Mechanism, Function, and Exploitation of Influenza A Virus-Activated Cell Death

PROJECT SUMMARY/ABSTRACT
Influenza A virus (IAV) triggers the death of most cell types in which it replicates, both in culture and in vivo. In
2016, we described an IAV-activated cell death pathway which accounts for almost all IAV-activated death in
infected pulmonary cells. Over that past funding cycle, we showed that this pathway is initiated when the host
sensor protein ZBP1 detects viral Z-RNAs and activates RIPK3 kinase. RIPK3 then triggers parallel, redundant
pathways of programmed necrosis (necroptosis) and apoptosis. These studies have outlined the dominant cell
death pathway activated by IAV in infected lungs. They have also demonstrated that ZBP1 is an RNA sensing
protein, and that the RNA structures it recognizes are Z-RNAs, which are unique, left-handed double-helical
RNA species that had not previously been thought to occur in nature. We have now made several intriguing
new discoveries that set the stage for this application. First, we found that the Z-RNAs produced during IAV
infections are not just viral in origin, but host cell-derived as well. Second, we observed that in alveolar
macrophages (AMs), unlike in all other lung cell types tested, ZBP1 activation does not result in cell death, but
instead in a non-cytolytic transcriptional program essential for protection against IAV lethality. Third, we found
that necroptosis during IAV infections is activated not in the cytoplasm, but in the nucleus, resulting in nuclear
rupture. Such `nuclear necroptosis' is highly inflammatory, and is a major driver of the pathogenesis during
severe influenza. Finally, we have obtained important new insight into the coevolution of IAV with the
necroptosis machinery in birds and bats, the two major natural hosts of these viruses. While birds simply do not
express ZBP1, we have found that bats possess a single amino acid alteration in RIPK3 which selectively
abrogates necroptosis (but not apoptosis) signaling, potentially explaining how these organisms tolerate such
a large diversity of IAV subtypes. To our knowledge, these discoveries provide the first evidence that
endogenous Z-RNAs may function as innate ligands for ZBP1 in anti-IAV immunity, that ZBP1 has non-cytolytic
functions during IAV infections, and that a IAV activates a unique, hyper-inflammatory form of necroptosis from
the nucleus. They also provide fresh evolutionary insight into how IAV strains are tolerated in their natural
reservoirs, but can activate pathogenic necroptosis in humans. Based these and other observations, the goals
of this proposal are to (1) to identify and characterize endogenous Z-RNA ligands for ZBP1, and to determine
their importance to ZBP1 activated cell death in IAV-infected cells; (2) to delineate the mechanism and function
of non-cytolytic ZBP1 signaling in AMs; and (3) to understand the role of nuclear necroptosis in influenza
pathogenesis and in evolution. These studies unite two labs with expertise in ZBP1 signaling (Balachandran)
and IAV pathogenesis (Thomas), and their successful completion stands to provide pioneering insight into the
mechanism and function of ZBP1-initiated cell death during IAV infections, with important clinical and
evolutionary ramifications.

Grant Number: 4R01AI135025-09
NIH Institute/Center: NIH
Principal Investigator: SIDDHARTH BALACHANDRAN