Retroviral Integrase: Difference between revisions
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==Function== | ==Function== | ||
[[Retroviral 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, | [[Retroviral 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 el al (2010)<ref name=Hare_2010>PMID:20118915</ref> 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. <br/> | ||
HIV Integrase inhibitors: Raltegravir, marketed as Isentress is currently approved as a therapeutic inhibitor of HIV integrase. It was approved on October 12, 2007. | HIV Integrase inhibitors: Raltegravir, marketed as Isentress is currently approved as a therapeutic inhibitor of HIV integrase. It was approved on October 12, 2007. | ||
[See below for a table of antiretroviral drugs with trade name, company, patents, and notes.] | [See below for a table of antiretroviral drugs with trade name, company, patents, and notes.] | ||
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==PFV Intasome Crystallization== | ==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. | To mimic the viral DNA ends of HIV-1, Hare ''et al'' (2010)<ref name="Hare_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. | 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. | ||
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===Crystallographic and Refinement Statistics=== | ===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. | Hare ''et al'' (2010)<ref name="Hare_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== | ==Overall Architecture & Components== | ||
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===Structure=== | ===Structure=== | ||
The overall structure of the assembled PFV intasome is a tetramer model based on two domain structures with a dimer-dimer interface. Previous intasome models depict a similar but more flexible structure while the PFV intasome has been shown to be highly constrained. Using homology modeling, Hare ''et al'' (2010) propose that shorter interdomain linkers may be a factor in flexibility, specifically in HIV-1 integrase. The inner subunits of the tetramer are implicated in the overall tetramerization and viral DNA binding. The catalytic core domains of the outer subunits may act as supports, but since the amino- and carboxy-terminal domains are unresolved in electron density maps, their function remains inconclusive. The catalytic core domain and carboxy-terminal domain linker adopts an extended conformation for most of its length, and are located parallel to the amino-terminal domain and catalytic core domain linker of the inner subunit. The interdomain linkers The interdomain linkers (CCD-CTD linker and NTD-CCD linker) bind both halves of the intasome together, and the structure is further stabilized by a pair of carboxy-terminal domains interacting with both inner catalytic core domains. | The overall structure of the assembled PFV intasome is a tetramer model based on two domain structures with a dimer-dimer interface. Previous intasome models depict a similar but more flexible structure while the PFV intasome has been shown to be highly constrained. Using homology modeling, Hare ''et al'' (2010)<ref name="Hare_2010" /> propose that shorter interdomain linkers may be a factor in flexibility, specifically in HIV-1 integrase. The inner subunits of the tetramer are implicated in the overall tetramerization and viral DNA binding. The catalytic core domains of the outer subunits may act as supports, but since the amino- and carboxy-terminal domains are unresolved in electron density maps, their function remains inconclusive. The catalytic core domain and carboxy-terminal domain linker adopts an extended conformation for most of its length, and are located parallel to the amino-terminal domain and catalytic core domain linker of the inner subunit. The interdomain linkers The interdomain linkers (CCD-CTD linker and NTD-CCD linker) bind both halves of the intasome together, and the structure is further stabilized by a pair of carboxy-terminal domains interacting with both inner catalytic core domains. | ||
===Integrase and DNA interactions=== | ===Integrase and DNA interactions=== | ||
