Characterization of Neuraminidases from the Highly Pathogenic Avian H5N1 and 2009 Pandemic H1N1 Influenza A Viruses

Jia Wu,Yonghui Zhang,Maorong Wang,Xiaobing Wu,Jianfang Zhou,Honglan Zhao,Jian Lu,Fengwei Zhang,Jingdong Song,Ping Zhao,Yue Wang,Jimin Gao,Chunqiong Xu,Wenjie Tan,Xiaojing Lin,Jianxin Lu,Ralph Tripp

doi:10.1371/journal.pone.0015825

Abstract

To study the precise role of the neuraminidase (NA), and its stalk region in particular, in the assembly, release, and entry of influenza virus, we deleted the 20-aa stalk segment from 2009 pandemic H1N1 NA (09N1) and inserted this segment, now designated 09s60, into the stalk region of a highly pathogenic avian influenza (HPAI) virus H5N1 NA (AH N1). The biological characterization of these wild-type and mutant NAs was analyzed by pseudotyped particles (pseudoparticles) system. Compared with the wild-type AH N1, the wild-type 09N1 exhibited higher NA activity and released more pseudoparticles. Deletion/insertion of the 09s60 segment did not alter this relationship. The infectivity of pseudoparticles harboring NA in combination with the hemagglutinin from HPAI H5N1 (AH H5) was decreased by insertion of 09s60 into AH N1 and was increased by deletion of 09s60 from 09N1. When isolated from the wild-type 2009H1N1 virus, 09N1 existed in the forms (in order of abundance) dimer>>tetramer>monomer, but when isolated from pseudoparticles, 09N1 existed in the forms dimer>monomer>>>tetramer. After deletion of 09s60, 09N1 existed in the forms monomer>>>dimer. AH N1 from pseudoparticles existed in the forms monomer>>dimer, but after insertion of 09s60, it existed in the forms dimer>>monomer. Deletion/insertion of 09s60 did not alter the NA glycosylation pattern of 09N1 or AH N1. The 09N1 was more sensitive than the AH N1 to the NA inhibitor oseltamivir, suggesting that the infectivity-enhancing effect of oseltamivir correlates with robust NA activity.

Highlights

Influenza A viruses cause seasonal epidemics and occasional pandemics [1,2,3]
We compared the sequences of the NA proteins from influenza A/Ohio/07/2009/H1N1 (#FJ969534) and influenza A/Anhui/1/2005/H5N1
The 09N1 stalk region contains a 20-aa segment that is not present in AH N1. This gap in the AH N1 stalk region sequence results in the loss of four potential glycosylation sites and a cysteine residue from AH N1

Summary

Introduction

Influenza A viruses cause seasonal epidemics and occasional pandemics [1,2,3]. The outbreak of a novel H1N1 influenza strain became a major global issue in April 2009 and, to date, this virus, here designated 2009H1N1, has been detected in 214 countries and has caused 17,919 deaths [4]. Influenza viruses contain eight negative-sense single-stranded RNA segments that together encode 11 proteins [2]. Two of these proteins, hemagglutinin (HA) and neuraminidase (NA), are large glycoproteins found on viral envelope [1,2,6]. Compared to the A/Gs/Gd/1/96/H5N1-like stalk region, the A/WSN/33/H1N1-like stalk region has a 16-amino acid (aa) deletion of residues 57–72, A/Puerto Rico/8/34/H1N1-like has 15-aa deletion of residues 63–77, A/Hong Kong/156/97/H5N1like has a 19-aa deletion of residues 54–72, A/chicken/Italy/ 1067/99/H7N1-like has 22-aa deletion of residues 54–75, and A/ chicken/Hubei/327/2004/H5N1-like has a 20-aa deletion of residues 49–68 [15] The extent of these deletions appears to have increased gradually; the biological impacts of variations in the NA stalk are not yet clear

Methods

Results

Conclusion