Abstract
BackgroundChicken Mx belongs to the Mx family of interferon-induced dynamin-like GTPases, which in some species possess potent antiviral properties. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. The normal cytoplasmic localisation of chicken Mx may influence its antiviral capacity. Here we report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV), an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We also report the consequences of re-locating chicken Mx to the nucleus.Methodology/Principal FindingsChicken Mx was tested in virus infection assays using NDV. Neither the Asn631 nor Ser631 Mx alleles (when transfected into 293T cells) showed inhibition of virus-directed gene expression when the cells were subsequently infected with NDV. Human MxA however did show significant inhibition of NDV-directed gene expression. Chicken Mx failed to inhibit a Thogoto virus (THOV) minireplicon system in which the cytoplasmic human MxA protein showed potent and specific inhibition. Relocalisation of chicken Mx to the nucleus was achieved by inserting the Simian Virus 40 large T antigen nuclear localisation sequence (SV40 NLS) at the N-terminus of chicken Mx. Nuclear re-localised chicken Mx did not inhibit influenza (A/PR/8/34) gene expression during virus infection in cell culture or influenza polymerase activity in A/PR/8/34 or A/Turkey/50-92/91 minireplicon systems.Conclusions/SignificanceThe chicken Mx protein (Asn631) lacks inhibitory effects against THOV and NDV, and is unable to suppress influenza replication when artificially re-localised to the cell nucleus. Thus, the natural cytoplasmic localisation of the chicken Mx protein does not account for its lack of antiviral activity.
Highlights
Mx proteins are interferon (IFN)-induced dynamin-like GTPases found in all vertebrate species examined so far
Activity of Chicken Mx against Newcastle Disease Virus Newcastle disease virus (NDV) was chosen as a candidate to test for sensitivity to the chicken Mx protein because (i) NDV is an RNA virus belonging to the Paramyxoviridae family, several members of which are sensitive to the human MxA protein [21,22,23,24,25,26]; (ii) NDV replicates exclusively in the cytoplasm of the host cell, where the chicken Mx protein is located and (iii) NDV is an important avian pathogen which poses a serious economic threat to the poultry industry
To determine whether NDV was sensitive to inhibition by the chicken Mx protein, NDV-directed gene expression was measured using flow cytometry in 293T cells that were transiently transfected with Mx expression plasmids
Summary
Mx proteins are interferon (IFN)-induced dynamin-like GTPases found in all vertebrate species examined so far. Murine Mx1 and human MxA mediate their anti-influenza effects via distinct mechanisms: Mx1 is nuclear and inhibits primary transcription of the virus genome [8], while the cytoplasmic human MxA protein [9] affects an ill-defined post-transcriptional step [10] probably via an interaction with the nucleoprotein [11]. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. We report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV), an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We report the consequences of re-locating chicken Mx to the nucleus
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