And development efforts herpes virus associated dna

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Unformatted text preview: a. •  Infects B ­lymphocytes –  Long term latent infecKon –  Licle to no progeny virions produced InfecKng B ­lymphocytes •  Two key receptor proteins for EBV –  CD21 (complement receptor) –  HLA class II (also know as MHC class II) Viral Envelope Protein g350/220 binds to CD21 EBV is uptaken in EndocytoKc vesicle Viral Envelope Protein gp42 binds to HLA class II, this direct viral entry gp42, gH, gL and gB mediate membrane fusion Gene Expression and Latency EBNA (Epstein Barr nuclear anKgens) and LMP (latent membrane protein) AnKviral Therapy Problems in the Discovery and Development of anti-herpetic Polymerase Inhibitors Viral polymerases are validated targets in drug discovery and development efforts. Herpes virus associated DNA polymerases are difficult to express/purify. Cell-based drug susceptibility assays are time-consuming, inaccurate, or not even available for each of the eight different viruses. Success in the development of assays that facilitate screening and assessment of polymerase inhibitors has therefore been limited. Objective Development of a surrogate system that facilitates the discovery and development of polymerase active site inhibitors. Similarities among Viral DNA polymerases and their Orthologs in Phages The polymerase of bacteriophage RB69 (gp43) can be expressed and purified at high yields for structural and biochemical studies. Yang et al, Biochemistry 2002 Structure ­Based Alignment of HCMV and Phage RB69 associated Polymerases Helix N A RB69 Helix P B C DGVIPTEIT VFN EH ~~~~A INR LLI SL AL HHV5(HCMV) VSVLSELLN WVS AV ~~~~E MAL VTC AF FT Approach: Design of Phage/Virus Chimeras Swap the natural substrate binding sites: Introduce helix N and helix P of herpesvirus polymerases against a backbone of gp43 Predicted properties: 1.  The chimeric enzymes can be expressed in bacteria (E.coli) 2.  Chimeras are sensitive to antiviral drugs RB69 gp43 is not sensiKve to PFA; The Chimera ABC is! ABC/ Q807A ABC PFA µM PFA µM PFA µM PFA µM Minus Mg2+ 0 0.4 1.2 3.7 11 33 100 0 0.4 1.2 3.7 11 33 100 0 0.4 1.2 3.7 11 33 100 0 0.4 1.2 3.7 11 33 100 gp43 ABC/ R784A p s gp43 IC50, µM ABC ABC/R784A ABC/Q807A > 100 2.9 27 29 Tchesnokov et al. , J Biol Chem 2009 Structural Data that Validate the Approach PFA Traps the gp43 ­UL54 Complex Pre ­TranslocaKon Zahn et al. JBC, 2011 A580 •  Structural alignments between HSV1 and RB69 shows that this residue is conserved between Herpes virus and Bacteriophage –  Not part of a moKf –  Residues before and aper are not conserved •  In the RB69 structure A580 is at the N terminal end of Q helix –  Part of the finger hinge •  The equivalent residue in HCMV A834, mutant of this residue to P produces GCV/ PFA/CDV resistance P helix Conserved glycine residue MoKf B A580 Q helix...
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This note was uploaded on 01/23/2014 for the course MIMM 324 taught by Professor Gregjmatlashewski during the Winter '12 term at McGill.

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