Molecular interactions of HIV-1 integrase (IN) with the viral DNA ends within the cytoplasmic preintegration complex (PIC) in virus infected cells are not clearly understood. Knowledge of these interactions is critical to understand strand transfer inhibitors (STI) directed against IN, drug-resistance to these inhibitors, and development of second generation inhibitors. Using purified HIV IN and viral DNA substrates, we have identified the transient synaptic complex (SC) on native agarose gels that shares properties associated with the PIC. We will use these new advancements for understanding concerted integration to: 1) determine the functional mechanisms of HIV SC assembly using purified IN monomers;2) explore conditions to produce homogenous HIV IN-DNA complexes using small DNA oligonucleotides (ODN) as substrates, without or in the presence of strand transfer inhibitors (STI);and 3) initiate co-crystallization studies of HIV IN-DNA complexes using state-of-the-art robotic technologies. The crystal structure of the prototype foamy virus (PFV) intasome (Cherepanov group) has greatly enhanced our understanding of IN subunit interactions with the cognate DNA ends to promote concerted integration. PFV IN is mainly a monomer that allows interdomain complementation between DNA ends to form the intasome. The PFV intasome and the HIV SC are functionally equivalent for concerted integration. Atomic resolution studies of the HIV SC are necessary because the amino acid identity between PFV and HIV IN is minimal outside their respective catalytic centers. A mystery existed for 20 years why recombinant HIV IN was not capable of efficiently using ODN for concerted integration. We have made two recent advances towards solving this mystery recently published this last October (Biochem. 50:9788-9796, 2011). One, we determined that our extensively studied HIV-1 IN exists as a highly soluble monomer. Two, we discovered that our purified HIV IN uses U5 LTR ODN (18 bp to 42 bp, 3'OH recessed or blunt) for highly efficient concerted integration activity where both IN and ODN are in the micromolar concentrations. Finally, we also recently identified several STI (>200 nM) that selectively produce a new IN-single DNA (ISD) complex using either blunt or recessed ended U5 substrates. In summary, we hypothesize that HIV IN monomers facilitate the assembly of the active tetramer onto viral DNA for concerted integration. We will explore conditions to produce soluble HIV IN-DNA complexes, without and with STI, for co-crystallization studies.

Public Health Relevance

Infection of humans by HIV-1 is generally lethal unless the patient is placed on highly active antiretroviral drug therapy (HAART). The recent addition of Raltegravir which inhibits the HIV-1 integrase to combinational drug therapies has significantly increased the success of HAART. Our proposed studies will provide biochemical and atomic resolution insights into the structural mechanisms associated with Raltegravir using wild type and drug-resistant HIV integrase mutants.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI100682-02
Application #
8495924
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Sharma, Opendra K
Project Start
2012-07-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
2
Fiscal Year
2013
Total Cost
$176,250
Indirect Cost
$58,750
Name
Saint Louis University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
050220722
City
Saint Louis
State
MO
Country
United States
Zip Code
63103