Glycine 184 in Nonstructural Protein NS1 Determines the Virulence of Influenza A Virus Strain PR8 without Affecting the Host Interferon Response

The nonstructural protein (NS1) of influenza A virus performs multiple functions in the virus life cycle. Proteomic screening for cellular proteins which interact with NS1 identified the cellular protein RAP55, which is one of the components of cellular processing bodies (P-bodies) and stress granules. To verify whether NS1 interacts with cellular P-bodies, interactions between NS1, RAP55, and other P-body-associated proteins (Ago1, Ago2, and DCP1a) were confirmed using coimmunoprecipitation and cellular colocalization assays. Overexpression of RAP55 induced RAP55-associated stress granule formation and suppressed virus replication. Knockdown of RAP55 with small interfering RNA (siRNA) or expression of a dominant-negative mutant RAP55 protein with defective interaction with P-bodies blocked NS1 colocalization to P-bodies in cells. Expression of NS1 inhibited RAP55 expression and formation of RAP55-associated P-bodies/stress granules. The viral nucleoprotein (NP) was found to be targeted to stress granules in the absence of NS1 but localized to P-bodies when NS1 was coexpressed. Restriction of virus replication via P-bodies occurred in the early phases of infection, as the number of RAP55-associated P-bodies in cells diminished over the course of virus infection. NS1 interaction with RAP55-associated P-bodies/stress granules was associated with RNA binding and mediated via a protein kinase R (PKR)-interacting viral element. Mutations introduced into either RNA binding sites (R38 and K41) or PKR interaction sites (I123, M124, K126, and N127) caused NS1 proteins to lose the ability to interact with RAP55 and to inhibit stress granules. These results reveal an interplay between virus and host during virus replication in which NP is targeted to P-bodies/stress granules while NS1 counteracts this host restriction mechanism.

Section: Discussionmentioning

confidence: 99%

The NS1 Protein of Influenza A Virus Interacts with Cellular Processing Bodies and Stress Granules through RNA-Associated Protein 55 (RAP55) during Virus Infection

Mok

Song

Wang

et al. 2012

“…At 9 h after influenza virus infection, the total RNA of infected cells was isolated using TRIzol reagent (Invitro-gen, USA). To determine the relative amounts of IFN-␤ pre-mRNA and mRNA, the total RNA was subjected to a reverse transcription reaction with an oligonucleotide complementary to sequence downstream of the poly(A) addition site of IFN-␤ pre-mRNA and an oligo(dT) primer using SuperScript III reverse transcriptase (Life Technologies, USA) (15,20), and quantitative PCR (qPCR) was conducted subsequently to detect the pre-mRNA of IFN-␤ and the mRNAs of IFN-␤ and ␤-actin using Kapa SYBR fast qPCR kits (Kapa Biosystems, USA) with primers described previously (15,20) and a Roche LightCycler 480II instrument.…”

Section: Methodsmentioning

confidence: 99%

“…The effector domains of NS1 proteins encoded by H3N2, H2N2, and seasonal H1N1 viruses strongly bind to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), thereby inhibiting the 3= end processing of host pre-mRNAs and the production of mature mRNAs, including beta interferon (IFN-␤) and other antiviral mRNAs (11)(12)(13). However, the NS1 proteins encoded by some strains of influenza A viruses, such as Puerto Rico/8/34/H1N1 (PR8) and the 2009 pandemic H1N1 viruses, do not efficiently bind CPSF30 (13)(14)(15). Consequently, these viruses depend solely on the ability of their NS1 proteins to efficiently suppress the activation of interferon regulatory factor 3 (IRF3) to inhibit the expression of type I IFN.…”

mentioning

confidence: 99%

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

Kuo

Lin

et al. 2016

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.

“…The minigenome expression plasmids were cotransfected into HEK293 cells together with influenza virus NP, PB1, PB2, and PA protein expression plasmids and with a transfection control renilla luciferase expression plasmid. Reporter gene activity was measured using a dual luciferase reporter assay (Promega, Madison, WI), as previously described (29). As a negative control, transfections were performed in the absence of the plasmid coding for NP.…”

mentioning

confidence: 99%

Recombinant Influenza A Viruses with Enhanced Levels of PB1 and PA Viral Protein Expression

Belicha-Villanueva

Rodríguez-Madoz

Maamary

et al. 2012

Influenza A viruses containing the promoter mutations G3A/C8U in a given segment express increased levels of the corresponding viral protein during infection due to increased levels of mRNA or cRNA species. The replication of these recombinant viruses is attenuated, and they have an enhanced shedding of noninfectious particles and are incapable of antagonizing interferon (IFN) effectively. Our findings highlight the possibility of increasing influenza virus protein expression and the need for a delicate balance between influenza viral replication, protein expression, and assembly.