Talk abstracts

Talk on Wednesday 03:45-04:00pm submitted by Brandon Crowe

Structural mechanisms utilized by retroviruses to tether and integrate into host chromatin

Brandon Crowe (Department of Chemistry and Biochemistry), Jocelyn Eidahl (Department of Pharmacy), Ross Larue (Department of Pharmacy), Mamuka Kvaratskhelia (Department of Pharmacy), Mark Foster (Department of Chemistry and Biochemistry)

Abstract:
Retroviruses must integrate their reverse transcribed DNA into host chromatin in order to replicate. However, sites of integration are not random and vary by retroviral genera. For example; lentiviruses, such as human immunodeficiency virus type I (HIV-1), target integration to actively transcribed genes, whereas γ-retroviruses, such as murine leukemia virus (MLV), target transcription start sites. Preferential site selection of retroviral integration is known to be mediated through interactions between retroviral integrase and different host transcription factors. My research seeks to understand the structural mechanisms behind this tethering. Lens epithelium-derived growth factor/p75 (LEDGF/p75) is the human protein that HIV-1 hijacks for integration. The binding of LEDGF/p75 to HIV-1 integrase occurs through a C-terminal domain termed the integrase binding domain (IBD), while binding of LEDGF/p75 to host chromatin occurs though the PWWP domain named for its conserved Pro-Trp-Trp-Pro motif. We determined the NMR solution structure of the LEDGF/p75 PWWP domain and have characterized the binding of this domain to chromatin by performing NMR titrations for binding to a peptide containing tri-methylated K36 from histone H3 (H3K36me3) or double-stranded DNA. The resulting chemical shift perturbations identified the sites of peptide and DNA binding. We have also determined the binding affinity for each by fluorescence anisotropy. Binding of the PWWP domain to H3K36me3 and DNA explains the site selection of HIV-1, since the H3K36me3 is an epigenetic mark for actively transcribed genes. In contrast to the findings for lentiviral integration, MLV hijacks a different transcription factor family, the bromodomain and extraterminal domain (BET) family. The BET family contains two N-terminal bromodomains, which bind and recognize acetylated lysines of histone tails, which are marks for transcription start sites, and an extraterminal (ET) domain which has recently been found to bind to MLV integrase directly. We used NMR chemical shift perturbations to determine the binding site on Brd4 ET domain of a peptide from MLV integrase. These studies have provided unique insights into the structural mechanisms retroviruses utilize to target integration to specific sites within human chromatin.

References:
Eidahl, J. O., Crowe, B. L., North, J. A., McKee, C. J., Shkriabai, N., Feng, L., Plumb, M., Graham, R. L., Gorelick, R. J., Hess, S., Poirier, M. G., Foster, M. P., and Kvaratskhelia, M. (2013) Nucleic acids research 41, 3924-3936
Larue, R. C., Plumb, M. R., Crowe, B. L., Shkriabai, N., Sharma, A., Difiore, J., Malani, N., Aiyer, S. S., Roth, M. J., Bushman, F. D., Foster, M. P., and Kvaratskhelia, M. (2014) Nucleic acids research

Keywords: Retrovirus integration, Epigenetics, NMR solution structure