co-immunoprecipitation-mouse-mx1-protein-with-influenza-virus

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Ghent University

Tyrosine phosphorylation is a hallmark for activation of STAT proteins, but their transcriptional activity also depends on other secondary modifications. Type I IFNs can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide RNAi screen, we identified the SIN3 transcription regulator homolog A (Sin3a) as an important mediator of this STAT3-targeted transcriptional repression. Sin3a directly interacts with STAT3 and promotes its deacetylation. SIN3A silencing results in a prolonged nuclear retention of activated STAT3 and enhances its recruitment to the SOCS3 promoter, concomitant with histone hyperacetylation and enhanced STAT3-dependent transcription. Conversely, Sin3a is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. The Sin3a complex therefore acts as a context-dependent ISGF3/STAT3 transcriptional switch.

The Sin3a repressor complex is a master regulator of STAT transcriptional activity.

Mx1 is a GTPase that is part of the antiviral response induced by type I and type III interferons in the infected host. It inhibits influenza virus infection by blocking viral transcription and replication, but the molecular mechanism is not known. Polymerase basic protein 2 (PB2) and nucleoprotein (NP) were suggested to be the possible target of Mx1, but a direct interaction between Mx1 and any of the viral proteins has not been reported. We investigated the interplay between Mx1, NP, and PB2 to identify the mechanism of Mx1's antiviral activity. We found that Mx1 inhibits the PB2-NP interaction, and the strength of this inhibition correlated with a decrease in viral polymerase activity. Inhibition of the PB2-NP interaction is an active process requiring enzymatically active Mx1. We also demonstrate that Mx1 interacts with the viral proteins NP and PB2, which indicates that Mx1 protein has a direct effect on the viral ribonucleoprotein complex. In a minireplicon system, avian-like NP from swine virus isolates was more sensitive to inhibition by murine Mx1 than NP from human influenza A virus isolates. Likewise, murine Mx1 displaced avian NP from the viral ribonucleoprotein complex more easily than human NP. The stronger resistance of the A/H1N1 pandemic 2009 virus against Mx1 also correlated with reduced inhibition of the PB2-NP interaction. Our findings support a model in which Mx1 interacts with the influenza ribonucleoprotein complex and interferes with its assembly by disturbing the PB2-NP interaction.

Interferon-inducible protein Mx1 inhibits influenza virus by interfering with functional viral ribonucleoprotein complex assembly.

Fifty years after the discovery of the mouse Mx1 gene, researchers are still trying to understand the molecular details of the antiviral mechanisms mediated by Mx proteins. Mx proteins are evolutionarily conserved dynamin-like large GTPases, and GTPase activity is required for their antiviral activity. The expression of Mx genes is controlled by type I and type III interferons. A phylogenetic analysis revealed that Mx genes are present in almost all vertebrates, usually in one to three copies. Mx proteins are best known for inhibiting negative-stranded RNA viruses, but they also inhibit other virus families. Recent structural analyses provide hints about the antiviral mechanisms of Mx proteins, but it is not known how they can suppress such a wide variety of viruses lacking an obvious common molecular pattern. Perhaps they interact with a (partially) symmetrical invading oligomeric structure, such as a viral ribonucleoprotein complex. Such an interaction may be of a fairly low affinity, in line with the broad target specificity of Mx proteins, yet it would be strong enough to instigate Mx oligomerization and ring assembly. Such a model is compatible with the broad "substrate" specificity of Mx proteins: depending on the size of the invading viral ribonucleoprotein complexes that need to be wrapped, the assembly process would consume the necessary amount of Mx precursor molecules. These Mx ring structures might then act as energy-consuming wrenches to disassemble the viral target structure.

Mx proteins: antiviral gatekeepers that restrain the uninvited.

Influenza viruses exist as a large group of closely related viral genomes, also called quasispecies. The composition of this influenza viral quasispecies can be determined by an accurate and sensitive sequencing technique and data analysis pipeline. We compared the suitability of two benchtop next-generation sequencers for whole genome influenza A quasispecies analysis: the Illumina MiSeq sequencing-by-synthesis and the Ion Torrent PGM semiconductor sequencing technique.

Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing.

The severity of influenza-related illness is mediated by many factors, including in vivo cell tropism, timing and magnitude of the immune response, and presence of pre-existing immunity. A direct way to study cell tropism and virus spread in vivo is with an influenza virus expressing a reporter gene. However, reporter gene-expressing influenza viruses are often attenuated in vivo and may be genetically unstable. Here, we describe the generation of an influenza A virus expressing GFP from a tri-cistronic NS segment. To reduce the size of this engineered gene segment, we used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. GFP and nuclear export protein coding information were fused in frame with the truncated NS1 open reading frame and separated from each other by 2A self-processing sites. The resulting PR8-NS1(1-73)GFP virus was successfully rescued and replicated as efficiently as the parental PR8 virus in vitro and was slightly attenuated in vivo. Flow cytometry-based monitoring of cells isolated from PR8-NS1(1-73)GFP virus infected BALB/c mice revealed that GFP expression peaked on day two in all cell types tested. In particular respiratory epithelial cells and myeloid cells known to be involved in antigen presentation, including dendritic cells (CD11c+) and inflammatory monocytes (CD11b+ GR1+), became GFP positive following infection. Prophylactic treatment with anti-M2e monoclonal antibody or oseltamivir reduced GFP expression in all cell types studied, demonstrating the usefulness of this reporter virus to analyze the efficacy of antiviral treatments in vivo. Finally, deep sequencing analysis, serial in vitro passages and ex vivo analysis of PR8-NS1(1-73)GFP virus, indicate that this virus is genetically and phenotypically stable.

Article	Year
The Sin3a repressor complex is a master regulator of STAT transcriptional activity. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22783022	2012
Interferon-inducible protein Mx1 inhibits influenza virus by interfering with functional viral ribonucleoprotein complex assembly. Journal of virology\| PubMed ID: 23015724	2012
Mx proteins: antiviral gatekeepers that restrain the uninvited. Microbiology and molecular biology reviews : MMBR\| PubMed ID: 24296571	2013
Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing. BMC genomics\| PubMed ID: 25758772	2015
A GFP Expressing Influenza A Virus to Report In Vivo Tropism and Protection by a Matrix Protein 2 Ectodomain-Specific Monoclonal Antibody. PloS one\| PubMed ID: 25816132	2015

Judith Verhelst

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