the full PA ORF, suggesting that PA-X could be the major determinant of host shutoff induced by influenza virus (12,15).…”

Section: Virus-induced Host Protein Shutoff Is Considered To Be a Majmentioning

confidence: 99%

Influenza A Virus Protein PA-X Contributes to Viral Growth and Suppression of the Host Antiviral and Immune Responses

Hayashi

MacDonald

Takimoto

2015

100

143

Influenza virus infection causes global inhibition of host protein synthesis in infected cells. This host shutoff is thought to allow viruses to escape from the host antiviral response, which restricts virus replication and spread. Although the mechanism of host shutoff is unclear, a novel viral protein expressed by ribosomal frameshifting, PA-X, was found to play a major role in influenza virus-induced host shutoff. However, little is known about the impact of PA-X expression on currently circulating influenza A virus pathogenicity and the host antiviral response. In this study, we rescued a recombinant influenza A virus, A/California/ 04/09 (H1N1, Cal), containing mutations at the frameshift motif in the polymerase PA gene (Cal PA-XFS). Cal PA-XFS expressed significantly less PA-X than Cal wild type (WT). Cal WT, but not Cal PA-XFS, induced degradation of host ␤-actin mRNA and suppressed host protein synthesis, supporting the idea that PA-X induces host shutoff via mRNA decay. Moreover, Cal WT inhibited beta interferon (IFN-␤) expression and replicated more rapidly than Cal PA-XFS in human respiratory cells. Mice infected with Cal PA-XFS had significantly lower levels of viral growth and greater expression of IFN-␤ mRNA in their lungs than mice infected with Cal WT. Importantly, more antihemagglutinin and neutralizing antibodies were produced in Cal PA-XFSinfected mice than in Cal WT-infected mice, despite the lower level of virus replication in the lungs. Our data indicate that PA-X of the pandemic H1N1 virus has a strong impact on viral growth and the host innate and acquired immune responses to influenza virus. IMPORTANCEVirus-induced host protein shutoff is considered to be a major factor allowing viruses to evade innate and acquired immune recognition. We provide evidence that the 2009 H1N1 influenza A virus protein PA-X plays a role in virus replication and inhibition of host antiviral response by means of its host protein synthesis shutoff activity both in vitro and in vivo. We also demonstrated that, while the growth of Cal PA-XFS was attenuated in the lungs of infected animals, this mutant induced a stronger humoral response than Cal WT. Our findings clearly highlight the importance of PA-X in counteracting the host innate and acquired immune responses to influenza virus, an important global pathogen. This work demonstrates that inhibition of PA-X expression in influenza virus vaccine strains may provide a novel way of safely attenuating viral growth while inducing a more robust immune response.

“…Due to the absence of preexisting immunity against avian influenza viruses among human populations, such viruses pose a serious threat of a global pandemic if they further adapt for human-to-human transmission. These adaptations include specific mutations in the hemagglutinin (HA), neuraminidase (NA), and internal genes as well as viral proteins that evolved to dampen host innate responses to the virus (8).…”

mentioning

confidence: 99%

Antigenic Fingerprinting of Antibody Response in Humans following Exposure to Highly Pathogenic H7N7 Avian Influenza Virus: Evidence for Anti-PA-X Antibodies

Khurana

Chung

Coyle

et al. 2016

Infections with H7 highly pathogenic avian influenza (HPAI) viruses remain a major public health concern. Adaptation of lowpathogenic H7N7 to highly pathogenic H7N7 in Europe in 2015 raised further alarm for a potential pandemic. An in-depth understanding of antibody responses to HPAI H7 virus following infection in humans could provide important insight into virus gene expression as well as define key protective and serodiagnostic targets. Here we used whole-genome gene fragment phage display libraries (GFPDLs) expressing peptides of 15 to 350 amino acids across the complete genome of the HPAI H7N7 A/Netherlands/33/03 virus. The hemagglutinin (HA) antibody epitope repertoires of 15 H7N7-exposed humans identified clear differences between individuals with no hemagglutination inhibition (HI) titers (<1:10) and those with HI titers of >1:40. Several potentially protective H7N7 epitopes close to the HA receptor binding domain (RBD) and neuraminidase (NA) catalytic site were identified. Surface plasmon resonance (SPR) analysis identified a strong correlation between HA1 (but not HA2) binding antibodies and H7N7 HI titers. A proportion of HA1 binding in plasma was contributed by IgA antibodies. Antibodies against the N7 neuraminidase were less frequent but targeted sites close to the sialic acid binding site. Importantly, we identified strong antibody reactivity against PA-X, a putative virulence factor, in most H7N7-exposed individuals, providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human influenza A virus infection. This knowledge can help inform the development and selection of the most effective countermeasures for prophylactic as well as therapeutic treatments of HPAI H7N7 avian influenza virus. IMPORTANCEAn outbreak of pathogenic H7N7 virus occurred in poultry farms in The Netherlands in 2003. Severe outcome included conjunctivitis, influenza-like illness, and one lethal infection. In this study, we investigated convalescent-phase sera from H7N7-exposed individuals by using a whole-genome phage display library (H7N7-GFPDL) to explore the complete repertoire of post-H7N7-exposure antibodies. PA-X is a recently identified influenza virus virulence protein generated by ribosomal frameshifting in segment 3 of influenza virus coding for PA. However, PA-X expression during influenza virus infection in humans is unknown. We identified strong antibody reactivity against PA-X in most H7N7-exposed individuals (but not in unexposed adults), providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human infection with pathogenic H7N7 avian influenza virus.A vian influenza viruses (AIVs) are mostly restricted to aquatic birds. On occasion, they acquire the capacity to directly infect the respiratory tract of poultry and mammals. Such cross-species transmission often results in a large number of sick birds (chickens and turkeys), as was reported for H7N1 viruses in Italy in 1999, H7N3 viruses in Canada in 2...