Characterization of Hemagglutinin Protein Structure of H5N1 Virus

Circulating in Indonesia based on Proteomic Analysis

M. Irfan Hadi

, Hanik Faizah

, Misbakhul Munir

, M Yusuf Alamudi

, Risa Purnamasari

and Nova Lusiana

UIN Sunan Ampel Surabaya, Indonesia

AI Lab-Yayasan Prof Nidom, Surabaya, Indonesia

risap1989@gmail.com, novalusiana@uinsby.ac.id

Keywords: characterization, hemagglutinin, H5N1, Indonesia, proteomics

Abstract: Bird flu virus or H5N1 is one of the infectious agents that still become a global problem. Based on data from

the World Health Organization (WHO) until June 15, 2017, more than 800 people were infected with the

H5N1 virus, and nearly 500 people died. Indonesia is the 2nd country in the world with the highest number

of infected bird flu virus that is 199 people infected and 167 people died, with more than 70% case fatality

rate. Influenza A especially H5N1 has eight genes, one of them is Hemagglutinin. Hemagglutinin (HA)

protein present on the surface of the virus. The presence of a "cleavage site" in HA protein will improve the

pathogenic nature of the AI virus. HA protein also plays a role in the process of viral infection into cells by

interacting directly with receptors on the surface of the host cell. Besides, HA protein also functions in the

movement of the virus from one cell to another cell. Through the accumulation of mutations in HA, the AI

virus can increase its infectious potency. The purpose of this study was to characterize the Hemagglutinin of

bird flu virus in Indonesia by using bioinformatics method. The study discovered the differences in the

characterization of bird flu virus circulating in Indonesia based on the proteomic analysis.

1 INTRODUCTION

Influenza is a strand-negative RNA virus belonging

to the Orthomyxoviridae family and has four types of

influenza viruses, i.e., Influenza A, influenza B,

influenza C, and thogoto virus. Influenza A can infect

hosts in a wide range including birds, poultry, and

mammals, with geographic coverage around the

world (Luke and Subbarao, 2006). According to

WHO (2017), the cumulative data of avian influenza

subtype H5N1 in human cases was 859 cases with

453 deaths overall occurred in 16 countries, while in

Indonesia there were 199 cases with 167 deaths.

Hemagglutinin (HA) is a glycoprotein having a

molecular weight of 76,000 kDa and a rod-shaped

molecule located in the layer of influenza virus. HA

protein plays an essential role in determining the

pathogenicity of influenza viruses, and this segment

often has spontaneous mutations that can lead to a

new pandemic and endemic influenza (Li et al.,

2004). Hemagglutinin consists of 5 antigenic sites

ranging from sites A, B, C, D, and E. The primary

function of these sites is as a receptor binding to sialic

acid from the target cell of influenza virus infection,

in an attempt to initiate the fusion process of virus

particle through the cell membrane (Bruce et al.,

2010; Lutz et al., 2005). The hemagglutinin consists

of two subunits: HA1 and HA2 bound by a disulfide

bridge. HA from the avian, horse and pig influenza

viruses have specificity to α (2,3) -linkage sialic acid

receptors, whereas HA from human influenza virus is

specific to α (2,6) -linkage sialic acid receptors. The

receptor of α (2, 3) -linkage sialic acid is found in the

mucosal tracts of avian, horse and certain marine

mammals, whereas α (2, 6) -linkage sialic acid

receptor is found in the human respiratory tract

mucosa. Specifically, in mucosal cells of the pig

trachea can be found both types of receptors, so pigs

are the only animals that can be attacked either by

human influenza virus or non-human influenza virus

(Brooks et al., 2010).

HA protein tends to change as a result of

mutations in gene encoding protein synthesis,

whereas HA protein is a major determinant of the

human immune system to recognize influenza

Hadi, M., Faizah, H., Alamudi, M., Munir, M., Purnamasari, R. and Lusiana, N.

Characterization of Hemagglutinin Protein Structure of H5N1 Virus Circulating in Indonesia based on Proteomic Analysis.

DOI: 10.5220/0008903700002481

In Proceedings of the Built Environment, Science and Technology International Conference (BEST ICON 2018), pages 95-98

ISBN: 978-989-758-414-5

antigens and produce specific antibodies against

influenza virus infection. As a result of changes in

HA, immune cells will not be able to recognize

influenza viruses that infect humans (Brooks et al.,

2010; Bruce et al., 2010; Lutz et al., 2005).

Proteolytic activation of hemagglutinin proteins

is an important factor for the infectivity and spread of

the virus throughout the body. Differences in HA VAI

protein sensitivity to host proteases will be associated

with the virulence levels (Puthavathana et al., 2005;

Shangguan et al., 1998). In addition to its role in

antigenic properties and viral pathogenicity level,

hemagglutinin proteins also play a role in the

specificity of VAI hosts. One of the factors that play

a role in this VAI infection is the compatibility

between the virus with the receptor on the surface of

the host cell. (Harvey et al., 2004). The study aimed

to characterize bird flu virus circulating in Indonesia

in 2008-2012.

2 METHOD

Hemagglutinin protein of H5N1 virus in 2008-2012

was collected from NCBI GenBank with acecesion

number of AKC43930, BAL61222, AJP13841,

AGC96167. The data of Hemagglutinin amino acids

from GenBank were analyzed using SwissProt

software and visualized using Pymol Software.

3 RESULTS AND DISCUSSION

This study was conducted to characterize the

Hemagglutinin protein of bird flu virus circulating in

Indonesia during 2008 to 2012. It was due to the bird

flu virus experienced a change from subclade 2.1.3 to

2.3.2 in those years. Based on the study conducted on

subclasses 2.1.3 and 2.3.2 from 2008 to 2012, the

Hemagglutinin of avian influenza virus had the form

of Homo-Trimer. Similar result was reported by

Stevens et al. (2006) and Zuo et al. (2015).

Hemagglutinin of influenza virus belongs to

transmembrane glycoprotein type 1 and is located on

the surface of the virus as a homotrimer.

Trimerisation was possible due to proteolytic

cleavage unfolding in HA0, as a precursor when the

folding process is in the monomer form.

Hemagglutinin has two chains: HA1 and HA2, each

monomer consists of a globular head (part of HA1)

and stem region (part of HA2) (Staneková and

Varečková, 2010; Velkov et al., 2013). HA belongs

to transmembrane glycoprotein type 1, the sequence

of signals that can be removed after translation. HA

also belongs to the membrane anchor domain near the

C terminal, and has a short cytoplasmic terminal

(Steinhauer, 1999), has a size of 13.5 nm and a

molecular weight of 76 kDa (Cheng et al., 2012a).

Hemagglutinin is a target molecule to neutralize

antibodies and is therefore considered as a primary

surface antigen (Ducatez et al., 2010). The primary

function of HA is initiation in infecting the host,

involving in the introduction of host cells and the

binding of the virus to host cell receptors, composed

of sialic acid (Cheng et al., 2012b; Edinger et al.,

2014).

The study was also obtained the number and type

of ligand in hemagglutinin on bird flu virus

circulating in Indonesia in 2008-2012 (table 1). There

were differences in the number and nature of ligands

in both subclasses 2.1.3 and 2.3.2. The study had a

similar result with the study conducted by Xu and

Wilson (2011). The ligand of GAL, NAG, and SIA

was associated with RBS (receptor binding site)

(Lazniewski et al., 2017). The study was also

obtained homologous protein from H5N1 (table 2).

Of H5N1 protein suspected H5N1 virus had the

possibility of part of Influenza virus more than one

subtype. It can be found about the possibility of the

origin of influenza virus especially H5N1 in

Indonesia although it requires a further study,

especially in the meta bioinformatics field.

Table 1:

Number and Type of Ligand in Hemagglutinin

Subclade

and year

of isolat

# of

Ligand

Type of

Ligand

Nature of

Ligand

2.3.2

(2012)

GAL (SUGAR (2-

MER) NAG

(SUGAR (N

ACETYL-D-

GLUCOSAMINE

)

Binding site not

conserved

Not biologically

relevant

2.1.3

(2012)

NAG (SUGAR

(N-ACETYL-D

GLUCOSAMINE

)

Binding site not

conserved

2.1.3

(2010)

NAG (SUGAR

(N-ACETYL-D

GLUCOSAMINE

) NAG (SUGAR

(2-MER) SIA

(SUGAR (2-

MER)

Clashing with

protein

Binding site not

conserved

Not biologically

relevant

Binding site not

conserved

2.1.3

(2008)

NAG (SUGAR

(N-ACETYL-D

GLUCOSAMINE

) NAG (SUGAR

(2-MER)

Clashing with

protein

Binding site not

conserved

Not biologically

relevant

Binding site not

conserved

BEST ICON 2018 - Built Environment, Science and Technology International Conference 2018

Table 2 : Homolog Protein

Subclade

dan Year of

isolation

Homolog Protein

2.3.2

(2012)

 H3 HAEMAGGLUTININ HA1 CHAIN

 Influenza B hemagglutinin (HA)

 PROTEIN (INFLUENZA

RECOMBINANT HA2 CHAIN)

 HEMAGGLUTININ FUSION

PEPTIDE G8A MUTANT

 uncharacterized protein

 Influenza H5 HA head domain VietNam

rdt mutations

 HEMAGGLUTININ FUSION

PEPTIDE G8A MUTANT

2.1.3

(2012)

 Influenza B hemagglutinin (HA)

 Influenza H5 HA head domain VietNam

rdt mutations

 HEMAGGLUTININ FUSION

PEPTIDE G8A MUTANT

2.1.3

(2010)

 H3 HAEMAGGLUTININ HA1 CHAIN

 Influenza B hemagglutinin (HA)

 Influenza H5 HA head domain VietNam

rdt mutations

 PROTEIN (INFLUENZA

RECOMBINANT HA2 CHAIN)

 Gp7-MYH7(1173-1238)-EB1 chimera

protein

 uncharacterized protein

2.1.3

(2008)

 Influenza B hemagglutinin (HA)

 Influenza H5 HA head domain VietNam

rdt mutations

 H3 HAEMAGGLUTININ HA2 CHAIN

 PROTEIN (INFLUENZA

RECOMBINANT HA2 CHAIN)

 uncharacterized protein

4. CONCLUSIONS

The study demonstrated that H5N1 virus

circulating in Indonesia had a form of homotrimer

based on the bioinformatic analysis. Besides, H5N1

virus circulating in Indonesia from 2008 to 2012 had

the number of ligands and homology based on protein

structure.

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