Influenza viruses are a major global health threat, causing disease both in humans and birds. Seasonal epidemics cause about half a million deaths each year and are a significant burden for health care systems and economies worldwide.
Every once in a while a reassortment event of two influenza viruses can occur, resulting in a novel virus with no previous exposure in the human population that can cause a global pandemic and considerable mortality.
Influenza viruses are enveloped single-stranded negative-sense segmented RNA viruses. The ends of each of these eight segments are bound by the viral polymerase consisting of the subunits PA, PB1 and PB2 and the remaining viral RNA is coated by viral nucleoprotein (NP). These complexes are called ribonucleoproteins or RNPs.
Originally an avian virus, influenza can cross the species barrier and infect humans and other mammalian species by acquiring certain adaptive mutations enabling efficient transmission and replication of the virus in a mammalian host.
Specifically, certain adaptive mutations in the viral polymerase are needed to enable avian viral genome replication in mammalian hosts and most of these adaptive mutations occur in or around a subdomain of the polymerase subunit PB2, called the 627-domain. Even though most domains of the viral polymerase have been studied and described in detail already, no function had been attributed to the 627-domain so far.
In this study we used newly available structural data for the viral polymerase to make structure-guided deletions of the PB2 subdomains and tested the functionality of these polymerases using a combination of in vitro and cellular assays.
First we showed that truncations of PB2 don’t affect expression or assembly of the polymerase in human cells and that the nuclear accumulation of the polymerase isn’t altered. Using purified polymerase we can study different functions of the polymerase in vitro by adding different nucleotides and primers to the reaction.
For complete genome replication the viral polymerase needs to carry out several different functions: The resident polymerase of incoming RNPs needs to be able to make mRNA from the viral genome by cleaving host capped pre-mRNAs and using the resulting short capped RNAs as primer for mRNA transcription (‘cap-snatching’). The polymerase needs to replicate the negative-sense viral RNA (vRNA) into an intermediate complementary positive-sense RNA (cRNA) and form a cRNP complex which then is used as a template for vRNA synthesis.
In vitro we were able to identify different structural requirements for mRNA, cRNA and vRNA synthesis and that the 627-domain is not required for any of these core polymerase activities. However, when testing the requirement of the 627-domain in a minireplicon system in human or avian cells, we found that the 627-domain is an essential component of the viral replication machinery. For replication to occur in a cell, the viral polymerase needs to be able to target and interact with replicating RNPs in order to assembly new RNPs from the nascently synthesised viral RNA.
Using a technique where we measure the amount of newly synthesised cRNA that transfected polymerase can ‘stabilise’ and protect in an infection as a measure for how efficiently these RNP-polymerase complexes are formed, we were able to determine that the 627-domain is needed for this ‘stabilising’ effect. This final result suggests that the 627-domain is needed for the polymerase to ‘interact’ with the replicating RNP in order to enable replication in the cell.
Author: Benjamin Nilsson
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