Abstract
BackgroundNon-structural protein NS1 of influenza A viruses harbours several determinants of pathogenicity and host-range. However it is still unclear to what extent each of its two structured domains (i.e. RNA-binding domain, RBD, and effector domain, ED) contribute to its various activities.MethodsTo evaluate the respective contributions of the two domains, we genetically engineered two variants of an H7N1 low pathogenicity avian influenza virus harbouring amino-acid substitutions that impair the functionality of either domain. The RBD- and ED-mutant viruses were compared to their wt- counterpart in vivo and in vitro, notably in chicken infection and avian cell culture models.ResultsThe double substitution R38A-K41A in the RBD dramatically reduced the pathogenicity and replication potential of the virus, whereas the substitution A149V that was considered to abrogate the IFN-antagonistic activity of the effector domain entailed much less effects. While all three viruses initiated the viral life cycle in avian cells, replication of the R38A-K41A virus was severely impaired. This defect was associated with a delayed synthesis of nucleoprotein NP and a reduced accumulation of NS1, which was found to reach a concentration of about 30 micromol.L− 1 in wt-infected cells at 8 h post-infection. When overexpressed in avian lung epithelial cells, both the wt-NS1 and 3841AA-NS1, but not the A149V-NS1, reduced the poly(I:C)-induced activation of the IFN-sensitive chicken Mx promoter. Unexpectedly, the R38A-K41A substitution in the recombinant RBD did not alter its in vitro affinity for a model dsRNA. When overexpressed in avian cells, both the wt- and A149V-NS1s, as well as the individually expressed wt-RBD to a lesser extent, enhanced the activity of the reconstituted viral RNA-polymerase in a minireplicon assay.ConclusionsCollectively, our data emphasized the critical importance and essential role of the RNA-binding domain in essential steps of the virus replication cycle, notably expression and translation of viral mRNAs.
Highlights
Non-structural protein NS1 of influenza A viruses harbours several determinants of pathogenicity and host-range
Our results showed that the double substitution in the RBD dramatically reduced the pathogenicity and replication potential of the virus, while the A149V substitution in the ED had only a modest effect
We introduced in the RNA-binding domain the double substitution R38A-K41A, which is considered to abrogate its RNA-binding properties [17, 20, 41]; on the other hand, the ED was modified by substitution A149V, which was previously shown to alter the IFN-antagonist activity of NS1 in avian cells [22, 24]
Summary
Non-structural protein NS1 of influenza A viruses harbours several determinants of pathogenicity and host-range. Influenza A viruses (IAVs) pose a permanent threat to human and animal health, as illustrated by the 2009 H1N1 pandemic and the frequent occurrence of epizootics with highly pathogenic avian influenza viruses (e.g. H5N1 and H5N8) across the world. Their genome consists of eight single stranded RNAs of negative polarity, which collectively code for up to fifteen proteins [1,2,3,4]. The non-structural protein NS1 of influenza viruses is considered as the major interferon antagonist of the virus This ~ 230-residue protein is highly expressed in the infected cell, where it exhibits several pro-viral activities [8, 9]. A linker region connecting the two structured domains [15, 16] confers plasticity to the quaternary structure, while the C-terminal tail (CTT, amino-acids 202-230) has the properties of an intrinsically disordered region [16]
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