Introduction

Avian influenza is an important infectious disease of birds ranged from mild to severe form of illness, caused by influenza A virus, 15 subtypes exists in nature. The pathogenicity of virus was multifactorial, depends on various surface encoding proteins. In addition to surface glycoproteins hemagglutinin and neuraminidase that determine recognition of host cell receptor, were also the main target of host immune response, other protein have contributed for the virulence of highly pathogenic H5N1 strains (Obenauer 2006). All the avian influenza viruses are classified by two surface proteins HA and NA. Till date, 16 HA subtype (H1-H16) and 9 NA subtypes (N1-N9) of influenza A viruses have been identified (Fouchier 2005). The three viral envelope proteins of influenza A virus are most medically important as these are essential viral proteins targeted by host antibody or antiviral drugs. The HA glycoprotein forms spikes at the virions, mediating attaching to host cell sialoside receptors and subsequent get entry by membrane fusion. The NA forms knoblike structures on the surface of virus particles and catalyzes the discharge from infected cells and allow virus to spread. Both proteins (HA and NA) are significant in constructing the phylogenic tree as they are present in the whole influenza virus. In 2003, outbreaks of highly pathogenic influenza A virus H5N1 was identified among the poultry in the republic of Korea (Lee et al 2005). Subsequently, outbreaks by antigenically related viruses were reported among poultry in Thailand, Vietnam, Japan, China, Cambodia, Malaysia and Indonesia, reason for this apparent simultaneous occurrence of H5N1 outbreaks in many Asian countries. However, H5N1 viruses have also been found in dead migratory birds, which may suggest a role of wild birds in the maintenance and spread of H5N1 virus in the region (Chen et al 2005). WHO (2005) reported an outbreaks of highly pathogenic influenza virus (H5N1) recently spread to nine Asian countries. Nucleotide sequences based analysis of H5N1 isolates from birds and humans showed two distinct clades with a non overlapping geographical distribution.

The bioinformatics prediction of protein subcellular localization had extensively been studied for prokaryotic and eukaryotic organisms but not for viruses whose proteins are often involved in extensive interactions at various subcellular localizations with host proteins, these predictions benefit the study of infectious disease to understand the role of proteins in host cells and thereby useful for designing improved therapeutic interventions (Scott et al 2006). Identification of a bacterial proteins subcellular localization (SCL) provides valuable clues regarding its biological function. Surface-exposed or secreted proteins are of primary interest due to their potential as vaccine candidates, diagnostic agents and the ease with which they may be accessible to drugs (Allan and Wren 2003; Mora et al 2003; Paine and Flower2002). Computational SCL analysis of the growing number of completed bacterial genomes or individual proteins allows researchers to screen for vaccine/drug candidates, automatically annotate gene products or select proteins for further study. Several tools were available for the prediction of bacterial, eukaryotic protein subcellular localization. Whereas, in case of virus limited number of bioinformatics tools were available for the prediction of subcellular localization of virus protein location in virus or in host cells. The better understanding of whole proteomes of influenza virus proteins plays a vital role in regulation of gene in virus.

Bioinformatics tools used to analyze and identify several motifs from the whole proteins of influenza virus. Motifs are typically 6-30 amino acids and correspond to its active site, substrate or ligand binding site and structurally important segment of proteins. Protein kinase C is a family of related serine/threonine kinases and plays a key role in cellular responses such as neurotransmission, gene expression, and cell growth and differentiation (Nishizuka 1984). Important motif amidation, a required post-translational modification for the bioactivation of many neuropeptides, is catalyzed by a bifunctional enzyme, peptidyl glycine- amidating monooxygenase, in a two-step reaction (Stewart and Klinman, 1988). RGD motif also plays the role in the host cell attachment. Recently, many motifs in the three proteins like hemagglutinin, neuramidinase and nucleoprotein if influenza virus were identified. Theses motifs were amidation, PKC, casein kinase 2, glycosylation, tyrosin kinase, myristoylation, ATP/GTP binding site.(Tamanna et al,2006) The present study was taken to demonstrate different source of influenza in the same country and computationally analyze the whole proteomes of influenza A virus subtype H5N1.

Material And Methods

Selection of Sequence data set

Blast

Phylogenetic Analysis

Prediction and identification of motifs

Results And Discussion

Total 36 hemagglutinin (~1700 bp) and 37 neuraminidase (~1410) nucleotide sequences were used to construct the phylogenetic tree. It was analyzed that different source of virus were present in the same country, four clades were obtained in each phylogenetic tree respectively. First clade showed the HA and NA sequence similarity and proximity with all different sources of isolates like cat, duck, goose, stone marten, whooper and swan, these were from Germany. It was concluded that same influenza virus was spread in entire host. All other three clades showed same sort of proximity (Fig.1 and Fig.2).

Figure 1

Figure 1: Phylogenetic tree based on the hemagglutinin nucleotide sequences from different host with accession number.

Figure 2

Figure 2: Phylogenetic tree based on the neuramidinase nucleotide sequences from different host with accession number.

Both trees showed good resemblance in the clades result. It concludes that this investigation will be helpful for knowing the taxonomy and evolution of newer influenza virus. A study was reported with comparison between antigenically distinct variants of dengue virus in that relationship between phylogenetic shows that DENV-1 was more homologous to DENV-3 as compared with other serotypes (Somvanshi and Seth, 2007).

The computational analysis of physicochemical properties of all proteins of influenza virus was done. The molecular weight and pI value indicates the stability of protein in that particular isoelectric point (pI). The prokaryotic and eukaryotic subcellular localization of protein were well documented but the virus subcellular localization prediction was not known. An online server (Virus-Ploc) was used to predict the virus protein localization within virus or targeting the host. The analysis of whole proteome and specific location of proteins of influenza virus were given (Table1).

Figure 3

Table 1: Physico-chemical properties and subcellular localization of whole proteomes of influenza A virus type H5N1

We investigate the extent of utilization of human cellular localization mechanisms by viral proteins and that appropriate subcellular localization predictors can be used to predict viral protein localization within the host cell. Motifs are typically 6-30 amino acids and correspond to the active site, substrate or ligand-binding site and structurally important segment of proteins. Several important motifs were identified in the study (Table2-9).

Figure 4

Table 2: Motifs in Neuraminidase representing the site name, sequences representative and position on sequences

Figure 5

Table 3: Motifs in Hemagglutinin representing the site name, sequences representative and position on sequences

Figure 6

Table 4: Motifs in Polymerase (PB2) representing the site name, sequences representative and position on sequences

Figure 7

Table 5: Motifs in Polymerase (PB1) representing the site name, sequences representative and position on sequences

Figure 8

Table 6: Motifs in Polymerase (PA) representing the site name, sequences representative and position on sequences

Figure 9

Table 7: Motifs in Matrix protein M2 representing the site name, sequences representative and position on sequences

Figure 10

Table 8: Motifs in Matrix protein M1 representing the site name, sequences representative and position on sequences

Figure 11

Table 9: Motifs in Nucleocapsid protein representing the site name, sequences representative and position on sequences

In this study, we identified nine motifs viz. N-glycosylation site, N- myristoylation site, protein kinase C phosphrylation site, casein kinase 2 phosphorylation site, cAMP and cGMP dependent protein kinase phosphorylation site, amidation, cell attachment sequences (RGD), tyrosine kinase, and prokaryotic membrane lipoprotein lipid attachment site, these motifs play important role in the function of proteome in influenza virus. cAMP is a second messenger used for intracellular signal transduction, such as transferring the effects of hormones like glucagon and adrenaline, which cannot get through the cell membrane. Its main purpose is the activation of protein kinases, also use to regulate passage of Ca2+ through ion channels (Francis and Corbin, 1999). Tyrosine kinase is an enzyme that transfers a phosphate group from ATP to a tyrosine residue in a protein. Phosphorylation of proteins by kinases is an important mechanism in signal transduction for regulation of enzyme activity. The Casein kinase 2 is a serine/threonine-selective protein kinase that is a tetramer of two alpha subunits and two beta subunits. The alpha subunits have the catalytic kinase domain. It has been implicated in cell cycle control, DNA repair, regulation of the circadian rhythm and other cellular processes (Burnett and Kennedy 1954; Bingham and Farrel 1974). Protein kinase C is a family of protein kinases consisting of ~10 isozymes, are activated through the same signal transduction pathway as phospholipase C. It is a family of related serine/threonine kinase and plays a key role in cellular responses such as neurotransmission, gene expression, and cell growth and differentiation (Nishizuka 1984). Myristoylation is an irreversible, post-translational protein modification found in animal, plant, fungi and viruses. It also occurs post-translationally viz. when previously internal glycine residues become exposed by caspase cleavage during apoptosis (Podell and Gribskov 2004). Important motif amidation is a required post-translational modification for the bioactivation of many neuropeptides, is catalyzed by a bifunctional enzyme, peptidyl glycine- amidating monooxygenase, in a two-step reaction (Stewart and Klinman, 1988). RGD motif also play role in the host cell attachment. Recently, Tamanna et al (2006) identified the several motifs (N-glycosylation site, N- myristoylation site, protein kinase C phosphrylation site, casein kinase 2 phosphorylation site, amidation, tyrosine kinase) in the three proteins of influenza virus like nucleoprotein, hemagglutinin and neuraminidase. It has been reported that several host cow, chicken, goose and human specific epitopes from the two significant surface proteins hemagglutinin and neuraminidase of Influenza A virus H5N1 for immunodiagnostic and vaccine development (Somvanshi et al 2007). However, this study may help to understand the whole proteome function, gene regulation and furthermore supports vaccine and antiviral drug target to inhibit the functioning of influenza at the specific position of predicted motifs.