Background: Ph. D. in Microbiology (University of Iowa, 1970)

Major Research Interests:
Retrovirus Integration: Investigating the structure-function relationships between retrovirus integrase (IN) and its viral DNA substrate in synaptic complexes that mediate full-site integration.

Current Research Projects:
Integration of the retroviral DNA genome into host chromosomes is an essential step in the virus replication cycle. Following the infection of cells and subsequent reverse transcription, the linear viral DNA (~10 kbp) with IN and other viral proteins produce cytoplasmic macromolecular structures termed preintegration complexes (PIC). The PIC is transported into the nucleus where integration occurs. This integration event is termed full-site integration.

We have developed an in vitro model for retrovirus integration. Our model uses synaptic complexes assembled in vitro whose capacity to mediate full-site integration is equivalent to PIC isolated from virus-infected cells. The schematic below illustrates the interaction between a synaptic complex and its DNA target. The synaptic complexes consist of purified avian myeloblastosis virus (AMV) or recombinant Rous sarcoma virus (RSV) IN (blue oval) with linear viral DNA substrates from 3.6 to 4.5 kbp in length. The DNA terminal attachment (att) sites (~20 bp) where IN specifically binds are necessary for full-site integration. The target for integration is circular DNA (red circle). The full-site integration products are resolved by agarose gel electrophoresis. We study the protein-protein and protein-DNA interactions responsible for the assembly of the IN-DNA synaptic complexes. These interactions are important prior to and for docking with the target. IN exhibits integration activity as a dimer. The multimeric structure of IN at the att sites for assembly and full-site integration are unknown but is represented here as a tetramer.

Numerous laboratories have made significant contributions for understanding the mechanisms involved in retrovirus integration over the last 27 years. But, our basic understandings of the PIC or synaptic complex are still limited. Our investigations are focused on studying the interactions between IN subunits within synaptic complexes. Independent but interlocking approaches that we have used include the full-site integration assay, att site mutagenesis, protein-protein crosslinking, DNaseI footprinting, site-directed mutagenesis of recombinant RSV IN, biophysical measurements and others. Recombinant RSV IN has the same specific activity as the virion-derived AMV IN. Our effort to investigate wild type and RSV IN mutants in the context of virus replication facilitates a further understanding of avian retrovirus PIC in cells.

Similar strategies are employed to understand human immunodeficiency virus type 1 (HIV-1) full-site integration. Our studies using nonionic detergent lysates of viable HIV-1 virions showed that all the proteins necessary for full-site integration are in virions. Recent studies have demonstrated that recombinant HIV-1 IN is fully capable of full-site integration that is similar to PIC. Cellular proteins may play a role in the stability and transport of the PIC across the nuclear membrane and directing the PIC to integrate into genomic regions that are actively being transcript. We are investigating the potential role of cellular host factors in integration using HIV-1 synaptic complexes.

Numerous laboratories are engaged in understanding the specificity of retroviral integration at the genomic level. Our basic research with avian and HIV-1 IN will hopefully provide valuable information that aids in the development of retrovirus vectors that are clinically safe for human gene therapy.

Drugs that inhibit HIV-1 IN will be a valuable accompaniment to the current inhibitors of HIV-1 replication in humans thus preventing HIV-1/AIDS. Several inhibitors directed against IN, effective in preventing HIV-1 replication in vivo and in animal models, are currently in human clinical trials. We are investigating the biochemical and biophysical aspects of this inhibition through use of the HIV-1 synaptic complexes.

Representative Publications:

Bera, S., Vora, A.C., Chiu, R., Heyduk, T., and Grandgenett, D. P. (2005). Synaptic complex formation of two retrovirus DNA attachment sites by integrase: A fluorescence energy transfer study. Biochemistry 44:15106-15114.

Grandgenett, D. P., and Bera, S. (2005). The Biological and Biochemical Machineries for Retrovirus Integration, in Encyclopedia of Molecular Cell Biology and Molecular Medicine, VCH-Wiley Press, vol. 12, p.399-412 (Invited Chapter Review).

Grandgenett, D. P. (2005). Symmetrical recognition of cellular DNA target sequences during retroviral integration. Proc. Nat. Acad. Sci. USA, 102:5903-5904 (Invited Commentary).

Pandey, K.K., Sinha, S., and Grandgenett, D. P. (2007). Transcriptional co-activator LEDGF/p75 modulates HIV-1 integrase mediated concerted integration. J. Virol. 81:3969-3979.

Pandey, K.K., Bera, S., Zahm, J., Vora, A., Stillmock, K., Hazuda, D., and Grandgenett, D. P. (2007). Inhibition of HIV-1 concerted integration by strand transfer inhibitors which recognize a transient structural intermediate. J. Virol. 81:12189-12199.

Pandey, K. K., and Grandgenett, D. P. (2008). HIV-1 integrase strand transfer inhibitors: Novel insights into their mechanism of action. Retrovirology: Research and Treatment 2:41-46.

Zahm, J., Bera, S., Pandey, K. K., Stillmock, K., Hazuda, D. and Grandgenett, D. P. (2008). Mechanisms of human immunodeficiency virus type-1 concerted integration as related to strand transfer inhibition and drug resistance. Antimicrob Agents Chemother. 52:3358-3368.

Grandgenett, D. P., Pandey, K. K., Bera, S., and Vora, A. C., Zahm, J., and Sinha, S. (2009). Biochemical and biophysical analyses of concerted (U3/U5) integration. In Mechanistic and Pharmacological Analyses of HIV-1Integration, ed. Engelman, A., Elesvier, (Invited Chapter), Methods 47:229-236.

Bera, S., Pandey, K.K., Vora, A. C., and Grandgenett, D.P. (2009) Molecular interactions between HIV-1 integrase and the two viral DNA ends within the synaptic complex that mediates concerted integration. J Mol. Biol. 389:183-198.

Grandgenett, D. P. (2010). PP32 is Hot. In HIV-1 integrase: Mechanism and inhibitor design. ed. Neamati, N. and Wang, G. Wiley Press, In press (Invited Book Chapter).

Grandgenett, D. P. and Korolev, S. 2010. Retrovirus integrase-DNA structure elucidates concerted integration mechanisms. Viruses 2: 1185-1189. (Invited Commentary).

Pandey, K.K., Bera, S., Vora, A. C. and Grandgenett, D.P. (2010) Physical trapping of the HIV-1 synaptic complex by different structural classes of integrase strand transfer inhibitors (Biochemistry, in press).

Bera, S., Pandey, K.K., Vora, A. and Grandgenett, D.P. (2011) HIV-1 integrase strand transfer inhibitors stabilize an integrase-single blunt-ended DNA complex. J. Mol. Biol. 410:831-846. PMC3123398.

Pandey, K. K., Bera, S., and Grandgenett, D. P. (2011) The HIV-1 integrase monomer induces a specific interaction with LTR DNA for concerted integration. Biochem. 50:9788-9796. PMID:21992419. PMC Journal-In progress.