J Proteomics Bioinform opg Journal of Proteomics & Bioinformatics 0974-276X OMICS Publishing Group India, USA 000063 Research Article Biochemistry Proteomics Bioinformatics Genomics Transcriptomics Biomarkers Comparative Modeling Of Viral Protein R (Vpr) From Human Immunodeficiency Virus 1 (Hiv 1) Renganathan Seenivasagan Renganathan Kasimani Parthiban Marimuthu Ramamoorthy Kalidoss Piramanayagam Shanmughavel * Department of Bioinformatics, DBT-BIF Facility, Bharathiar University, Coimbatore – 641 046 * To whom correspondence should be addressed: Dr. P. Shanmughavel, Ph.D., Department of Bioinformatics, DBT-BIF Facility, Bharathiar University, Coimbatore – 641 046, Ph: +91-422-2422655; Fax : +91-244-2422387; E-mail: shanvel_99@yahoo.com 05 2008 20 05 2008 1 2 073 076 21 04 2008 14 05 2008 Copyright: © 2008 Seenivasagan R, etal. 2008

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During Human Immunodeficiency Virus infection interactions take place between host and the pathogen. This interaction mainly determines the efficiency of viral infection and the disease progression. Human Immunodeficiency Virus infected cells responds to several antiviral immune mechanisms such as innate, cellular and humoral immune antiviral defense. This virus has also gained resistance to suppress these host cellular responses. Out of many self resistance mechanisms, Viral protein R (VpR) occupies a significant role, such as nuclear transport of the viral pre-integration complex, activation of viral transcription, induction of cell cycle and apoptosis of the host cells. Apart from these specific roles, the function of VpR remains mystery for the structural bioinformatics. The comparative modeling approach is used to predict the structure of a VpR using NMR structure of the HIV-1 regulatory protein as the template (PDB ID: 1ESX: A). The theoretical structure of VpR is generated using Modeller9v1, a program for comparative modeling of protein using special restraints. This theoretical structure believes to paves the way for the novel lead synthesis.

 Viral protein R (VpR) Human immunodeficiency virus-1 (HIV-1) Structural Bioinformatics Comparative modeling Structure Based Drug Design citation Renganathan S, Renganathan K, Parthiban M, Ramamoorthy K, Piramanayagam S (2008) Comparative Modeling Of Viral Protein R (Vpr) From Human Immunodeficiency Virus 1 (Hiv 1) Introduction

Human immunodeficiency virus (HIV) belongs to retroviral family which leads to Acquired Immuno Deficiency Syndrome which ultimately leads to failure in immune response. There are two types of HIV infecting humans: HIV-1 and HIV-2. HIV-1 is more a virulent and easily transmitted than in case o HIV-2 which is less transmitted. HIV-1 possess the following virion particle is Vif, VpR, Nef, p7 and viral protease. VpR strands for, “Viral Protein R” a 96 residue polypeptide of 14 kDa protein, (Hoch J. et al.1995), (Lang SM et al. 1993). VpR plays an important role in regulating nuclear import of the HIV-1 preintegration complex (Heinzinger NK et al. 1994). VpR is an integral part of viral particles suggesting an important role in early stages of infection (Cohen EA et al, 1990), (Yu XF et al, 1990), (Lu YL et al, 1993), (Emerman M, 1996) which is required for viral replication in non-dividing cells such as macrophages (Agostini I et al, 2002) and also induces cell cycle arrest and apoptosis in proliferating cells, which leads to immune dysfunction (He J et al, 1995), (Jowett JB et al, 1995), (Re F et al, 1995), (Rogel ME et al, 1995). Recombinant, over expressed VpR forms ion channels in E. coli which is permeable to Na+ ions (Piller SC et al, 1999), (Piller SC et al, 1999). Different cellular proteins are reported to interact with VpR : transcription factor Sp1 (Wang L et al, 1995), uracil DNA glycosylase (UNG) (Bouhamdan M et al, 1996), HHR23A – a protein implicated in DNA repair (Withers- Ward ES et al, 1997), importin – a nuclear pore protein Nsp1p (Vodicka MA et al, 1998), and many others. VpR play multiple functions such as, cell cycle progression during the virus life cycle, regulation of apoptosis, and the transactivation of the HIV-LTR as well as host cell genes (Le Rouzic E, Benichou S, 2005). VpR is conserved in both HIV-1 and HIV-2.

The NMR structure of the template protein has revealed the structural information of VpR using comparative modeling technique. The template structure is characterized by a well defined gamma turn (14-16) - alpha helix (17-33)- turn (34-36) followed by a alpha helix (40-48) –loop(49-54)- alpha helix (55- 83) domain and ends with a very flexible C terminal sequence (Wecker K et al, 2002).

 Materials and Methods Sequence Alignment

The protein sequence of VpR was retrieved form from the NCBI database (P0C1P5) which has 96 amino acids. The target sequence was searched for similar sequence using the BLAST (Basic Local Alignment Search Tool), against Protein Database (PDB). The BLAST results yielded NMR structure of HIV-1 regulatory protein R VpR with 85% similarity to our target protein.

 Comparative Modling

The theoretical structure of VpR is generated using Modeller-9v1 for comparative modeling of protein structure prediction (Eswar N et al, 2006), (Martí-Renom MA et al, 2000). It implements comparative structural modeling by conforming special restraints (Sali A, Blundell TL. 1993),(Fiser A et al, 2000).

 Modle Refinement

Theoretical structure of VrP is verified for the steroschemical clashes by subjecting the model to SwissPDB Viewer. The energy minimization with a harmonic constraint of 100kj/mol/A2, was applied for all protein atoms, using the Steepest- Decent and Conjugate Gradient technique to eliminate bad contacts between protein atoms. Computations were carried out with the GROMOS96 in Swiss-PDB Viewer. Backbone conformation was evaluated by inspecting Psi/Phi Ramachandran plot was obtained from the PROCHECK analysis.

 Result and Discussion

VpR involved in key regulation of nuclear import of HIV-1 preintegration complex, a significant role in early stages of integration, viral replication in non-dividing cells, induces cell cycle arrest as well as apoptosis in proliferating cells.

Model Building

Based on the results obtained from the BLAST program VpR from HIV-1 was selected as template. The use of several independent third generation algorithms of secondary structure prediction suggests that the theoretical structure of VpR comprises of a helical structure. We used the approach of comparative modeling based on the template of a known structure. This approach is best suitable when a homolog of the target is known. The prediction of typical VpR exhibited 85% similarity which inturn means that the true accuracy of our prediction could be a little higher identity of the prediction when compared with the template. Energy minimizations were performed using Deep View with GROMOS96 parameters which did not significantly modify the initial models.

 Secondary Structure Prediction

The secondary structure of VpR protein was predicted by SOPMA (Self Optimized Prediction Method with Alignment) which correctly predicts 69.5% of amino acids for a three-state description of the secondary structure prediction (alpha helix, beta-sheet, and coil) (Geourjon C, Deléage G. 1995). The most of the regions are Alpha helix (HH): 45.83%, the extended strands (EE):11.46% and the random coils (CC): 42.71%. SOPMA resulting for both target and template has more or less similar secondary structural elements except two places. Where 45th and 46th place in case of target forms continious helix where in case of template it forms extended sheet. The position at 48 to 51 where in target forms coil, in case of template which is a turn (Figure 1),(Figure 2).

Comparative modeled protein structure showed in PyMol (protein structure viewer). Protein which forms alpha helices (17-33), (40- 48), (55-83), turns and coil was showed. Sequence alignment of both target and template sequence with SOPMA secondary structural prediction. Amino acids with red color are different from the template sequence.SOPMA secondary structure prediction of V pr protein. Ramachandran Plot Analysis

The theoretical structure of VpR was energy minimized and subjected to procheck web interface for sterochemical analysis. (Figure 3). The most of the residues were in favored regions [A,B,L] -68 (85 %); residues in additional allowed regions [a, b, l, p] – 9 (11.2 %); residues in generously allowed regions [-a, -b, -l, -p ] - 2 (2.5 %) ; residues in disallowed region 1 (1.2 %); number of non-glycine and non-proline residues -80 (100 %) number of end residues (excl. Gly and Pro) – 2; Number of glycine residues (shown as triangles) – 9; number of proline residues– 5; Total number of residues – 96.

Secondary structure prediction of VpR protein. Sequence alignment of both template and target sequence with SOPMA secondary structure prediction. Amino acids with red color were showed changes in template from target sequence. Conslusion

Based on the Template structure it is clearly observed that the theoretical structure generated is structurally similar to the template structure which is highly sufficient for the development of specific ligand for VpR. Our model of VpR is only a predictive, and needed to be confirmed experimentally, testing in vivo the activity of ligand candidates which could constitute an important first step towards the validation of our model as tools for the discovery of novel VpR agonist. This modeled structure can be used to predict the molecular function, the active sites which haven’t studied much. The predicted 3D model of the VpR of HIV will be very useful in wet laboratory while studying the real structure of the protein.

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