COMPUTATIONAL ANALYSIS OF CONSERVED AND MUTATED AMINO ACIDS IN GP160 PROTEIN OF HIV TYPE-1

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1 Journal of Cell and Tissue Research Vol. 10(3) (2010) ISSN: (Available online at Original Article COMPUTATIONAL ANALYSIS OF CONSERVED AND MUTATED AMINO ACIDS IN GP160 PROTEIN OF HIV TYPE-1 SUMIT KUMAR, VAISHAKHI, T. 1, PRATIK, C. 1 Biotechnology Department, VVP Engineering College, Rajkot ; 1 Bioinformatics Programme, Applied Mathematics Department, M. S. University of Vadodara, Baroda E mail: btsumit@gmail.com Received: October 1, 2010; Accepted: October 30, 2010 Abstract: The multiple sequence analysis of 300 sequences of gp160 of HIV-1 using Clustal yielded sites having amino acids showing varying degree of conservation. The positive selection of mutation in terms of polar, non-polar, hydrophilic and hydrophobic amino acids for the selected sites showed great degree of variation. The findings of this work could assist in the better understanding of selective pressure exerted on the conservation of certain amino acids at the selected regions in the gp160 protein. This in turn could contribute towards designing of potential drugs to combat the menace of HIV infection and AIDS. Key words: HIV type-1, Bioinformatics, Conservation, Mutation, gp160.? INTRODUCTION Human immunodeficiency virus (HIV) is a lent virus which cause acquired immunodeficiency syndrome (AIDS). World statistics of HIV/AIDS by UNAIDS in 2008 says roughly 33.4 million people living with HIV infections. Estimated number of people living with HIV infection in India is 2.31 million [1]. The major problem for unavailability of drugs or vaccine against HIV infection is high mutation rate of HIV [2,3]. Mutation is well observed in laboratory experiments and clinical studies but still the exact mechanism of mutation is not known. Many hypotheses are proposed to explain mutation rate in HIV. One theory is that error-prone Reverse Transcriptase (RT) is responsible for generation of variation after each cycle of replication. So when RT composes pro-viral DNA, there will be nucleotide mis-transmission due to substitution, deletion, addition etc [4,5]. Another theory explains high diversity of HIV as recombination between each unique HIV virion in infected individual during life span of infection [6,7]. Whatever the mechanism HIV likes to apply for generation of diversity but the concern for human is failure of any traditional way of disease prevention. Apart from regular mutational diversity, there exists some major kind of diversity/species in HIV called HIV-1 and HIV-2. HIV-1 is the virus that was initially discovered and termed lymphadenopathy-associated virus (LAV) [8]. HIV-1 is more virulent, more infective and is the cause of the majority of HIV infections globally while HIV-2 is limited to West Africa due to its low infectivity [9]. Based on phylogenetic relationship, HIV-1 is further divided into 3 sub types/clades called M, N and O. Type M of HIV-1 is predominant and includes further 9 subtypes: A, B, C, D, F, G, H, J and K which are genetically diverging by % in env gene and % in gag gene [10,11]. Type N is found only in Cameroon and has very low global predominance. HIV-1 variants not included in type-m or type-n are classified as type-o [12]. For the better understanding of the mechanism and nature of HIV mutation and persistence, structural and bioinformatics analysis is carried out by some research groups but still a strong conclusion about mechanism and drug failure is remained left due to complexity of study. Therefore, the present study aims to analyze the conserved and mutated amino acids in the gp160 protein of HIV-1 from the resources available on various websites. 2359

2 J. Cell Tissue Research MATERIALS AND METHODS Collection of gp160 protein sequence of HIV Type-1: The human immunodeficiency virus is known for its frequent mutation and variation, resulting in the great diversity in genome sequences as well amino acid sequences of produced proteins. The genome sequences and their translated amino acid sequences are available freely on various websites. For the present investigation, about 300 translated amino acid sequences of gp160 protein were collected from different HIV type-1 genomes including different subtypes like A1, B, and C from HIV sequence database available at mainpage.html. These retrieved genome sequences were stored using FASTA format. Multiple sequence alignment using Clustal X: The Clustal X software freely available from website was used for the multiple sequence alignment of the collected amino acid sequences of gp160 protein of HIV type-1. The alignment was facilitated by loading the FASTA file in Clustal X and by using its multiple sequence alignment function. The use of different colour tags assisted in locating the proper alignment among the similar sequences and in the finding of mismatched sequences if any. The results of these alignments were used to identify the variation in amino acids of various gp160 proteins of HIV-1. Positive selection of mutation: After multiple sequence alignment of amino acid sequences of gp160 proteins, on the basis of colour tags, variation in amino acids at the same position was identified. The regions with less than 30% similarities were selected to determine the maximum variation in amino acids among the selected sequences. The variations in amino acids at same position in different sequences were grouped into polar amino acids versus non-polar amino acids and hydrophilic versus hydrophobic amino acids, using the features of MS-Excel Determination of conserved amino acids: Among the aligned amino acid sequences of gp160 proteins, the conserved amino acids were indicated by using colour tags at the same position. The regions with 100% similarities at same position were selected from among all sequences. This analysis was done manually using the features of MS-Excel The results thus obtained were subjected to statistical analysis to draw the useful conclusions. Statistical analysis: It was performed to determine per cent mutation, per cent conservation using the features of MS-Excel The data obtained from statistical analysis were used making result tables and graphs to arrive at significant conclusions. RESULTS AND DISCUSSION Conserved amino acids in HIV-1 subtype A1: The multiple sequence alignment for the selected sites in gp160 protein of HIV-1 subtype A1 has yielded first conserved site is a region between 125 to 132 (of 8 amino acids) flanked by two different conserved sites of two amino acids having only one gap, the second conserved site between 282 to 287 (of 6 amino acids) with only one gap at 281 and a conserved region of two amino acids at as shown in figure 1(a) and (b). The conserved region between 282 and 287 is present at the exposed surface of gp120 envelope protein, as shown in figure 3. From the literatures, it was found that the great variability in HIV-1 antigenic epitopes is one of the major mechanisms used by the virus to evade the host immune response. To elicit broadly neutralizing antibody (NAb) against HIV-1, one or more conserved epitopes should be recognized to overcome the extensive antigenic diversity. The major targets of HIV-1 neutralizing antibodies are located on the surface gp120, whose diverse antigenic epitopes mediate receptor and co receptor binding, and on the transmembrane gp41, which causes membrane fusion and allows the virus to gain entry into host cells. The antigenic diversity of HIV-1 is a major tool used by the virus as an immune evasion strategy, and this poses a major obstacle for the development of an effective HIV-1 vaccine. Therefore, a major focus of vaccine developers has been the difficulty in the elicitation of a broadly neutralizing antibody response. In this work we have studied a limited number of conserved amino acid sequences, which can be treated as epitopes on the envelope glycoprotein of HIV-1 primary isolates. The data thus obtained from this study would assist in the design of synthetic peptides, with a greater potential to combat the menace of AIDS. 2360

3 Sumit Kumar et al. 2361

4 J. Cell Tissue Research A1: The selected sites in the gp160 protein of HIV- 1 subtype A1 include different amino acid positions such as 32, 137, 441 and 915. The multiple sequence alignment for these selected positions showed variation in more than 15 amino acids at a unique position. The aligned sequences of amino acids showed replacement of amino acids of same group. The variation in the same amino acid group was noticed in more than 70% cases, in terms of polar, non-polar, hydrophilic and hydrophobic amino acids, as represented in figure 4. In the present study, it is found that a few amino acid sites undergo positive selection and play a critical role in adapting the HIV-1 virus against the host immune response. It is found that at site-32, out of 87 selected sequences of HIV-1 of subtype A1, 76 showed positive selection in being the polar amino acids while 09 showed selection in being the nonpolar amino acids. Similarly, at site-441, out 87 sequences, 59 showed positive selection in being the polar and 28 being the non-polar amino acids. At site- 137, out of 87 sequences, 62 showed positive selection in being the non-polar and 24 being the polar amino acids. At site-915, out of 87 sequences, only 2 showed in being the polar while 80 being the non-polar amino acids. For these selected sites, the hydrophilic amino acids constituted 87.35%, 65.51%, 16.09% and 3.44% respectively, while the hydrophobic amino acids constituted 10.34%, 32.18%, 82.75% and 90.80% respectively. Conserved amino acids in HIV-1 subtype B: The multiple sequence alignment for the selected sites in gp160 protein of HIV-1 subtype B has yielded first conserved site is a region between 45 to 55 (of 11 amino acids) with no gap and the second site 288 to 295 (of 8 amino acids) with no any gap as represented in the figure 1 (c) and (d). It is determined that the second conserved region is present at the surface of gp120 protein of HIV-1 as shown in figure 3. B: The selected sites in the gp160 protein of HIV-1 subtype B include different amino acid positions such as 95, 146, 442, 710 and 599. The aligned sequences of amino acids showed replacement of amino acids of same group. The variation in the same amino acid group was noticed in more than 70% cases, in terms of polar, non-polar, hydrophilic and hydrophobic amino acids, as represented in figure 5. In the present study, it is found that a few amino acid sites undergo positive selection and play a critical role in adapting the HIV-1 virus against the host immune response. It is found that at site-32, out of 87 selected sequences of HIV-1 of subtype A1, 76 showed positive selection in being the polar amino acids while 09 s howed selection in being the nonpolar amino acids. Similarly, at site-441, out 87 sequences, 59 showed positive selection in being the polar and 28 being the non-polar amino acids. At site- 137, out of 87 sequences, 62 showed positive selection in being the non-polar and 24 being the polar amino acids. At site-915, out of 87 sequences, only 2 showed in being the polar while 80 being the non-polar amino acids. For these selected sites, the hydrophilic amino acids constituted 87.35%, 65.51%, 16.09% and 3.44% respectively, while the hydrophobic amino acids constituted 10.34%, 32.18%, 82.75% and 90.80% respectively. Conserved amino acids in HIV-1 subtype B: The multiple sequence alignment for the selected sites in gp160 protein of HIV-1 subtype B has yielded first conserved site is a region between 45 to 55 (of 11 amino acids) with no gap, the second conserved site is between 288 to 295 (of 8 amino acids) with no any gap as represented in the figure-1 (c) and (d). Its determined that the second conserved region is present at the surface of gp120 protein of HIV-1. This conserved site is same as that of at the surface of gp120 protein of HIV-1 subtype A1 at site 2, as shown in figure-3. B: The selected sites in the gp160 protein of HIV-1 subtype B include different amino acid positions such as 95, 146, 442, 710 and 599. The aligned sequences of amino acids showed replacement of amino acids of same group. The variation in the same amino acid group was noticed in more than 70% cases, in terms of polar, non-polar, hydrophilic and hydrophobic amino acids, as represented in figure 5. In the present study, it is found that a few amino acid sites undergo positive selection and play a critical role in adapting the HIV-1 virus against the host immune response. It is found that at site-95, out of 2362

5 Sumit Kumar et al. Fig.4: Nature of amino acids at the four selected sites in gp160 of HIV-1 subtype A Site-32 Site-441 Site-137 Site-915 Number of polar amino acids Number of non-polar amino acids Number of hydrophilic amino acids Number of hydrophobic amino acids Fig. 5: Nature of amino acids at the five selected sites in gp160 of HIV-1 subtype B Number of polar amino acids Number of non polar amino acids Number of hydrophilic amino acids Number of hydrophobic amino acids 0 Site-95 Site-146 Site-442 Site-599 Site-710 Fig 6: Nature of amino acids at the four selected sites in gp160 of HIV-1 subtype C Number of polar amino acids Number of non-polar amino acids Number of hydrophilic amino acids Number of non-polar amino acids 0 Site-372 Site-383 Site-430 Site-491 Site sequences, 70 showed positive selection in being the non-polar while 30 being the polar amino acids, at site-146, out of 100 selected sequences, 71 showed positive selection in being the polar amino acids while rest 28 showed selection in being the non-polar amino acids. Similarly, at site 442, out 100 sequences, 80 showed positive selection in being the polar and 20 being the non-polar amino acids. At site-710, out of 100 sequences, 99 showed positive selection in being the polar and only 1 being the non-polar amino acids. At site 599, out of 100 sequences, 98 showed positive selections in being the non-polar while only 2 being the polar amino acids. For these selected sites, the hydrophilic amino acids constituted 30%, 71%, 73%, 97% and 2% respectively, while the hydrophobic amino acids constituted 70%, 28%, 27%, 3% and 98% respectively. Conserved amino acids in HIV-1 subtype C: The multiple sequence alignment for the selected sites in gp160 protein of HIV-1 subtype C has yielded 2363

6 J. Cell Tissue Research first conserved site is a region between 421 to 4264 (of 6 amino acids) with no gap, the second conserved site is between 582 to 587 (of 6 amino acids) with no gap as shown in the figure-2 (a) and (b). C: The selected sites in the gp160 protein of HIV-1 subtype C include different amino acid positions such as 372, 383, 430, 491 and 699. The aligned sequences of amino acids showed replacement of amino acids of same group. The variation in the same amino acid group was noticed in major cases, in terms of polar, non-polar, hydrophilic and hydrophobic amino acids, as represented in figure 5. It is found that at site-372, out of 100 selected sequences of HIV-1 of subtype C, all 100 showed positive selection in being the polar amino acids while no single amino acid showed selection in being the non-polar amino acids. Similarly, at site-383, out of 100 sequences, 81 showed positive selection in being the polar and 19 being the non-polar amino acids. At site 430, out of 100 sequences, 79 showed positive selection in being the polar and 31 being the nonpolar amino acids. At site 491, out of 100 sequences, 93 showed positive selections in being the polar while only 7 being the non-polar amino acids. At site-699, out of 100 sequences, 87 showed positive selections in being the polar while 13 being the non-polar amino acids. For these selected sites, the hydrophilic amino acids constituted 99%, 81%, 78%, 93% and 83% respectively, while the hydrophobic amino acids constituted 1%, 19%, 32%, 7% and 17% respectively. CONCLUSION The positive selection of mutation in terms of polar, non-polar, hydrophilic and hydrophobic amino acids for the selected sites showed great degree of variation. Multiple sequence analysis of gp120 also yielded conserved amino acids throughout 300 sequences. Comparison between these conserved sites and already determined structure of gp120 showed that one of the conserved sites is available on surface of gp120 protein. Another finding of this work could assist in the better understanding of selective pressure exerted on the conservation of certain amino acids at the selected regions in the gp160 protein. This in turn could contribute towards designing of potential drugs to combat the menace of HIV infection and AIDS. FUTURE PROSPECTIVE The outcomes of this work can be exploited for developing antibodies against the most conserved regions of gp160 protein of HIV-1 as well as designing of potential drugs to combat the menace of HIV infection. The work will also help to understand the selective pressure exerted on the conservation of polar, non-polar, hydrophilic or hydrophobic amino acids of gp160 protein. ACKNOWLEDGEMENTS The authors wish to acknowledge Dr. Dhanesh Patel of Applied Mathematics Department of M. S. University of Baroda for providing the necessary infrastructure as well as technical support required for this work. REFERENCES [1] UNAIDS: AIDS epidemic update : November 2009: (2009). [2] Hutchinson, J.F.: Annual Rev. Anthropol., 30: (2001). [3] Ho, D.D., Neumann, A.U., Perelson, A.S., Chen, W., Leonard, J.M. and Markowitz, M.: Nature, 373: (1995). [4] Dougherty, J.P. and Temin, H.M.: J. Virol., 62(8): (1988). [5] O Neil, P.K., Sun, G., Yu, H., Ron, Y., Dougherty, J.P. and Preston, B.D.: J. Biol. Chem., 277 (41): (2002). [6] Ramirez, B.C., Simon-Loriere, E., Galetto, R. and Negroni, M.: Virus Res., 134: (2008). [7] Kijak, G.H. and McCutchan, F.E.: Current Infectious Disease Reports, 7: (2005). [8] Basavapathruni, A. and Anderson, K.S.: The FASEB J., 21: (2007). [9] Gilbert, P.B., McKeague, I.W., Eisen, G., Mullins, C., Gueye-NDiaye, A., Mboup, S. and Kanki, P.J.: Statistics Med., 22: (2003). [10] Kantor, R., Katzenstein, D.A., Efron, B., Carvalho, A.P., Wynhoven, B., Cane, P., Clarke, J., Sirivichayakul, S., Soares, M.A., Snoeck, J., Pillay, C., Rudich, H., Rodrigues, R., Holguin, A., Ariyoshi, K., Bouzas, M.B., Cahn, P., Sugiura, W., Soriano, V., Brigido, L.F. et.al.: PLoS Med., 2(4): (2005). [11] Geretti, A.M.: Current Opinion in Infectious Diseases, 19: 1-7 (2006). [12] Gurtler, L.G., Hauser, P.H., Eberle, J., Brunn, A.V., Knapp, S., Zekeng, L., Tsague, J.M. and Kaptue, L.: J. Virol., 68 (3): (1994). 2364

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