Duane P. Grandgenett, Ph.D.
Molecular Microbiology and Immunology
Ph.D. in Microbiology, University of Iowa, 1970
The Retrovirus Integrase:
Following infection of a cell by a retrovirus and subsequent reverse transcription of the viral RNA, the linear viral DNA coupled with integrase (IN) and other viral proteins produce a cytoplasmic macromolecular structure termed the preintegration complex (PIC). The PIC is transported into the nucleus where the viral DNA ends are inserted in a concerted fashion by IN into the host chromosomes.
Numerous research laboratories and pharmaceutical companies have made significant contributions towards understanding the mechanisms involved in retrovirus integration over the last 38 years. Integration of human immunodeficiency virus type 1 (HIV-1) DNA into human chromosomes is essential for viral replication. Unchecked replication of HIV-1 in humans causes AIDS. Integration of the HIV-1 DNA is mediated by the viral IN. The FDA has approved three effective IN strand transfer inhibitors (STIs) which are used in combination with other inhibitors directed against the viral reverse transcriptase and protease to prevent AIDS. As of April 2015, there are five recommended regimens for antiretroviral therapy for treatment-naïve HIV-1 infected patients: four STI-based regimens and one ritonavir-boosted protease inhibitor-based regimen (http://aidsinfo.nih.gov/guidelines).
The further development of retrovirus vectors for human gene therapy requires a deep
understanding of the biological and biochemical properties of retrovirus INs.
Current Research Projects
We are currently using purified recombinant HIV-1 and Rous sarcoma virus (RSV) IN with viral DNA substrates to reconstruct a complex, termed intasome, which mimics the ability of the PIC to promote concerted integration. The assembly pathways for the intasome using HIV-1 and RSV IN are being investigated. Clinical HIV-1 strand transfer inhibitors are also being studied. Crystallography studies of RSV and HIV-1 intasomes as well as IN bound to viral/target DNAs are being pursued. We were recently successful in determining the atomic structure of the RSV strand transfer complex that contains viral/target DNA sequences (Nature 530:361-366, 2016).
To investigate the structure-functional relationship between retrovirus IN and its viral DNA substrate necessary to mediate the biological relevant concerted integration reaction.
Grandgenett, D.P., and Aihara, H. (2018) Oligomerization of retrovirus integrases. In Virus Protein and Nucleoprotein Complexes, Edited J. R. Harris and D. Bhella. Springer Subcellular Biochemistry series (Invited Book Chapter).
Major Point: Integration of the reverse-transcribed viral cDNA into the host’s genome is a critical step in the lifecycle of all retroviruses. Retrovirus integration is carried out by integrase (IN), a virus-encoded enzyme that forms an oligomeric ‘intasome’ complex with both ends of the linear viral DNA to catalyze their concerted insertions into the backbones of the host’s DNA. Different species of retrovirus INs also form complexes with different host proteins, which guides the intasome to preferred regions of the chromosome. Recent 3-dimensional structural studies have revealed remarkable diversity as well as conserved features among the architectures of the intasome assembled from five different genera of retroviruses. The minimal oligomeric form of IN for concerted integration is a tetramer but other higher-order octameric, dodecameric and hexadecameric IN structures are found in these different retroviruses. This chapter reviews how IN oligomerizes to achieve its function, with particular focus on alpharetrovirus including the 3-dimensional structure of its octameric intasome and biology of the avian retrovirus Rous sarcoma virus.
Pandey, K.K., Bera, S., Shi, K., Aihara, H., and Grandgenett, D.P. (2017) A C-Terminal "Tail" Region in the Rous Sarcoma Virus Integrase Provides High Plasticity
of Functional Integrase Oligomerization during Intasome Assembly. J Biol Chem. 2017 Mar 24;292(12):5018-5030.
Major Point: The assembly processes for forming Rous sarcoma virus intasomes capable of concerted integration of the retrovirus DNA into a target DNA are unknown. We have determined that the conversion of an intasome containing integrase (IN) tetramers to an intasome containing IN octamers is controlled by the C-terminal last 18 residues or "tail region" of IN. The results suggest a high degree of plasticity for functional multimerization and reveal a critical role of the C-terminal tail region of IN in higher order oligomerization of intasomes, potentially forming future strategies to prevent HIV-1 integration.
Yin, Z., Shi., K., Banerjee, S., Pandey K.K., Bera, S., Grandgenett, D.P., and Aihara, H. (2016) Crystal structure of the Rous sarcoma virus intasome. Nature. 530: 362-366.
Major Point: The crystal structure of a three-domain IN from Rous sarcoma virus (RSV) in complex with viral and target DNAs was determined. The structure shows an octameric assembly of IN, in which a pair of proximal IN dimers engage viral DNA ends for catalysis while another pair of distal IN dimers, bridged between the proximal IN dimers, help capture target DNA. The individual domains of the eight IN molecules play varying roles to hold the complex together, making extensive networks of protein-DNA and protein-protein contacts distinct from those previously observed for prototype foamy virus (PFV) intasome, a tetrameric IN complex. Our work highlights diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.
A movie depicts the RSV intasome structure included in the Nature article. The viral DNA is black while the target DNA is blue/yellow. The intasome contains eight IN subunits individually colored. The N-terminal domain (NTD), catalytic core domain (CCD), and C-terminal domain (CTD) are also depicted. The movie was produced by Dr. Hideki Aihara (University of Minnesota). v530 n7590.
Grandgenett, D.P., Pandey, K.K., Bera, S. and Aihara, H. (2015) Multifunctional facets of retrovirus integrase. World J. Biol. Chem. 6:83-94.
Major Point: This review examines the multifunctional properties of retrovirus integrase (IN) besides its key function of integrating the viral DNA into host chromosomes. IN also has a major role in the maturation of the virus, reverse transcription and nuclear transport of the preintegration complex. IN binds to cellular cofactors for uncoating of the viral core and to other cellular proteins that guide the preintegration complex to prefer regions on the host genome for integration. Understanding these IN functions has resulted in the production of three clinical IN strand transfer inhibitors to prevent HIV/AIDS and development of retrovirus vectors for human gene therapy.
Pandey, K. K., Bera, S., Korolev, S., Campbell, M., Yin, Z., Aihara, H., and Grandgenett, D. P. (2014) Rous sarcoma virus synaptic complex capable of concerted integration is kinetically trapped by human immunodeficiency virus integrase strand transfer inhibitors. J. Biol. Chem. 289: 19648-19658.
Major Point: RSV IN concerted integration activity is effectively inhibited by HIV IN strand transfer inhibitors (STIs) at low concentrations.
Shi, K., Pandey, K. K., Bera, S., Vora, A., Grandgenett, D. P., and Aihara, H. (2013)
A possible role for the asymmetric C-terminal domain dimer of Rous sarcoma virus integrase in viral DNA binding. Plos One 8:e56892.
Major Point: Crystal structure of the 3-domain Rous sarcoma virus IN was resolved at 1.86 Å. The binding of Rous sarcoma virus IN to viral DNA may be different than that observed with the 4-domain prototype foamy virus IN.
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. Biochemistry 50:9788-9796.
Major Point: The HIV-1 IN monomer selectively interacts with the viral DNA ends for concerted integration and appears to be the precursor of the IN tetramer necessary for assembly of the synaptic complex (intasome). The monomer may be more suitable than dimers of IN for producing crystals of IN/DNA complexes.
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.
Major Point: Various strand transfer inhibitors at high concentrations promote the formation of HIV-1 IN on a single viral DNA molecule.
Grandgenett, D. P. (2011). pp32 is Hot. In HIV-1 integrase: Mechanism and inhibitor design.ed., Neamati, N. and Wang, G. Wiley Press, June (Invited Book Chapter).
Major Point: A description of our research efforts that led to the discovery of the avian retrovirus IN in 1978.
Grandgenett, D. .P, Korolev, S. (2010) Retrovirus Integrase-DNA structure elucidates concerted integration mechanisms.Viruses. 2:1185-1189.
Major Point: Review of prototype foamy virus IN bound to viral DNA ends in a crystal structure.
Pandey, K.K., Bera, S., Vora, A. C. and Grandgenett, D.P. (2010) Physically Trapping of the HIV-1 synaptic complex by different structural classes of integrase strand transfer inhibitors. Biochemistry 49:8376-8387.
Major Point: Clinical strand transfer inhibitor Raltegravir and others are able to “trap” or stabilize the HIV-1 synaptic complex (intasome) by binding to the IN-DNA complex.
"Assembly of HIV Intasomes"
06/15/2016 - 05/31/2019
"Structural studies of DNA-processing enzymes"
09/01/2016 - 06/30/2021
Role: Subaward PI