Apoptosis, cytokine and chemokine induction by non-structural 1 (NS1) proteins encoded by different influenza subtypes

Chan, Lam Wy, Yeung

Description

Influenza pandemic remains a serious threat to human health. Viruses of avian origin, H5N1, H7N7 and H9N2, have repeatedly crossed the species barrier to infect humans. Recently, a novel strain originated from swine has evolved to a pandemic. This study aims at improving our understanding on the pathogenic mechanism of influenza viruses, in particular the role of non-structural (NS1) protein in inducing pro-inflammatory and apoptotic responses. Methods Human lung epithelial cells (NCI-H292) was used as an in-vitro model to study cytokine/chemokine production and apoptosis induced by transfection of NS1 mRNA encoded by seven infleunza subtypes (seasonal and pandemic H1, H2, H3, H5, H7, and H9), respectively. Results The results showed that CXCL-10/IP10 was most prominently induced (> 1000 folds) and IL-6 was slightly induced (< 10 folds) by all subtypes. A subtype-dependent pattern was observed for CCL-2/MCP-1, CCL3/MIP-1α, CCL-5/RANTES and CXCL-9/MIG; where induction by H5N1 was much higher than all other subtypes examined. All subtypes induced a similar temporal profile of apoptosis following transfection. The level of apoptosis induced by H5N1 was remarkably higher than all others. The cytokine/chemokine and apoptosis inducing ability of the 2009 pandemic H1N1 was similar to previous seasonal strains. Conclusions In conclusion, the NS1 protein encoded by H5N1 carries a remarkably different property as compared to other avian and human subtypes, and is one of the keys to its high pathogenicity. NCI-H292 cells system proves to be a good in-vitro model to delineate the property of NS1 proteins.

Sujets

• Influenza pandemic
• Avian influenza
• NS1 Influenza Protein
• Inflammation
• Cytokine storm
• Apoptosis
• Pathogenesis

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 4
• Langue: English

Extrait

Lam et al. Virology Journal 2011, 8:554
http://www.virologyj.com/content/8/1/554
RESEARCH Open Access
Apoptosis, cytokine and chemokine induction by
non-structural 1 (NS1) proteins encoded by
different influenza subtypes
1 1 1,2*WY Lam , Apple CM Yeung and Paul KS Chan
Abstract
Background: Influenza pandemic remains a serious threat to human health. Viruses of avian origin, H5N1, H7N7
and H9N2, have repeatedly crossed the species barrier to infect humans. Recently, a novel strain originated from
swine has evolved to a pandemic. This study aims at improving our understanding on the pathogenic mechanism
of influenza viruses, in particular the role of non-structural (NS1) protein in inducing pro-inflammatory and
apoptotic responses.
Methods: Human lung epithelial cells (NCI-H292) was used as an in-vitro model to study cytokine/chemokine
production and apoptosis induced by transfection of NS1 mRNA encoded by seven infleunza subtypes (seasonal
and pandemic H1, H2, H3, H5, H7, and H9), respectively.
Results: The results showed that CXCL-10/IP10 was most prominently induced (> 1000 folds) and IL-6 was slightly
induced (< 10 folds) by all subtypes. A subtype-dependent pattern was observed for CCL-2/MCP-1, CCL3/MIP-1a,
CCL-5/RANTES and CXCL-9/MIG; where induction by H5N1 was much higher than all other subtypes examined. All
subtypes induced a similar temporal profile of apoptosis following transfection. The level of apoptosis induced by
H5N1 was remarkably higher than all others. The cytokine/chemokine and apoptosis inducing ability of the 2009
pandemic H1N1 was similar to previous seasonal strains.
Conclusions: In conclusion, the NS1 protein encoded by H5N1 carries a remarkably different property as compared
to other avian and human subtypes, and is one of the keys to its high pathogenicity. NCI-H292 cells system proves
to be a good in-vitro model to delineate the property of NS1 proteins.
Keywords: Pandemic influenza, Avian influenza, NS1, Inflammation, Hypercytokinemia, Apoptosis, Pathogenesis
Background following the initial detection in Mexico and United
Influenza A viruses are major animal and human patho- States in April 2009, resulting in another influenza pan-
gens with potential to cause pandemics. Avian subtypes demic as declared by the World Health Organization
H5N1, H7N7 and H9N2 have repeatedly crossed the (WHO) on June 11 2009 [10]. Although most of the
species barrier to infect humans [1-8]. Since 2003, there infections are associated with a mild, self-limiting influ-
have been repeated outbreaks of H5N1 in poultries and enza-like illness; the fact that some severe and even fatal
sporadic human infections associated with high mortal- outcomes have been observed in individuals without
underlying medical conditions poses concerns regardingity [8,9]. The recently emerged swine-origin influenza A
virus (2009 pandemic H1N1 influenza virus - 2009 the pathogenesis of 2009 pdmH1N1 [11,12].
pdmH1N1) has spread globally within a few months Previous data on human infection with avian influenza
virus indicate that cytokine storm is a key mediator, as
* Correspondence: [e-mail] well as a predictor, for adverse clinical outcomes; espe-
1Department of Microbiology, Faculty of Medicine. The Chinese University of cially the haemophagocytic syndrome commonly seen in
Hong Kong, 1/F Clinical Sciences Building, Prince of Wales Hospital, Shatin,
severe human influenza A H5N1 infections [4,13-16]New Territories, Hong Kong Special Administration Region of the People’s
Republic of China The preferential infection of deeper lung cells and the
Full list of author information is available at the end of the article
© 2011 Lam et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.Lam et al. Virology Journal 2011, 8:554 Page 2 of 9
http://www.virologyj.com/content/8/1/554
prompt induction of apoptosis may also explain the 504/2004) (H7N3/2004) which caused conjunctivitis and
rapid deterioration in lung function [17]. In short, influ- mild upper respiratory tract infection in humans in
enza infection can go through a direct pathogenic path- Canada, an H9N2 isolate (A/Duck/Hong Kong/Y280/
way by inducing apoptosis, and hence cell death and 1997) (H9N2/1997) that was closely related to those
loss of critical function; and alternatively or most prob- strains found in human H9N2 infections in Hong Kong,
ably at the same time through an indirect pathogenic and two previous circulating seasonal influenza strains
pathway by inducing excessive cytokine/chemokine pro- isolated in 2002 (A/HongKong/CUHK-13003/2002)
duction from the infected cells. The state of hypercyto- (H1N1/2002) and 2004 (A/HongKong/CUHK-22910/
kinaemia will then trigger adverse consequences such as 2004) (H3N2/2004). Stocks of these viruses grown in
haemophagocytic syndrome [18]. Mandin-Darby canine kidney (MDCK) cells were used.
The virulence of influenza A virus is a polygenic trait.
Multiple molecular interactions are involved in deter- Cell cultures
mining the outcome of an influenza infection in certain The bronchial epithelial cell line, NCI-H292, derived
host species [19-28]. The genome of influenza virus is from human lung mucoepidermoid carcinoma cells
segmented, consisting of eight single-stranded, negative (ATCC, CRL-1848, Rockville, MD, USA), were grown
sense RNA molecules, which encode eleven proteins as monolayers in RPMI-1640 medium (Invitrogen,
[29]. These are polymerase basic protein 1 (PB1), PB1- Carlsbad, CA) supplemented with 10% fetal bovine
F2 protein, polymerase basic protein 2 (PB2), polymer- serum (FBS), 100 U/mL penicillin and 100 μg/mL
ase acidic protein (PA), hemagglutinin (HA), nucleopro- streptomycin (all from Gibco, Life Technology, Rock-
tein (NP), neuraminidase (NA), matrix protein 1 (M1), ville, Md., USA) at 37°C in a 5% CO incubator. NCI-2
matrix protein 2 (M2), non-structural protein 1 (NS1) H292 cells were used to study the host cellular
and non-structural protein 2 (NS2) [17]. This study response to NS1 proteins.
focused on NS1 protein which carries multiple functions
including the control of temporal synthesis of viral-spe- In-vitro transcription of NS1 mRNA
cific mRNA and viral genomic RNAs [30,31], and inter- Viral NS1 mRNA was prepared from an in-vitro tran-
action with the cellular protein phosphatidylinositol-3- scription system. Total RNA was extracted from virus-
kinase (PI3-kinase) [32-34]; which may cause a delay in infected cell lysates using the TRIzol-total RNA extrac-
virus-induced apoptosis [35]. NS1 protein also has an tion kit (Invitrogen). The extracted RNA was eluted in
ability to circumvent the host cell antiviral responses by 30 μL of nuclease-free water, and stored in aliquots at
blocking the activation of RNaseL [36], limiting the -80°C until used. In order to avoid contamination with
induction of interferon (IFN)-b [37-39], interacting with genomic DNA, the extracted preparation was treated
the cellular protein retinoic acid-inducible gene product with DNA-Free DNase (Invitrogen). The quality of
I (RIG-I) [40-42], blocking host cell mRNA polyadenyla- extracted RNA preparation was assessed by measuring
tion [43,44], blocking the double-stranded-RNA-acti- optical density at 260/280 nm with the NanoDrop ND-
vated protein kinase (PKR)-mediated inhibition of 1000 spectrophotometer (NanoDrop Technologies, Wil-
protein synthesis [31,45], and interacting with cellular mington, DE). cDNA were reversely transcribed from
PDZ-binding proteins [46]. Furthermore, it has been RNA using the SuperScript™ III reverse transcriptase
shownthatNS1proteinpreventsthematurationof (Invitrogen). DNA fragments containing the coding
human primary dendritic cells, thereby limiting host T- region of the NS1 gene were linked to a T7 promoter
cell activation [47]. sequence, with or without “hexa histidine-tag” and a
To improve our understanding on the pathogenic polyA tail was created at the end of the fragment by
®mechanism of the newly emerged pandemic strain as PCR. The PCR was performed using the Platinum Taq
well as for influenza viruses in general, we set on this DNA polymerase high fidelity (Invitrogen). The primers
study to examine the property of NS1 proteins encoded used for the amplification are listed in Table 1. In-vitro
by different influenza virus subtypes. transcription was performed using the mMESSAGE
mMachine T7 Ultra kit (Ambion, Austin, TX, USA)
Methods with 2 h incubation at 37°C. The mRNA was TURBO
Virus isolates DNase treated for 15 min at 37°C. Polyadenylation was
This study examined the NS1 proteins encoded by seven also performed. The mRNA products were then purified
strains of influenza A viruses including the newly using the MEGAclear kit (Ambion). The quantity and
emerged 2009 pandemic H1N1 (A/Auckland/1/2009) quality of capped mRNA produced was analyzed by
(2009 pdmH1N1), an H2 subtype (A/Asia/57/3) (H2N2), measuring optical density at 260/280 nm with the Nano-
an H5N1 isolated from a fatal case in Thailand (A/Thai/ Drop ND-1000 spectrophotometer (NanoDrop Technol-
KAN1/2004) (H5N1/2004), an H7N3 isolate (A/Canada/ ogies) and by denaturing gel electrophoresis.Lam et al. Virology Journal 201