Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin

Naganori Nao,Asako Shigeno,Noriko Kishida,Manabu Igarashi,Rashid Manzoor,Wakako Furuyama,Junya Yamagishi,Aiko Ohnuma,Masahiro Kajihara,Yoshimi Tsuda,Reiko Yoshida,Ayato Takada,Hiroko Miyamoto,Mary K Estes

doi:10.1128/mbio.02298-16

Naganori Nao, Asako Shigeno + Show 12 more

Open Access

https://doi.org/10.1128/mbio.02298-16

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Abstract

ABSTRACTHighly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes.

Highlights

Pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site
Our findings suggest that the RNA sequence determining the HA cleavage site amino acid motif has a key role in inducing viral polymerase slippage, which increases the frequency of nucleotide insertions, and that this mechanism contributes to the acquisition of additional codons for basic amino acids to create the polybasic HA cleavage site
Since the firefly luciferase gene that followed the 28- or 29-nucleotide linkers was out of frame, the luciferase was expected to be expressed only when nucleotides were inserted into the linker region of mRNA, cRNA, and/or viral RNA to make the linker sequence in frame with the open reading frame (ORF) of the reporter gene

Summary

Introduction

Pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. This mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site This mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. The polybasic HA cleavage site is known to be generated by multiple nucleotide insertions/ substitutions to create codons for basic amino acids [17, 24,25,26] or by recombination with cellular or viral RNAs [27,28,29] and is considered to be the primary virulence marker of HPAI viruses [16, 30]

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