Mutation of influenza A virus PA-X decreases pathogenicity in chicken embryos and can increase the yield of reassortant candidate vaccine viruses

Abstract

word count: 242 24 Main text word count: 5308 25Abstract 26 27 The PA-X protein of influenza A virus has roles in host cell shut-off and viral pathogenesis. 28While most strains are predicted to encode PA-X, strain-dependent variations in activity have 29 been noted. We found that PA-X protein from A/PR/8/34 (PR8) strain had significantly lower 30 repressive activity against cellular gene expression compared with PA-Xs from the avian 31 strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of 32 normal PA-X expression, either by mutation of the frameshift site or by truncating the X-33 ORF, had little effect on the infectious virus titre of PR8 or PR8 7:1 reassortants with T/E 34 segment 3 grown in embryonated hens' eggs. However, in both virus backgrounds, mutation 35 of PA-X led to decreased embryo mortality and lower overall pathology; effects that were 36 more pronounced in the PR8 strain than the T/E reassortant, despite the low shut-off activity 37 of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of HA to 38 NP and M1 compared to their WT counterparts, suggesting altered virion composition. When 39 the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine 40 reassortant analogues containing the HA and NA segments from H1N1 2009 pandemic 41 viruses or an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 42 PA-X protein may harbour a function unrelated to host cell shut-off and that disruption of the 43 PA-X gene has the potential to improve the HA yield of vaccine viruses. 44 45 IMPORTANCE Influenza A virus is a widespread pathogen that affects both man and a 46 variety of animal species, causing regular epidemics and sporadic pandemics with major 47 public health and economic consequences. A better understanding of virus biology is 48 therefore important. The primary control measure is vaccination, which for humans, mostly 49 relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of 50 3 interest. However, not all reassortants replicate well enough to supply sufficient virus antigen 51 for demand. The significance of our research lies in identifying that mutation of the PA-X 52 gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the 53 pathogenicity of the virus in embryonated eggs. 54 55 ectodomain of the CVV glycoprotein genes (13-21), or introducing a wild-type (WT) virus-101 derived segment 2 (21-29). Our approach was to manipulate expression of an accessory 102 protein virulence factor, PA-X (30). Segment 3, encoding PA as the primary gene product, 103 also expresses PA-X by low-level ribosomal shifting into a +1 open reading frame (ORF) 104 termed the X ORF ( Fig 1A) (30). PA-X is a 29 kDa protein that contains the N-terminal 105 endonuclease domain of PA, and in most isolates, a 61 amino acid C-terminus from the X 106 ORF (30-32). It has roles in shutting off host cell protein synthesis and, at the whole animal 107 le...

Contribution of Segment 3 to the Acquisition of Virulence in Contemporary H9N2 Avian Influenza Viruses

“…6A and B ). For UDL-01 WT, introduction of the E26K and/or FS mutation (individually or together) ablated shutoff activity, indicating that, as we and others have shown, segment 3 shutoff activity maps to PA-X ( 19 , 34 , 35 , 37 , 38 ). Conversely, as WF10 WT segment 3 had no shutoff activity, the FS mutant alone had no additional effect but ablated the increased shutoff activity seen when combined with WF10 K26E.…”

“…We initially investigated the combined effect of position and FS mutations on host shutoff in mammalian and avian cells ( Figure 6A,B). For UDL-01 WT introduction of either the E26K or FS mutation individually or together ablated shutoff activity, indicating as we and others have shown, that segment 3 shutoff activity maps to PA-X (19,34,35,37,38).…”

“…Briefly, cells were cotransfected with expression plasmids containing the different segment 3s along with a β-gal reporter plasmid, followed by enzymatic readout of β-gal activity to give a measure of host gene expression in the transfected cells and thus the ability of the different segment 3 plasmids to cause host shutoff. Previous work has suggested that the majority of influenza host cell shutoff comes from expression of PA-X rather than that of PA ( 19 , 34 , 35 ).…”

“…To assess shutoff activity further, in the context of viral infection rather than transfection and overexpression, we performed both radioactive and non-radioactive metabolic labelling experiments. To test the shutoff activity in avian cells, primary chicken embryonic fibroblast (CEF) cells were infected with a high MOI of virus containing mutations in segment 3 and were subsequently pulsed with 35 S methionine then lysed and run on SDS-PAGE. Autoradiography was performed and densitometry used to measure the abundance of the highly abundant host protein, actin.…”

“…The changes at PA position 26 are responsible for attenuation of WF10 in vitro and in vivo; these correlated better with the in vitro host cell shutoff activity of PA-X than with polymerase activity. To investigate whether the E26K polymorphism exerted its in vivo phenotypic effect via PA-X rather than through PA, we introduced a well characterized set of nucleotide substitutions into the frameshift site (FS mutant) of PA which has previously been shown to inhibit expression of PA-X (19,34,35). We further combined this FS mutation with the reciprocal mutants at position 26.…”