In new study led by @bdadonaite.bsky.social, we show many influenza HAs (H5, H7, H9, H1, H2, H3) can use avian or human MHC-II to enter cells. We then use novel combo of deep mutational scanning & cryoEM to define how H5 HA binds to avian MHC-II. Preprint: doi.org
As background, influenza long known to use sialic acid as entry receptor. ~7 yrs ago was shown bat flu can enter cells via MHC-II (nature.com); later shown for some H2 & H3 strains. We set out to assess how common MHC-II usage is & define how HA interacts w MHC-II.
www.nature.com
We first measured ability of two H5 HAs to enter cells via sialic acid or MHC-II. As shown below, both HAs could use tufted duck & to lesser extent human MHC-II. [Note: experiments used pseudoviruses, which can only undergo single round of cell entry, providing safe way to study HA]
We tested 80 H5 HAs: most but not all could enter cells via tufted duck & to lesser extent human MHC-II. Note MHC-II highly variable within and between species. See dms-vep.org for interactive version of below plot.
To understand how HA interacts w MHC-II, we measured how all H5 HA mutations affect pseudovirus entry via sialic acid or tufted duck MHC-II. Identified loss-of-function mutants that could only use MHC-II or sialic acid.
The mutations that reduced MHC-II entry clustered in a region on HA head defining the MHC-II binding surface. We also directly measured how HA mutations affect binding to tufted duck MHC-II, and identified same binding surface.
To determine structure, we produced H5 HA protein w mutations that increased binding to tufted duck MHC-II. This HA increased fraction of particles bound to MHC-II in ns-EM, and we were able to use it solve cryo-EM structure of H5 HA bound to tufted duck MHC-II.
To validate structure, we used inverted pseudotyping deep mutational scanning to measure how mutations to tufted duck MHC-II affect binding to H5 HA. Most mutations with big impact in alpha chain, but beta-chain mutations near peptide-binding groove also have effect.
So although structure only 4.8 A, it is corroborated by deep mutational scanning of both HA and MHC-II showing that sites in both proteins that affect binding are at structural interface. (Sites where mutations decrease binding are red in structure below)
Structure & deep mutational scanning suggest identity of peptide bound to MHC-II could influence interaction of HA & MHC-II. Also, HA binding would likely block ability of MHC-II to interact with T-cell (perhaps analogous to how EBV gp42 can bind to MHC-II to block T cell activation).
We also showed H7 HA binds tufted duck MHC-II similarly to H5 HA, & some H1, H2, H3, & H9 HAs also can use avian or human MHC-II. But patterns vary among strains. For instance, an avian influenza HA and the 1918 HA can use tufted duck MHC-II, but later human strains cannot.
Although breadth of MHC-II usage among HAs suggests evolutionary selection in some strains/hosts, further work needed to understand biological relevance. Hypotheses include that it could impact cell entry or immunogenicity in actual infection of some hosts. All our data is available along with rich set of interactive plots: dms-vep.org
dms-vep.org
Note our study used pseudoviruses and conditionally replicative virions to ensure biosafety, and reports deep mutational scanning only for HA usage of tufted duck MHC-II to limit any information hazard concerns.
Please see the preprint for additional details: doi.org Thanks to @bdadonaite.bsky.social for leading study, and Annie Dosey, @jahn0.bsky.social, @timyu.bsky.social, Sara Sunshine, Ariana Farrell, and @kinglabipd.bsky.social for valuable contributions.
doi.org