Retroviral Integrase: Difference between revisions

==Introduction==

Integrase is an essential retroviral enzyme that binds to viral DNA and inserts it into a host cell chromosome.  The reverse transcribed cDNA of human immunodeficiency virus type 1 (HIV-1) is inserted in the host cell genome in order increase pathogen fitness and virulence. Integrase is produced by a class of retrovirus (like HIV) and is used by the virus to incorporate its genetic material into the host cell DNA.  The host cellular machinery then produces mRNA and then protein from the incorporated genetic material, thus replicating the virus.  Although several integrase inhibiting drugs have been investigated, the mechanism responsible for strand-transfer inhibition action remains to be elucidated. However, Hare ''et al'' (2010) determined the structural constituents of retroviral integration. Further elucidation of the complete structure of the retroviral integrase, and its application to regulate functional and enzymatic activities could potentially enable researchers to delay the progression of retroviral diseases. Moreover, study of HIV-1 integration could lead to a promising new target, and contribute to the generation pharmacophore models for antiviral therapy.

[See below for a table of antiretroviral drugs with trade name, company, patents, and notes.]

==Integrase Mechanism of Action==

[[Image:GAViralLifeCycle.jpg|thumb|alt=Alt text|Caption]]

==HIV and AIDS==  

[[Image:HIV_Stats.jpg|thumb|alt=Alt text|In 2010, there are more than 25 million people have died of AIDS, and it is estimated that approximately 33 million people are living with HIV.]]

To date, more than 25 million people have died of AIDS and it is estimated that approximately 33 million people are living with HIV today.

==Impact of Structure==

While existing antiretroviral agents improve the quality of life as well as extending the life of many patients, it fails to eradicate the disease. Studies in integrase inhibitors show that combination with other antiretroviral drugs diminish viral adaptations, and may have the potential to be used for ''salvage therapy'' for patients who have acquired resistance to other drugs. For more, please see [[User:Eric_Martz/Molecular_Playground/HIVDrug|AIDS Before Protease Inhibitors & HIV Protease Inhibitors: A Breakthrough]].

==PFV Intasome Crystallization==

To mimic the viral DNA ends of HIV-1, Hare ''et al'' (2010) utilized soluble and fully functional prototype foamy virus (PFV) intasome preparations, obtained using recombinant PFV integrase and double-stranded oligonucleotides.  

The remarkable stability of the integrase-DNA complexes were determined by observing the ''in vitro'' strand transfer reactions, which were classified into three modes of deproteination migration: (1) single concerted events: linearized target plasmid; (2) multiple concerted events: smear; (3) half-site events: open circular DNA. Further characterization of the PFV intasome also exhibited structural substantiality which implied strong protein-protein and protein-DNA interactions despite prolonged incubation under high ionic strength conditions. Comprehensive crystallization assays effected a viable crystal configuration that diffracted X-rays to 2.9 Angstroms resolution. A three-dimensional structure was ultimately determined. The asymmetric unit contained a single integrase dimer with a stably bound viral DNA molecule, and a pair of integrase dimers consociated with symmetry, which formed an oblong tetramer. The dimer interface is stabilized by intermolecular amino terminal and catalytic core domains (inner subunit-outer subunit) interactions. The overall shape of the oblong tetramer is unique albeit bearing semblances to previously reported HIV-1 integrase complexes.

===Crystallization Technique===

The protein-DNA complexes were formed using the full-length, wild-type PFV IN and synthetic dsDNA that modeled the viral end.

The intasome was crystallized utilizing the vapor-drop hanging diffusion technique.  The reservoir solution consisted on 1.35 M ammonium sulphate, 25% (v/v) glycerol, 4.8% (v/v) 1,6-hexanediol, and 50mM 2-(N-morpholino) ethanesulphonic acid (MES) at pH 6.5.  The protein-DNA crystals were also soaked in the presence of MK0518, GS9137, Mg(II), and/or Mn(II).  The crystal structure was solved using molecular replacement.

===Crystallographic and Refinement Statistics===

Hare ''et al'' (2010) have published data on seven crystal structures.  These data include the PFV IN complex (apo form) and six additional structures, including the complex bound to Mg, Mn, Mg/MK0518, Mn/MK0518, Mg/GS9137, and Mn/GS9137.  All seven structures belong to the P41212 space group.  They have been refined to between 2.85 and 3.25 Å resolution.

==Overall Architecture & Components==

===Active Site===

==Mechanism for strand-transfer inhibition action==

[[Image:Strand Inhibition.jpg|thumb|alt=Alt text|Above is a JMOL image of MK-0518 blocking the 3' end of the viral DNA from binding the active site.  Notice the metal chelating oxygen atoms in MK-0518 interacting with the magnesium cations found in the active site.]]

==Integrase Inhibitors==  

{| class="wikitable"

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==References==

1.Hare, Stephen; Gupta, Saumya Shree; Valkov, Eugene; Engelman, Alan & Cherepanov, Peter (2010) Retroviral intasome assembly and inhibition of DNA strand transfer. ''Nature'' 2010/01/31/online doi:10.1038/nature08784 <http://www.nature.com/nature/journal/vaop/ncurrent/full/nature08784.html> {{STRUCTURE_3l2q |  PDB=3l2q  |  SCENE=  }}

6.James F. Braun, DO, Ruth J. Cronje, PhD, Marnie G. Henderson (2008) HIV-1 Integrase Inhibitors.  www.prn.org  Volume 13, Pages 1–9

==Further Reading==

* GEN News Highlights [http://www.genengnews.com/news/bnitem.aspx?name=74423430&nc=1 "Scientists Solve 3-D Crystal Structure of Retroviral Integrase Bound to Viral DNA"], [http://www.genengnews.com Genetic Engineering & Biotechnology News] February 1, 2010.