Influenza A virus defective viral genomes are inefficiently packaged into virions relative to wild-type genomic RNAs

Abstract

Deletion-containing viral genomes (DelVGs) are commonly produced during influenza A virus infection and have been implicated in influencing clinical infection outcomes. Despite their ubiquity, the specific molecular mechanisms that govern DelVG formation and their packaging into defective interfering particles (DIPs) remain poorly understood. Here, we utilized next-generation sequencing to analyze DelVGs that form de novo early during infection, prior to packaging. Analysis of these early DelVGs revealed that deletion formation occurs in clearly defined hotspots and is significantly associated with both direct sequence repeats and enrichment of adenosine and uridine bases. By comparing intracellular DelVGs with those packaged into extracellular virions, we discovered that DelVGs face a significant bottleneck during genome packaging relative to wild type genomic RNAs. Surprisingly, packaged DelVGs exhibited no signs of enrichment for specific deletion characteristics suggesting that all DelVGs are equally limited in packaging efficiency. Our data provide the first unbiased, high-resolution portrait of the diversity of DelVGs that are generated by the IAV replication machinery and shed light on the mechanisms that underly DelVG formation.

Library-based analysis reveals segment and length dependent characteristics of defective influenza genomes

“…similar finding; that HA readily accumulates deletions within cells, but that these deletions are generally lost during packaging (Alnaji et al, 2021). Our study, which provides length variation above that found in natural populations, provides an explanation in the form of divergent requirements in the amount of sequence necessary for efficient packaging.…”

“…The divergence in packaging between PB1 and HA also underscores the capacity of this step in the viral lifecycle to shape the distribution of defective viral segments. A recent report described a similar finding; that HA readily accumulates deletions within cells, but that these deletions are generally lost during packaging ( Alnaji et al, 2021 ). Our study, which provides length variation above that found in natural populations, provides an explanation in the form of divergent requirements in the amount of sequence necessary for efficient packaging.…”

“…The first question we wished to address with these libraries was whether length, independent of other features of the viral life cycle, influences genome replication. There are conflicting reports concerning whether shorter segments replicate faster, and thus outcompete their longer counterparts ( Laske et al, 2016 ; Alnaji et al, 2021 ). To make these measurements, we co-transfected libraries with plasmids encoding the minimal influenza genome replication machinery—PB2, PB1, PA, and NP—and measured barcode abundance in viral genomes after 24 hours.…”

“…In addition to an advantage during genome replication, it has also been posited that segments bearing deletions might outcompete their full-length counterparts in packaging, although recent work has suggested that the opposite may be true ( DUHAUT and MCCAULEY, 1996 ; Alnaji et al, 2021 ). To explore aspects of the viral lifecycle uncaptured in our genome replication assay, we transfected cells with our libraries and the viral replication machinery and waited 24 hours.…”

The influenza virus RNA polymerase as an innate immune agonist and antagonist

“…4B) [22,98]. Such polymerase translocations might be affected or directed by A/U-rich sequences, which have been observed near DVG breakpoints [108,109]. It is possible that such A/U-rich sequences facilitate separation of the template and nascent strand prior to translocation.…”