Comparing the functions of equine and canine influenza H3N8 virus PA-X proteins: Suppression of reporter gene expression and modulation of global host gene expression

Abstract

The influenza PA-X protein is translated from the PA open reading frame from frameshifting and suppresses cellular gene expression due to its ribonuclease activity. We further defined the functional roles of PA-X by comparing PA-X proteins from two related viruses – equine influenza (EIV) and canine influenza (CIV) H3N8 – that differ in a C-terminal truncation and internal mutations. In vitro reporter gene assays revealed that both proteins were able to suppress gene expression. Interestingly, EIV PA-X demonstrated ~50% greater activity compared to CIV PA-X, and we identified the mutations that caused this difference. We used RNA-seq to evaluate the effects of PA-X on host gene expression after transfection into cultured cells. There were no significant differences in this property between EIV and CIV PA-X proteins, but expression of either resulted in the up-regulation of genes when compared to controls, most notably immunity-related proteins, trafficking proteins, and transcription factors.

PA-X: a key regulator of influenza A virus pathogenicity and host immune responses

“…Moreover, unlike the specific role played by PA in cutting single-stranded (ss)RNA, PA-X is capable of digesting both ssRNA and double-stranded (ds)RNA, with a preference for ssRNA substrates, such as poly r(A) or poly r(U), which suggests that PA-X has a wide-spread degradation ability for various host RNAs [42]. Consistent with the results from both these studies, by comparing the PA-X proteins from EIV and CIV H3N8 viruses, Feng et al found that position 231 (Ser) and the C-terminal elongated tail both contribute to the stronger host-shutoff ability of EIV PA-X relative to CIV PA-X [37]. Collectively, these studies reveal that both the N-and C-termini of PA-X contribute to its overall shutoff ability.…”

“…Using global transcriptional profiling, they further demonstrated that down-regulation of PA-X expression leads to an accelerated global host response in virus-infected mouse lungs, notably inflammatory, apoptotic, and T lymphocyte responses [11]. Many later studies have also reported on PA-X-mediated host-shutoff activity in human H1N1 virus [23,26,27,29,[32][33][34], avian H9N2 virus [35], avian H5N1 virus [24,36], equine influenza virus (EIV) and canine influenza virus (CIV) of the H3N8 subtype [37], swine H1N2 influenza virus [38], and triple-reassortment (TR) H1N2 swine influenza virus (SIV) [39]. Therefore, the universal expression of PA-X in IAVs and the relatively conservative genetic characteristics of the X-ORF suggest that the shutoff ability of the PA-X protein may also be a common functionality in all influenza virus strains.…”

Interplay of PA-X and NS1 Proteins in Replication and Pathogenesis of a Temperature-Sensitive 2009 Pandemic H1N1 Influenza A Virus

“…3A). Several studies have reported that the PA-X protein is involved in the inhibition of host protein expression (15,16,18,20,35,42,43). In order to generate a PA-X-deficient construct, the frameshift sequence was changed to two different sequences to potentially inhibit PA-X expression (UCC UUC AGA or AGC UUC AGA) (Fig.…”

“…In order to generate a PA-X-deficient construct, the frameshift sequence was changed to two different sequences to potentially inhibit PA-X expression (UCC UUC AGA or AGC UUC AGA) (Fig. 3A) (15,16,18,20,35,42,43). Importantly, none of the approaches used for the abolition of the ribosomal frameshift motif changed the PA amino acid sequence.…”

“…It has been previously shown that the PA-X and NS1 proteins from pH1N1 can and cannot, respectively, suppress host protein expression, and a correlation between their individual function and viral replication and pathogenesis has also been described (34,35,43). Studies on the contribution of PA-X to viral pathogenicity showed IAV strainspecific differences (15,20,22,35,42,43). Jagger et al showed that PA-X expression reduced the viral pathogenicity of 1918 H1N1 IAV (15).…”