Abstract
Whether chicken Mx inhibits influenza virus replication is an important question with regard to strategies aimed at enhancing influenza resistance in domestic flocks. The Asn631 polymorphism of the chicken Mx protein found in the Shamo (SHK) chicken line was previously reported to be crucial for the antiviral activity of this highly polymorphic chicken gene. Our aims were to determine whether cells from commercial chicken lines containing Asn631 alleles were resistant to influenza virus infection and to investigate the effects that other polymorphisms might have on Mx function. Unexpectedly, we found that the Asn631 genotype had no impact on multicycle replication of influenza virus (A/WSN/33 [H1N1]) in primary chicken embryo fibroblast lines. Furthermore, expression of the Shamo (SHK) chicken Mx protein in transfected 293T cells did not inhibit viral gene expression (A/PR/8/34 [H1N1], A/Duck/England/62 [H4N6], and A/Duck/Singapore/97 [H5N3]). Lastly, in minireplicon systems (A/PR/8/34 and A/Turkey/England/50-92/91 [H5N1]), which were highly sensitive to inhibition by the murine Mx1 and human MxA proteins, respectively, Shamo chicken Mx also proved ineffective in the context of avian as well as mammalian cell backgrounds. Our findings demonstrate that Asn631 chicken Mx alleles do not inhibit influenza virus replication of the five strains tested here and efforts to increase the frequency of Asn631 alleles in commercial chicken populations are not warranted. Nevertheless, chicken Mx variants with anti-influenza activity might still exist. The flow cytometry and minireplicon assays described herein could be used as efficient functional screens to identify such active chicken Mx alleles.
Highlights
Mx proteins are interferon (IFN)-induced dynamin-like GTPases found in all vertebrate species examined so far, and they exhibit a range of antiviral capabilities [10]
Our findings demonstrate that Asn631 chicken Mx alleles do not inhibit influenza virus replication of the five strains tested here and efforts to increase the frequency of Asn631 alleles in commercial chicken populations are not warranted
One focus for selective breeding approaches has been the Asn631 polymorphism of the chicken Mx protein, which was reported by Ko et al to confer antiviral activity on the Mx protein [14]
Summary
Mx proteins are interferon (IFN)-induced dynamin-like GTPases found in all vertebrate species examined so far, and they exhibit a range of antiviral capabilities [10]. Certain chicken Mx proteins have been shown to inhibit replication of vesicular stomatitis virus (VSV) [14], and artificial mutation has demonstrated that the amino acid at position 631 of the chicken Mx protein is a crucial determinant of anti-VSV activity (Asn631 is active and Ser631 is inactive against VSV) [15]. The purpose of this study was to validate cell culture assays that could be used to identify Mx alleles from commercial lines of chickens with activity against influenza virus and to determine the functional contributions of polymorphisms in addi-. HN HN a The amino acid residues at the 15 reported polymorphic sites of the chicken Mx protein are given for the CEF and DF-1 cell lines, with reference to those of the. The bottom row shows breed identifiers, as given in reference 14, for Mx alleles that differ from the SHK amino acid at the positions shown but which are active against VSV. The bottom row shows breed identifiers, as given in reference 14, for Mx alleles that differ from the SHK amino acid at the positions shown but which are active against VSV. (The allele WLK-2 is from a strain of White Leghorn, and the HN allele is from the Hinaidori breed.) b Data from reference 14. c Accession number Z23168
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