Multiple Anti-Interferon Actions of the Influenza A Virus NS1 Protein

Abstract

The replication and pathogenicity of influenza A virus (FLUAV) are controlled in part by the alpha/beta interferon (IFN-␣/␤) system. This virus-host interplay is dependent on the production of IFN-␣/␤ and on the capacity of the viral nonstructural protein NS1 to counteract the IFN system. Two different mechanisms have been described for NS1, namely, blocking the activation of IFN regulatory factor 3 (IRF3) and blocking posttranscriptional processing of cellular mRNAs. Here we directly compare the abilities of NS1 gene products from three different human FLUAV (H1N1) strains to counteract the antiviral host response. We found that A/PR/8/34 NS1 has a strong capacity to inhibit IRF3 and activation of the IFN-␤ promoter but is unable to suppress expression of other cellular genes. In contrast, the NS1 proteins of A/Tx/36/91 and of A/BM/1/18, the virus that caused the Spanish influenza pandemic, caused suppression of additional cellular gene expression. Thus, these NS1 proteins prevented the establishment of an IFN-induced antiviral state, allowing virus replication even in the presence of IFN. Interestingly, the block in gene expression was dependent on a newly described NS1 domain that is important for interaction with the cleavage and polyadenylation specificity factor (CPSF) component of the cellular pre-mRNA processing machinery but is not functional in A/PR/8/34 NS1. We identified the Phe-103 and Met-106 residues in NS1 as being critical for CPSF binding, together with the previously described C-terminal binding domain. Our results demonstrate the capacity of FLUAV NS1 to suppress the antiviral host defense at multiple levels and the existence of strain-specific differences that may modulate virus pathogenicity.The genome of influenza A virus (FLUAV) consists of eight RNA segments that encode nine structural proteins and two nonstructural proteins, called NS1 (41) and PB1-F2 (11). NS1 is a virulence factor of FLUAV by virtue of conferring resistance to the antiviral effects of the host interferon (IFN) system (21,40,63). Previous studies with recombinant FLUAV carrying deletions in the NS1 gene (delNS1) showed a strong attenuation in IFN-competent systems, whereas the NS1-deleted virus replicated to levels similar to those of wild-type virus in cell culture and in mice with a defect in the IFN system (16,23,39).The expression of type I IFNs (IFN-␣/␤) is induced in response to viral infection. Viral single-stranded and doublestranded RNAs (dsRNAs) with phosphorylated 5Ј ends are among the viral products that induce IFN-␣/␤ (32, 42, 55). These viral RNA molecules activate a variety of cellular signaling pathways, resulting in the activation of transcription factors, such as the IFN regulatory factors (IRFs) and the stress-induced transcription factors NF-B and c-Jun/ATF2 (34, 60, 64). Upon activation, these latent transcription factors move from the cytoplasm into the nucleus and initiate the expression of type I IFNs. IFN-␣/␤ subtypes bind to a common type I IFN receptor, thus activating the JAK-STAT signaling ...

SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

“…Thus, the expression construct for the fusion protein coded for amino acid residues 1 to 87 of NS1 followed by the full-length amino acid sequence of Ubc9. Because it was shown previously that most of the IFN-blocking activity mediated by PR8 NS1 is associated with the RNA binding activity of its N-terminal domain (40), we also developed a mutant form of the NS1-Ubc9 construct containing two amino acid substitutions known to prevent RNA binding by its NS1 RBD portion, namely, an R-to-A substitution at position 38 and a K-to-A substitution at position 41 (R38AK41A) (41). Both the NS1-Ubc9 fusion carrying the wild-type (wt) NS1 sequence and the NS1-Ubc9 fusion carrying the amino acid substitutions preventing RNA binding were shown to enhance the SUMOylation of full-length wild-type NS1 when coexpressed by transfection in mammalian cells, specifically by enhancing SUMOylation at position K219 in PR8 NS1, and did not appear to exhibit self-SUMOylating activity.…”

“…To test this hypothesis, we aimed to develop recombinant viruses carrying the non-SUMOylatable form of NS1 using a previously reported influenza A/WSN/1933 (here referred to simply as WSN33) 12-plasmid reverse genetic system (38). However, since WSN33 NS1 lacks the second SUMOylation site (K219), and PR8 NS1 exhibits strong and well-characterized IFN-blocking activity (40,55,56), we replaced WSN33 NS with PR8 NS but maintained the original 5= and 3= UTRs of the WSN33 NS gene segment.…”

An engineered avian-origin influenza A virus for pancreatic ductal adenocarcinoma virotherapy

“…Indeed, no recombinant IAV of any origin has been appraised so far for PDA treatment, and the relative small number of previous investigations concerning the use of IAV for virotherapy were focused on the human strain H1N1 A/Puerto Rico/8/34 (Bergmann et al, 2001;Muster et al, 2004;Sturlan et al, 2010;Wolschek et al, 2011). Importantly, the choice of a particular viral isolate is likely to affect its efficacy as an OV because IAVs differ in their ability to counteract host IFN response (Geiss et al, 2002;Hayman et al, 2006;Kochs et al, 2007), induce apoptosis (Kasloff et al, 2014) and in their sensitivity to host ISGs (Dittmann et al, 2008).…”

“…The greater impact of NS1 truncation on the ability of the H7N3 virus to control IFN induction implies that this NS1 protein relies on its C-terminal ED to post-transcriptionally limit processing of host pre-mRNAs (Geiss et al, 2002;Hayman et al, 2006;Kochs et al, 2007), whereas the NS1 protein of H1N1 PR8 virus does not due to amino acid substitutions that block its interaction with the cleavage and polyadenylation specificity factor 30 (CPSF30) (Kochs et al, 2007;Steidle et al, 2010). To monitor the expression of a gene transcribed by host-cell machinery, 293T cells were transfected with pCAGGSLuc plasmid, which directs polymerase II mediated expression of firefly luciferase.…”

Inhibition of host innate immune responses and pathogenicity of recombinant Newcastle disease viruses expressing NS1 genes of influenza A viruses

“…A major function of the NS1 protein is to antagonize host innate immune responses. However, the mechanisms and targets for the NS1 protein vary among influenza A viruses (Kochs et al, 2007). The length of the NS1 protein is strain-specific.…”