Asparagine 631 variants of the chicken Mx protein do not inhibit influenza virus replication in primary chicken embryo fibroblasts or in vitro surrogate assays

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.

FIG. 1.

Multicycle influenza virus (A/WSN/33) replication in CEF lines. CEFs were infected with A/WSN/33 (MOI of 0.01). Supernatants were harvested at the times indicated, and the virus yields were titered on MDCK cells. The means (and standard deviations) of three independent experiments are shown. The amino acids at position 631 of the Mx alleles of the different lines are indicated. *, titer for C1 significantly different from A1, B1, or A2 (P < 0.01).

FIG. 2.

Flow cytometric detection of influenza vRNPs in transfected 293T cells. 293T cells were cotransfected with Mx-expressing plasmids (or pcDNA3) (1.5 μg) and pEGFP-C1 (0.5 μg) and infected after 48 h with A/PR/8/34, A/Duck/England/62, or A/Duck/Singapore/97. Fourteen hours postinfection, the cells were stained for vRNP and analyzed by flow cytometry. Panel A shows the level of A/PR/8/34 antigen in GFP-positive cells that had been cotransfected with the indicated plasmids. Panel “a” shows the background staining detected in uninfected, pcDNA3-transfected cells and was used to set the fluorescence threshold marker (M1), which demarcates between antigen-positive and -negative cells. In panel B, the data are derived from independent experiments (A/PR/8/34, n = 5; A/Duck/England/62, n = 3; A/Duck/Singapore/97, n = 3) and the bar heights represent the percentages of antigen-positive cells expressed relative to that for the pcDNA3 control group for each virus. The means (and standard deviations) are shown for either GFP-positive or GFP-negative cell populations from wells that had been transfected with the constructs as shown. *, P of <0.01 relative to GFP-positive pcDNA3-transfected cells.

FIG. 3.

Activity of chicken Mx proteins in influenza virus minireplicon systems. (A) Effect of chicken Mx on an A/PR/8/34 minireplicon system. 293T cells were cotransfected with plasmids encoding A/PR/8/34 PB1, PB2, PA, and NP, a huPolI Luc minigenome plasmid, and a SEAP-expressing plasmid, together with pcDNA3 or a plasmid expressing the indicated Mx protein (chicken Mx proteins SHK [Asn631] and 8.1 [Ser631], murine Mx1 and its mutant Mx1 K49A, and human MxA and its mutant MxA T103A). The ratio of pcDNA3/Mx plasmid to each of the 3P/NP plasmids was 10:1. Forty-eight hours posttransfection, luciferase activity was measured and is shown as SEAP-normalized relative light units (rlu). The means (and standard deviations) of three independent experiments are shown. *, P of <0.01 relative to pcDNA3. (B) Effect of chicken Mx on the Turkey/England/50-92/91 minireplicon system in chicken DF-1 cells. DF-1 cells were transfected with plasmids encoding A/Turkey/England/50-92/91 PB1, PB2, PA, and NP (25 ng each), A/PR/8/34 NS1 (125 ng), a chicken PolI Luc minigenome plasmid (100 ng), and a SEAP-expressing plasmid (50 ng) together with pcDNA3 or a plasmid expressing the indicated Mx protein (125 ng). The ratio of pcDNA3/Mx plasmid to each of the 3P/NP plasmids was 5:1. Forty-eight hours posttransfection, luciferase activity was measured and is shown as relative light units (rlu). The means (and standard deviations) of six independent experiments are shown. *, P of <0.01 relative to pcDNA3.