Sequence: LANA [Human herpesvirus 8]

Gene:HHV8GK18_GP81     Conserved Domain     Related Protein 3D Structures

AID	Name	Type	Comment
2629	Fluorescence Polarization Cell-Free Homogeneous Primary HTS to Identify Inhibitors of the LANA Histone H2A/H2B Interaction	screening	Primary HTS
2659	Summary of Broad Institute MLPCN Kaposi's Sarcoma Herpes Virus Latent Infection Project	summary	Project summary

Primary Collaborators:
Kenneth Kaye,Brigham & Womens,Boston MA,kkaye@rics.bwh.harvard.edu,617-525-4256
Chantal Beauchemin,Brigham & Womens,Boston MA,cbeauchemin@rics.bwh.harvard.edu,617-525-4256

Keywords: KSHV, LANA, fluorescence polarization, nucleosomes, viral persistence

Assay Overview:
Screening for inhibitors that reduce or prevent the binding of LANA to the acidic region of the H2A/H2B histone dimer interface. Synthetic LANA peptide is labeled with an amino terminal FITC fluorophore linked via a beta alanine residue. The peptide contains the first 23 amino acids of the LANA protein that is essential for binding to the H2A/H2B interface. Nucleosomes were purifed from chicken Erythrocytes as a source of intact H2A/H2B dimers. Fluorescence is measured in the S and P planes with a Perkin Elmer Viewlux after 1 hour incubation.

Expected Outcome:
Compounds will be identified that inhibit the interaction of LANA 1-23 peptide with purified nucleosomes (containing H2A/H2B histone dimers). Inhibitors will have a reduced mP value.

LANA 1-23 peptide containing the first 23 amino acids of the LANA protein from Kaposi's sarcoma herpesvirus (KSHV) was synthesized with an N-terminal FITC via a beta alanine linker and HPLC purified. Purified chicken Erythrocyte nucleosomes are provided by the assay provider and purified from chicken red blood cells. They are stored at -80C at 30 uM.

Assay buffer (TEN-BT) is 10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 2.5 mM NaCl, 5 mM Beta-mercaptoethanol, 0.01% Triton-X-100

LANA peptide is diluted to a final concentration of 50 nM in TEN-BT buffer with 100 nM of purified nucleosomes in black 1536 well plates with 10 uL per well. 7.5 nL of compounds are pinned per well. The reaction is incubated at room temperature for 60 minutes (but is stable for up to 2 hours). The assay is read on a Viewlux plate reader using a 480 nM excitation filter, 535nM S and P emission filters and D505fp/D535 dichoric mirror. mP value for FP measurement = 1000*(S-G*P)/(S+G*P) where S= , P=, G= G-factor. The G Factor = 1.

LANA 1-23 peptide containing the first 23 amino acids of the LANA protein from Kaposi's sarcoma herpesvirus (KSHV) was synthesized with an N-terminal FITC via a beta alanine linker and HPLC purified. Purified chicken Erythrocyte nucleosomes are provided by the assay provider and purified from chicken red blood cells. They are stored at -80C at 30 uM.

Assay buffer (TEN-BT) is 10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 2.5 mM NaCl, 5 mM Beta-mercaptoethanol, 0.01% Triton-X-100

LANA peptide is diluted to a final concentration of 50 nM in TEN-BT buffer with 100 nM of purified nucleosomes in black 1536 well plates with 10 uL per well. 7.5 nL of compounds are pinned per well. The reaction is incubated at room temperature for 60 minutes (but is stable for up to 2 hours). The assay is read on a Viewlux plate reader using a 480 nM excitation filter, 535nM S and P emission filters and D505fp/D535 dichoric mirror. mP value for FP measurement = 1000*(S-G*P)/(S+G*P) where S= , P=, G= G-factor. The G Factor = 1.

Grant Number: 1 R21 NS061738-01