Since the beginning of the project, we have setup the lab with all the necessary equipment and reagents to operate as a molecular biology research lab. We have established our SOPs and optimised techniques around the in vitro imaging of influenza A virus RNA, RNA handling including cellular fractionation and RNA half-life experiments, RNA-binding protein isolation and identification, and finally establishing how to NGS and proteomics datasets at our host institute.
The lab has recruited 2 PhD students and 2 postdocs, who have carried out the bulk of the work since the inception of the project. This has spanned 4 key project thus far, with each described in detail below.
1. TDP-43 and influenza A virus
In mammalian cells, RNA-binding proteins (RBPs) are associated co-transcriptionally to mRNAs and they control the fate of the bound RNA. These regulatory processes include through splicing, trafficking, translation and stability. RNA viruses are reliant on the host cell machinery, and the utilisation of host RBPs to regulate viral mRNAs is crucial to complete viral replication. Recently, several techniques around cross-linking and proteomics have emerged to identify RBPs crucial for the replication of RNA viruses. However, these methods have predominantly focused on positive-stranded RNA viruses like SARS-CoV-2. Influenza viruses, on the other hand, are negative-stranded RNA viruses. Their mRNA molecules undergo unique capping and polyadenylation mechanisms facilitated by the viral polymerase. Despite this, the cellular RBPs associated with influenza mRNAs have not been thoroughly explored. Our study marks the first attempt to elucidate the composition and function of influenza mRNA ribonucleoprotein complexes (mRNPs). It sheds light on the crucial role played by the viral polymerase in assembling viral mRNPs. Additionally, our findings supplement the limited data available on mRNP composition in human cells.
2. MKRN2 and influenza A virus mRNA export
Influenza A virus undergoes transcription and genome replication in the nucleus. This poses problems in the export of the multiple unspliced mRNAs from the nucleus to the cytoplasm for onward translation. Influenza A virus mRNAs likely must recruit additional host factors to serve as trafficking partners to be efficiently exported across the nuclear membrane. We sought to use a previously published technique, termed RNA-interactome capture (RIC), to uncover novel host RNA-binding proteins (RBPs) that could play a role in the post-transcriptional regulation of viral mRNAs. Taking advantage of the fact that influenza A mRNAs have been shown to occupy a large proportion of the total poly(A)+ pool of RNA within an infected cell, we identified the RBPome for mRNAs in influenza virus infected cells, which up until now had never been reported. Through this analysis we hit upon 1 RBP in particular, MKRN2, which positively regulates viral replication via an interaction with viral mRNAs. Through further experimentation we uncovered that MKRN2 translocates to the nucleus of infected cells and regulates IAV mRNA export.
3. m6A mapping on influenza A virus mRNA
IAV utilises host-cell machinery to post-transcriptionally modify bases across its viral transcripts and genome. Data from our lab and others have shown IAV transcripts to be extensively modified, but the precise location, number and effect of these modifications remains unclear. We sought to identify the location of two RNA modifications across IAV transcripts, N-6-methyladenosine (m6A) and pseudouridine (Ψ), the latter of which has not yet been investigated for IAV. m6A was mapped across IAV transcripts by way of DART-Seq coupled with Nanopore direct RNA sequencing. By comparing different experimental conditions with this method, we were able to define several ‘high confidence’ m6A sites.
4. Identification of influenza A vRNPs associating proteins that are involved in cytoplasmic trafficking
The Rab11a-based endosomal recycling pathway is exploited by respiratory RNA viruses like IAV, RSV, and potentially coronaviruses, aiding their dispersal into the airways from the apical surface of polarized epithelial cells. Late in infection, Rab11a-containing vesicles have been shown to specifically transport viral ribonucleoprotein (vRNP) complexes to the surface before packaging and budding. Rather than utilising traditional Rab11-positive recycling endosomes, virus-infected cells seem to generate remodelled Rab11a-containing vesicles, as has been observed during IAV infection. Besides Rab11a, no other conserved host co-factors have been identified among these various vRNP trafficking vesicles. Here we uncover and confirm the association of a novel transmembrane protein with IAV vRNPs in the cytoplasm, co-localizing with Rab11a, during late stages of infection. This protein recruits co-factors, which in turn are involved in endosomal biogenesis, to these unique vRNP trafficking endosomes, highlighting a pivotal role for our novel protein candidate in viral replication.
