Investigating the complexity of retroviral nuclear import

Investigation the complexity of retroviral nuclear import: from HIV PPT to IN NLS, including components of NPC (TR-SR2): 

Investigation the complexity of retroviral nuclear import: from HIV PPT to IN NLS, including components of NPC (TR-SR2) MD/PhD student Sergey Shytyakov Wuerzburg. 2009

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HIV PPT part HIV IN and TR-SR2 part HIV IN Phosphorylation and folding part Parts of presentation

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Retrovirus life cycle:

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4) (-) strand DNA terminates at the primer binding site 5) When (-) strand elongation passes the polypurine tract (ppt) region, the RNA template escapes digestion by RNase H and serves as a primer for (+) strand synthesis by DNA dependent DNA polymerization (DDDP) Reverse transcription

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6) (+) strand synthesis then continues back to the U5 region with the (-) strand DNA as the template and terminates after copying the first 18 nt of the primer tRNA and stops, forming the (+) strand strong stop product (+ssDNA) Reverse transcription

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7) The tRNA is then removed by RNase H activity of RT 8) The exposed PBS anneals to the PBS sequence at the 3’ end of the (-) strand DNA, allowing the second template exchange. Product of the second template exchange is a circular DNA molecule with overlapping 5’ ends. Reverse transcription

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Reverse transcription J-J Kupiec, P Sonigo, JGV 1996

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Cloning strategy

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Peters et al., 2008 Cell-free virus secretion into the supernatant Intracellular expression levels of Gag and Pol Transduction rate of pMD9 mutants Transfected cells Transducted cells

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2.87:1:3.05:2.11:1.92:2.93 Gag p71/p68 Env p130 Physical titre evaluation in different PFV mutants by Env, Gag measurements

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EC50 of different viral titres calculated by non-linear regression function

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M. Sherman, W. Greene., MicInf, 2002

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M Stevenson , Nature Medicine , 2000

Enzymatic Proteins: Integrase: 

Enzymatic Proteins: Integrase Integrates retroviral DNA into host genome Endonuclease activity Drugs being developed

Transportins Serine/Arginine rich (SR1, SR2) : 

Transportins Serine/Arginine rich (SR1, SR2) Basic ’classical’ NLS Non-basic NLS Importin alpha

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TR-SR2 model was made by using ESyPred3D automated homology modeling software. In-build neural network created enhanced multiple alignmen ing performances on the basis of modeling package MODELLER. TR-SR1 (A) and TR-SR2 (B) homology models. Both molecules represent α-helical structures with two arches. The N-terminal arch is important for the Ran-GDP binding and C-terminal arch for the cargo binding. (A) (B)

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Statistic index Observed Expected # res in phipsi core 785 (88%) 798 (90%) # res in phipsi allowed 83 (9%) 62 (7%) # res in phipsi generous 15 (1%) 9 (1%) # res in phipsi outside 2 (0%) 0 (0%) Free energy of folding -839.83 -862.11 stereochemical quality of TR-SR2 protein structure

TR-SR1(gray) and TR-SR2(blue) superimposition: 

TR-SR1(gray) and TR-SR2(blue) superimposition

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The rigid docking of HIV-1 IN CCD (A) and TR-SR2 (B). Protein domains painted in different colours: HIV-1 IN CCD (162 aa) is green; NLS (13 aa) is red; Ran-GDP BD (1-303 aa) is grey; Acidic loop (304-378 aa) is yellow; CBD (379-887 aa) is blue, RMSD=1.6 Å.

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Close interaction of TR-SR2 Ran-GDP BD fragment (11-QQVLQLLKDSQSPNTATQR-29) in grey and HIV-1 IN CCD NLS (115-IIGQVRDQAEHLK-127). There are various stick residues interacting with each other. TR-SR2 fragment consists of two α-helices and one ‘hinge’ region.

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HIV-1 IN NLS Area Acc. (%) Ile115 157.887 92.2 Ile116 118.882 69.4 Gly117 61.635 71.7 Gln118 138.416 74.0 Val119 71.374 48.0 Arg120 93.843 38.5 Asp121 128.365 79.2 Gln122 125.090 66.9 Ala123 50.419 44.6 Glu124 151.511 80.5 His125 127.643 66.6 Leu126 122.689 69.8 L ys127 226.537 100.0 TR-SR2 Fragment Area Acc. (%) Gln11 172.366 92.2 Gln12 97.244 52.0 Val13 85.211 57.2 Leu14 106.186 60.4 Gln15 109.943 58.8 Leu16 53.422 30.4 Leu17 93.744 53.3 Lys18 183.996 86.9 Asp19 87.537 54.0 Ser20 46.466 100.0 Gln21 77.776 41.6 Ser22 92.172 100.0 Pro23 83.142 57.5 Asn24 25.791 16.3 T hr25 55.044 38.2 A la26 83.684 74.0 T hr27 101.880 70.7 G ln28 42.686 22.8 A rg29 163.078 66.8 Residue area and accessibility of docked molecules

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The first conformation of HIV-1 IN NLS (A) and TR-SR2 Ran-GDP BD fragment (B) flexible docking. Labeled amino acids are forming H-bonds between two molecules. Molecules are colored according to their atom composition. Wire frame spheres are displayed on the atoms with the hydrogen bonds.

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AA residues that could be involved in H-bonds formation obtained from AUTODOCK 4.0 donor 'ligand'-> acceptor 'receptor' residues in close contact donor 'receptor'-> acceptor 'ligand' residues in close contact Gln122-> Leu17 Lys127-> Gln21, Ser20, His125-> Asp19 Gln21-> Lys127 Asp19-> His125 Ser20-> Lys127 Leu17-> Gln122

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TR-SR2 Ran-GDP Fragment (A) and HIV-1 IN NLS amino acids (B). There is a correlation between residue’s accessibility and hydrophobicity of those amino acids which have tendency to H-bond formations. Highly accessible amino acids have very low hydrophobicity index.

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Viral Variant Nuclear Localization Signals Predicted KBM HIV-1 (YU-2) LKKIIGQV RDQAEHLK TAV Tyrosine kinase binding motif HIV-1 (cl. A) LKKIIGQV REQAEHLK TAV Tyrosine kinase binding motif SIV/CPZ (US) LKKIIGQI RDQAEHLK TAV Tyrosine kinase binding motif HIV-2 (cl. A) LKNQISRIREQANTZETIV No motif found SIV/SMM LKNQISRIREQANSZETIV No motif found SIV/AGM (VER) LKEIIG KIRDDCQY TETAV Tyrosine kinase binding motif SIV/AGM (SAB) LKEIIGQI RDDAERLE TAV Tyrosine kinase binding motif SIV/SYK LKEAISQIRDDVTHLQTAV No motif found SIV/L'HOEST LKKIIGQV RDQAEHL KTAV Tyrosine kinase binding motif PFV ARPA SLR PRWHKPSTVLKVLNPR Protein kinase C binding motif SV40 (T-Ag) MPKK KRKVEDPG T Tyrosine kinase binding motif Different viral NLS contain different KBMs (KBMs are in red).

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Interactions in proteins Bonded interactions are required to maintain the integrity of the structure. Non-bonded interaction

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Molecular dynamics Employs Newton’s Laws of physics: Dynamical simulation  Deterministic . Generates a sequence of configurations or states (an ensemble) as a function of time. Requires force field to compute forces on atoms . Both thermodynamical and dynamical quantities can be computed from the simulation.

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CTHL (Cys, Thr, His, Leu)-EEEE (Extended conformation) ETGQ (Glu, Thr, Gly, Gln)-CCHH (Coil-Helix) ETAY (Glu, Thr, Ala, Tyr)-HHHH (Alpha helix) TTVK (Thr, Thr, Val, Lys)-HHHH (Alpha helix) YSAG (Tyr, Ser, Ala, Gly)-EEHH (Extended Conf-Helix) http://pred.ngri.re.kr/PredPhospho.htm

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MDS of non-phosphorylated and phosphorylated peptides CTHL (Cys, Thr, His, Leu)-EEEE (Extended conformation) Torsion angle: phi: -62, psi: 77 ( total energy : 475.713030 kJ/mol) Torsion angle: phi: 66, psi: -79 ( total energy : -416.334966 kJ/mol) Torsion angle (cryst): phi: -129, psi: 146 ETGQ (Glu, Thr, Gly, Gln)-CCHH (Coil-Helix) Torsion angle: phi: -73 , psi: -48 ( total energy : -60.933959 kJ/mol) Torsion angle: phi: -77, psi: -64 ( total energy : -882.084298 kJ/mol Torsion angle (cryst): phi: -145, psi: 152 ETAY (Glu, Thr, Ala, Tyr)-HHHH (Alpha helix) Torsion angle: phi:-62, psi: -33 ( total energy : 218.030897 kJ/mol) Torsion angle: phi: -64, psi: -47 ( total energy : -252.003679 kJ/mol Torsion angle (cryst): phi: -54, psi: -49 TTVK (Thr, Thr, Val, Lys)-HHHH (Alpha helix) Torsion angle: phi: -64, psi: -33 ( total energy : 373.268748 kJ/mol) Torsion angle: phi: -46, psi: -24 ( total energy : -440.040907 kJ/mol) Torsion angle (cryst): phi: -64, psi: -40 YSAG (Tyr, Ser, Ala, Gly)-EEHH (Extended Conf-Helix) Torsion angle: phi: -68, psi: 92 ( total energy : 350.075199 kJ/mol) Torsion angle: phi: -70, psi: 113 ( total energy : -470.023203 kJ/mol) Torsion angle (cryst): phi: -94, psi: 162

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HIV IN phosphorylated form HIV IN non-phosphorylated form Superimposition HIV IN crystal structure NLS-domain? Regulatory domain?

Acknowledgments to: 

Thank you for your attention Acknowledgments to Institute of Virology and Immunobiology, University of Wuerzburg Prof. Dr Axel Rethwilm PD. Dr Carsten Scheller Tatiana Wiktorowicz Daniel Matthes Dept of Bioinformatics, Biocenter, University of Wuerzburg Prof. Dr Thomas Dandekar Dr. Karin Schleinkofer Dept of Structural Bioinformatics, EMBL, Heidelberg Dr. Rob Russell