Homology modelling and insilico analysis of neuraminidase protein in H1N1 Influenza A virus

AGRICULTURE, AGRIBUSINESS AND BIOTECHNOLOGY

Homology modelling and insilico analysis of neuraminidase protein in H1N1 Influenza A virus

Abhilash Manohar^*

Department of Biotechnology Engineering, Oxford College of Engineering, Bangalore - India

ABSTRACT

In this work, modelling of Neuraminidase protein of Influenza A virus (A/Himeji/1/2009(H1N1)) neuraminidase (NA) protein was done using Modeller 9V2. Modelled structure was submitted to protein model database and could be downloaded using accession number PM0075830. The modelled protein structure was subjected to In silco analysis using various bioinformatics tools. Two anti-influenza drugs currently being used to treat infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. Hence the modelled structure of H1NI Neuraminidase could be very useful for in silico analysis of potential neuraminidase inhibitors.

Key words: H1N1, Neuraminidase, Modelling, in silico analysis

INTRODUCTION

The 2009 flu pandemic has been a global outbreak of a new strain of influenza A virus subtype H1N1, identified in April 2009 and commonly referred to as swine flu, which infects and is transmitted between humans. It is thought to be a mutation - more specifically, a reassortment - of four known strains of influenza A virus subtype H1N1: one endemic in humans, one endemic in birds, and two endemic in pigs (swine). Swine influenza (also called swine flu, hog flu, and pig flu) is an infection of a host animal by any one of several specific types of microscopic organisms called "swine influenza virus". A June 10, 2009 update by the United Nation's World Health Organization (WHO) states that "74 countries have officially reported 27,737 cases of influenza A (H1N1) infection, including 141 deaths".WHO officially declared the outbreak to be a "pandemic" on June 11, but stressed that the new designation was a result of the global "spread of the virus," not its severity. The WHO stated the pandemic appeared to have moderate severity in comparatively well-off countries however, it would be prudent to anticipate a bleaker picture if the virus spread to areas with limited resources, poor health care, and a high prevalence of underlying medical problems. The case fatality rate (CFR) of the pandemic strain was estimated at 0.4% (range 0.3%-1.5%).

A swine influenza virus (SIV) is any strain of the influenza family of viruses that is usually hosted by (is endemic in) pigs. As of 2009, the known SIV strains was the influenza C virus and the subtypes of the influenza A virus known as H1N1, H1N2, H3N1, H3N2, and H2N3. Swine influenza is common in pigs in the United States (particularly in the midwest and occasionally in other states), Mexico, Canada, South America, Europe (including the United Kingdom, Sweden, and Italy), Kenya, and eastern Asia (namely China, Taiwan, and Japan). The 2009 swine flu outbreak in humans was due to a new strain of influenza A virus subtype H1N1 that contained genes closely related to swine influenza (Trifonov et al., 2009). The origin of this new strain is unknown. However, the World Organization for Animal Health (OIE) reported that this strain had not been isolated in pigs (Maria Zampaglione, 2009). This strain can be transmitted from human to human, and causes the normal symptoms of influenza (Myers et al., 2007). Pigs can become infected with human influenza, and this appears to have happened during the 1918 flu pandemic and the 2009 swine flu outbreak.

Virus characteristics

The virus is a novel strain of influenza from which human populations have been neither vaccinated nor naturally immunized. The Centers for Disease Control and Prevention (or CDC), after examining the virus samples from suspected cases in Mexico, matched the strain with those from cases in Texas and California, and found no known linkages to either to animals or one another. It was also determined that the strain contained genes from four different flu viruses: North American swine influenza; North American avian influenza; human influenza; and two swine influenza viruses typically found in Asia and Europe. Further analysis showed that several proteins of the virus were most similar to strains that cause dmild symptoms in humans. Scientists in Winnipeg completed the first full genetic sequencing of the virus on 6 May 2009.

Influenza A

Swine influenza is known to be caused by the influenza A subtypes H1N1 (Shin et al., 2006), H1N2 (Shin et al., 2006), H3N1 (Shin et al., 2006), H3N2 (Ma et al., 2007), and H2N 3(Ma et al., 2007). In pigs, three influenza A virus subtypes (H1N1, H3N2, and H1N2) are the most common strains worldwide (Ma et al., 2007). In the United States, the H1N1 subtype was exclusively prevalent among the swine populations before 1998; however, since late August 1998, H3N2 subtypes have been isolated from the pigs. As of 2004, H3N2 virus isolates from US swine and turkey stocks were triple reassortants, containing genes from human (HA, NA, and PB1), swine (NS, NP, and M), and avian (PB2 and PA) lineages (Gramer et al., 2007).

Virus origins

In early June, Oxford University's Department of Zoology, reported test results that showed that this strain has been circulating among pigs, possibly among multiple continents, for many years prior to its transmission to humans. The research team that worked on this report also reported that it was derived from several viruses circulating in swine, and that the initial transmission to humans occurred several months before recognition of the outbreak. The team concluded that despite widespread influenza surveillance in humans, the lack of systematic swine surveillance allowed for the undetected persistence and evolution of this potentially pandemic strain for many years (Smith et al., 2009). According to the findings, the movement of live pigs between Eurasia and North America seemed to have facilitated the mixing of diverse swine influenza viruses, leading to the multiple reassortment events associated with the genesis of the (new H1N1) strain (Lindstrom et al., 2004).

Transmission of swine influenza virus from pigs to humans is not common and does not always cause human influenza, often only resulting in the production of antibodies in the blood. The meat of the animal poses no risk of transmitting the virus when properly cooked. If transmission does cause human influenza, it is called zoonotic swine flu. People who work with pigs, especially people with intense exposures, are at increased risk of catching swine flu. In the mid-20th century, the identification of influenza subtypes became possible, which allows accurate diagnosis of transmission to humans. Since then, fifty confirmed transmissions have been recorded.

Rarely, these strains of swine flu can pass from human to human. In humans, the symptoms of swine flu are similar to those of influenza and of influenza-like illness in general, namely chills, fever, sore throat, muscle pains, severe headache, coughing, weakness and general discomfort (Kimura et al., 2008).

Treatment

Antiviral drugs

According to the CDC, the antiviral drugs can be given to treat those who become severely ill. These antiviral drugs are prescription medicines (pills, liquid or an inhaler) and act against influenza viruses, including the 2009 pandemic virus. There are two such medications that are recommended for use against the 2009 H1N1 swine flu virus, oseltamivir (Tamiflu) and zanamivir (Relenza) (Antonovics et al., 2006). CDC has noted that as the flu pandemic spreads, antiviral drugs such as oseltamivir (Tamiflu) and zanamivir (Relenza) might become in short supply. Therefore, the drugs would be given first to those people who have been hospitalized or are at high risk of complications(Olsen, 2002).The drugs work best if given to the patient within two days of becoming ill , but might be given later if illness became severe or to those at a high risk for complications.

Neuraminidase

Neuraminidase is an enzyme on the surface of influenza viruses that enables the virus to be released from the host cell. When influenza virus reproduces, it moves to the cell surface with a hemagglutinin molecule on the surface of the virus bound to a sialic acid receptor on the surface of the cell. In order for the virus to be released free from the cell, neuraminidase must break apart (cleave) the sialic acid receptor.

Function

The enzyme helps viruses to be released from a host cell. Influenza virus membranes contain two glycoproteins: haemagglutinin and neuraminidase. While the hemagglutinin on the surface of the virion is needed for infection, its presence inhibits the release of the particle after budding. It also mediates cell-surface sialic acid receptor binding to initiate virus infection. Viral neuraminidase cleaves the terminal neuraminic acid (also called sialic acid) residues from glycan structures on the surface of the infected cell. This promotes the release of progeny viruses and the spread of the virus from the host cell to uninfected surrounding cells. Neuraminidase also cleaves sialic acid residues from viral proteins, preventing the aggregation of viruses.

MATERIAL AND METHODS

Influenza A virus (A/Himeji/1/2009(H1N1)) segment 6 neuraminidase (NA) sequence with accession number GQ261273 Submitted (15-JUN-2009) by Horikawa et al., from National Institute of Technology and Evaluation (NITE), Tokyo, Japan was selected for in silico analysis.

The Sequence selected for the analysis

Tool used for modelling of neuraminidase protein was Modeller 9V2.

Tools used for neuraminidase protein analysis

• Gor IV secondarystructure prediction tool (Garnier et al., 1978): The GOR (Garnier- Osguthore- Robson) method uses both information theory and Bayesian statistics for predicting the secondary structure of proteins.

• Pep tool (Version 2.0): Pep Tool is a suite of applications for comprehensive analysis of peptide and protein sequences.

RESULTS AND DISCUSSION

The MODELLER was used for homology or comparative modeling of three-dimensional protein structures. The Alignment of a sequence to be modelled was provided with the known related structures and the MODELLER automatically calculated a model containing all non-hydrogen atoms. The MODELLER implemented the comparative protein structure modeling by satisfaction of spatial restraints, and could perform many additional tasks, including de-novo modeling of loops in protein structures, optimization of various models of protein structure with respect to a flexibly defined objective function, multiple alignment of protein sequences and/or structures, clustering, searching of sequence databases, comparison of protein structures, etc.

The Structure of modeled protein was visualized using Rasmol (Structure visualization tool) (Figure 1). Modelled structure was submitted to protein model database (PMDB) a repository for three dimensional protein models obtained by structure prediction methods. The Submitted, Modelled H1N1 Neuraminidase protein could be downloaded from PMDB using accession number PM0075830.

Protein Structure Analysis

The Secondary structure prediction of the modelled neuraminidase virulence protein was carried out using GOR IV (Garnier-Osguthore-Robson) secondary structure prediction tool (Figure 2).

Amino acid frequency plot (Figure 3), plot of charge vs pH (Figure 4), Beta staircase model (Figure 5), Helical wheel model (Figure 6) and molecular properties calculation (Table 1) of the neuraminidase protein of H1N1 Influenza virus was obtained using pep tool a comprehensive protein analysis software.

Beta staircase Model

The beta staircase graphically displays (Figure 5) the disposition of amino acid side chains about an assumed alpha helix. The view is always along the central axis of the helix from N to C-terminus. The helical wheel is an effective method for displaying the symmetry of hydrophobic/hydrophilic side chains of BBI C-II. It is useful for observing how the amino acids are positional in relation to one another (Khot et al., 2004)

CONCLUSIONS

Novel H1N1 (Referred to as "Swine flu" early on) is a new influenza virus causing illness in people. This new virus was first detected in people in the US in April 2009. A June 10, 2009 update by the WHO state that 74 countries had officially reported 27,737 cases of influenza A (H1N1) infection, including 141 deaths. Two anti-influenza drugs currently being used to treat the infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. This project aimed at designing structure of Neuraminidase of H1N1 which will be useful for designing the novel Neuraminidase inhibitors which might help to combat H1N1 pandemic.

Received: July 18, 2009; Revised: September 17, 2009; Accepted: April 16, 2010.

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Publication Dates

• Publication in this collection
  03 Mar 2011
• Date of issue
  Feb 2011

History

• Accepted
  16 Apr 2010
• Reviewed
  17 Sept 2009
• Received
  18 July 2009