Analysis of PB2 protein from H9N2 and H5N1 avian flu virus

Influenza A viruses of subtype H9N2 are wide spread among poultry and other mammalian species. Crossing the species barrier from poultry to human occurred in recent years creating a pandemic of H9N2 virus. It is known that the pathogenicity of H9N2 is lower than H5N1. Nonetheless, it is important to establish the molecular functions of H9N2 viral proteins. We studied mutations in the polymerase protein PB2 of H9N2 from different strains and compared it with the highly pathogenic H5N1. The mutation M294T was found to be important in the N-myristoylation domain of Ck/UP/2573/India/04(H9N2) isolate. Prediction of secondary structures and PROSITE motif assignments were performed for PB2 to gain functional insight. Subsequently, the effect of mutations in secondary structures among strains is discussed.


Background:
Influenza viruses belong to the member of orthomyxo-viridae family, having a single-standard, negative sense, segmented RNA genome in an enveloped virion [1-6]. The genome of influenza viruses have 8 RNA segments encoding 10 proteins including two surface glycoprotein, haemaggulutinin (HA), neuraminidase (NA), nucleoproteins (NP), three polymerase proteins (PA,PB1,PB2) two matrix (M1,M2) and nonstructural proteins (NS1,NS2). Influenza viruses are classified as types A, B and C based on the antigenic properties of nucleoprotein (NP) and matrix (M1) [4, 5]. Avian influenza is caused by a type-A virus and it is further classified into subtype based on two surface glycoprotein, haemagglutinin (HA) and neuraminidase (NA). There are about 16 known HA and 9 known NA in type-A viruses [7]. The polymerase protein PB2 is 759 residues long influenza-A viruses. The polymerase complex is formed protein subunit interactions with the PB1 subunit [11]. PB2 interacts with NP and not with PA [12,13]. This subunit plays an important role in transcription of mRNA by binding with the 5' methylated cap of pre-mRNA in host cells for providing primers to viral mRNA synthesis [14]. Two hypothetical domains (residues 242-280 and 538-577) identified in PB2 are hypothesized for viral binding to host cells [15]. Here, we describe the effect of mutations in PB2 from different strains of H9N2. The analysis is extended to that in H5N1 for comparison.

Methodology: Data set
The PB2 protein sequences of subtypes H5N1 and H9N2 were downloaded from Genome directory INFLUENZA at NCBI [23] and summary of sequence data given in Table 1 (see supplementary material). The dataset consists of PB2 sequences from 3 H9N2 strains and 2 H5N1 strains. We used CLUSTALW [16] for generating a multiple sequence alignment MSA of 5 PB2 sequences from H9N2 strains and H5N1 strains.

Mutational analyses
The MSA for 5 PB2 sequences from H9N2 strains and H5N1 strains was used to identify mutations in PB2 with reference to Ck/UP/India/04. We used the amino acids sequences 151 to 690 for the analyses.  On comparing the data we found that all the mutations except 340(R→K) and 478(V→I) in A/goose/MN/5733-1/80(H9N2), and 667(V→I) in A/HK/213/03(H5N1). All the mutations that were observed are synonymous and will not change the chemical and structure properties of this protein.
Only one non synonymous mutation, M294T in Ck/UP/2543/India was found to change the chemical property from Hydrophobic to Hydrophilic at the respective site. The most important finding, observed, was the identification of N-myristoylation domain. This domain (GGvrTV) was found at positions 290-295 due to mutation at 294 (M→T) in A/Ck/UP/2543/India/04 (H9N2) as shown in Table 3 (supplementary material). Moreover, mutations at position N265S and M570I in A/Ck/UP/2543/India/04(H9N2) were observed in binding regions of PB2 protein.

Secondary structure prediction
Secondary structure changes due to mutations were also found. Mutations, M294T in Ck/UP/2543/India/04 and I478V in Gs/Mn/5733-1/80 responsible for change in secondary structure from helix to strand, mutation M570I in U.P isolate altered the secondary structure form strand to helix (

Sequence alignment
Amino acid sequences of PB2 protein of H9N2 and H5N1 subtypes were aligned by using ClustalW program. Sequence analyses of PB2 protein (AA from 151 to 690) of H9N2 and H5N1 isolates revealed that PB2 protein of these two subtypes were similar by 97% (Figure 1). This study also revealed that the change in secondary structure occurred, is due to mutations as discussed in Table 2 (supplementary material). These mutations not only change the secondary structure conformation at the respective site but also alter the nearest side chain conformation as predicted by GORIV methods. Sequence of PB2 protein of these two subtypes was showing similarity (Figure 1) among each other, raising the possibility that exchange of internal segments between these two subtypes have been occurred [21,22].

Conclusion:
The PB2 subunit plays an important role in transcription regulation in viral mRNA synthesis. We showed mutational difference in PB2 in different strains of H9N2. Mutations are seen in the predicted secondary structures of PB2. These findings have implications in understating PB2 function in viral multiplication and infection.