Altered Nucleoprotein Binding to Influenza Virus RNA Impacts Packaging Efficiency and Replication

Abstract

24The genome of Influenza A viruses consists of eight negative-sense RNA segments that 25 are bound by viral nucleoprotein (NP). We recently showed that NP binding is not uniform along 26 the segments but exhibits regions of enrichment as well as depletion. Furthermore, genome-wide 27 NP binding profiles are distinct even in strains with high sequence similarity, such as the two 28 H1N1 strains A/WSN/1933 and A/California/07/2009. Here, we performed interstrain segment 29 swapping experiments with segments of either high or low congruency in NP binding, which 30 suggested that a segment with a similar overall NP binding profile preserved replication fitness of 31 the resulting virus. Further sub-segmental swapping experiments demonstrated that NP binding 32 is affected by changes to the underlying nucleotide sequence, as NP peaks can either become 33 lost or appear de novo at mutated regions. Unexpectedly, these local nucleotide changes in one 34 segment not only affect NP binding in cis, but also impact the genome-wide NP binding profile on 35 other segments in a vRNA sequence-independent manner, suggesting that primary sequence 36 alone is not the sole determinant for NP association to vRNA. Moreover, we observed that sub-37 segmental mutations that affect NP binding profiles can result in reduced replication fitness, which 38 is caused by defects in vRNA packaging efficiency and an increase in semi-infectious particle 39 production. Taken together, our results indicate that the pattern of NP binding to vRNA is 40 important for efficient virus replication. 41 3 Author Summary 42 Each viral RNA (vRNA) segment is bound by the polymerase complex at the 5′ and 3′ 43 ends, while the remainder of the vRNA is coated non-uniformly and non-randomly by 44 nucleoprotein (NP). To explore the constraints of NP binding to vRNA, we used high-throughput 45 sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) of mutant H1N1 46 strains with exchanged vRNA sequences and observed that NP binding can be changed based 47 on vRNA sequence. The most striking observation of our study is that nucleotide changes in one 48 segment can have genome-wide effects on the NP binding profile of other segments. We refer to 49 this phenomenon as the 'butterfly effect' of influenza packaging. Our results provide an important 50 context in which to consider future studies regarding influenza packaging and assembly.51 4 Introduction 52The segmented nature of influenza A virus (IAV) genomes poses a logistical challenge for 53 viral replication, as all of the eight negative-sense single-stranded RNA segments must find their 54 way into a budding virion to give rise to an infectious particle [1, 2]. Following nuclear export, viral 55 ribonucleoprotein complexes (vRNP) containing newly synthesized viral RNA (vRNA) assemble 56 on recycling endosomes en route to the plasma membrane for packaging into virions [3, 4]. An 57 accumulating body of evidence suggests that the intracellular pre-assembly process of vRNP 58 trafficking is media...