Retroviral integrases are key drug targets that mediate the integration of viral cdna into the host chromosome. Retroviral intasome assembly and inhibition of dna strand. Retroviral integrase in is an enzyme produced by a retrovirus such as hiv that integratesforms covalent links betweenits dna genetic information into that of the host cell it infects. We recently reported cocrystal structures of the prototype foamy virus pfv intasome with raltegravir and elvitegravir, establishing the general insti binding mode. Cherepanovretroviral intasome assembly and inhibition of dna strand transfer. Although mostly defective and inactive, some of the hervs may be activated under certain physiological and pathological conditions. The improved biophysical properties of intasomes assembled with ledgf. Clinical inhibitors preferentially target the dnabound form of integrase as compared with the free protein, highlighting the critical requirement for detailed understanding of hiv1 intasome structure and. Solution conformations of prototype foamy virus integrase. In the nucleus, in catalyzes dna strand transfer using the reactive 3. Frontiers inhibition of htlv1 infection by hiv1 first. Crystallization of the pfv intasome the majority of characterized ins predominantly promote the insertion of. Retroviral integrase in recognizes and acts upon the termini of the.
Binding of strand transfer inhibitors displaces the reactive viral dna end from the active site, disarming the viral nucleoprotein complex. Integrase is an essential retroviral enzyme that binds both termini of linear viral dna and inserts them into a host cell chromosome. Due to the importance of human immunodeficiency virus type 1 hiv1 integrase as a drug target, the biochemistry and structural aspects of retroviral dna integration have been the focus of intensive research during the past three decades. Inhibition of htlv1 infection by hiv1 first and second. The structure of fulllength retroviral integrase, either separately or in complex with dna, has been lacking. Hiv integrase as a target for antiretroviral therapy. The viral integrase enzyme catalyzes this process and is a major drug target. Retroviruses create permanently integrated proviruses that exist in the host genome. Retroviral integrase proteins and hiv1 dna integration.
When performing the strand transfer reaction in hepes ph 7, b strongly stimulates the concerted integration activity of htlv1 in. Pdf retroviral intasome assembly and inhibition of dna. While interference with rag1 and 2 recombination is associated with a negative impact on immune function, the inhibition of metnase or htlv. Cryoem structures and atomic model of the hiv1 strand. As alluded to above, catalysis under these conditions occurs in large aggregate assemblies more so than discrete soluble complexes 35, 62. A peptide derived from lens epitheliumderived growth factor.
These feature a catalytic triad of negatively charged amino acids, dde or ddd, that bind divalent metal ions and are brought together by an rnasehlike fold. Retroviral dna integration takes place in the context of the intasome nucleoprotein complex. Yoder 1 1 department of cancer biology and genetics, the ohio state university college of medicine. In cleaves a dinucleotide from each 30 end of the reversetranscribed viral doublestranded dna, in a process termed 30 processing, to prepare the dna for integration into the host genome in a consequent second step termed strand transfer, the latter step being the target of the current in strand transfer inhibitors instis. Assembly and purification of prototype foamy virus.
The outer monomers would be good candidates to form. A structure of the htlv1 or related deltaretroviral intasome bound to instis is needed. Insight into the ervk integrase propensity for dna damage. In vitro dna cleavage assays with purified t216ad249a transposase showed the nts strand remained intact. Retroviruses and ltr retrotransposons are transposable elements that encapsidate the rnas that are intermediates in the transposition of dna copies of their genomes proviruses, from one cell or one locus to another. Hare s, gupta ss, valkov e, engelman a, cherepanov p. The 5 overhang of the nontransferred dna strand is threaded between the ctd and ccd of the monomer that also binds the 3 end of the transferred strand of the same dna molecule figure 1. Our findings define the structural basis of retroviral dna integration, and will allow modelling of the hiv1 intasome to aid in the development of antiretroviral drugs.
Novel hiv1 integrase inhibitors derived from quinolone. Processing and strand transfer catalysed by retroviral. With larger size viral dna substrates 1 kb in complexes capable of concerted integration, the length of viral dna protected by hiv1 in varies from. Nih public access eugene valkov alan engelman, and peter. Structural role of the flanking dna in mariner transposon excision jacqueline dornan. Stephen hare, saumya shree gupta, eugene valkov, alan engelman, peter cherepanov. Because ral and related compounds preferentially inhibit dna strand transfer activity, the drugs are referred to as in strand transfer inhibitors instis. The whole elongated assembly of the two monomers is organized around two dna molecules and is unlikely to exist in a dnafree form.
The complex multistep life cycle of hiv allows it to proliferate within the host and integrate its genome in to the host chromosomal dna. The intasome is the basic recombination unit of retroviral integration, comprising the integrase protein and the ends of the viral dna made by reverse transcription. Valkov e, engelman a, cherepanov p 2010a retroviral intasome assembly and inhibition of dna strand transfer. Mechanistic similarities in dna transposase enzymes and retroviralretrotransposon integrases underscore the close evolutionary relationship among. Pdf molecular modeling of quinolinebased compounds as. Cherepanov, retroviral intasome assembly and inhibition of dna strand transfer. Structural role of the flanking dna in mariner transposon. This provirus can remain dormant for an indefinite period. Integration of viral dna into cellular dna is an essential step in the replication of hiv1 and other retroviruses. The retroviral integrase enzyme acts on the linear doublestranded viral dna product of reverse transcription. Both viral dna cleavage and dna strand transfer display inversion at chiral. This cited by count includes citations to the following articles in scholar. Pro236 is a conserved aminoacid that could contribute to the inhibition activity of rt even help to preserve the activity with. Retroviral intasome assembly and inhibition of dna strand transfer.
The ledgf also stimulates hiv1 in dna strand transfer activity and. Differential sensitivities of retroviruses to integrase. We show that pfv strand transfer complexes stc can be assembled on product dna without going through the normal forward reaction pathway. Migration of dna species in the gel is indicated on the right of the gel. The mvv intasome was assembled using wild type integrase proteins and. While clinical strand transfer inhibitors disarm the in active site, allosteric inhibition of enzyme activity through the disruption of inin protein interfaces holds great therapeutic potential. Molecular mechanisms of retroviral integrase inhibition. In the 11 march 2010 issue of nature, peter cherepanov and colleagues from the imperial college, london, determined the crystal structure of the prototype foamy virus pfv integrase in in complex with its viral dna. This addition could help with the activity of dual inhibitors of rt and in.
An essential step in the life cycle of lentiviruses such as hiv1 is when viral dna integrates into the host genome, establishing a permanent infection of the host cell. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus pfv retroviral intasome searches for an integration site by onedimensional 1d rotationcoupled diffusion along dna. Retroviral intasome assembly and inhibition of dna. The finding that a retroviral stc can be assembled in this way may provide a powerful tool to alleviate the obstacles that impede structural studies of nucleoprotein intermediates in hiv. Structurebased modeling of the functional hiv1 intasome and its. Human endogenous retroviruses hervs are genomic sequences of retroviral origin which were believed to be integrated into germline chromosomes millions of years ago and account for nearly 8% of the human genome.
Our findings define the structural basis of retroviral dna integration and will allow modeling of the hiv1 intasome to aid in the development of antiretroviral drugs. The ones marked may be different from the article in the profile. The catalytically competent cleaved synaptic complex csc intasome, which contains free. A novel assay for the dna strandtransfer reaction of hiv1 integrase. As followup of our past publication 1, we propose that quinolones as part of the pyridinone family are capable to increase the number of interactions with hiv reverse transcriptase rt or integrase in by adding a halogen in position c8 of aromatic portion of the quinolones. The development of hiv integrase in strand transfer inhibitors instis and our understanding of viral resistance to these molecules have been hampered by a paucity of available structural data. Once a target site is identified, the time between pfv strand transfer events is 470 ms. Retroviral intasomes search for a target dna by 1d. The retroviral intasome is the fundamental nucleoprotein complex that catalyses integration into a target dna 12. Insights into the functions of a molecular swiss army knife.
Pdf retroviral intasome assembly and inhibition of dna strand. In contrast, under optimal conditions pipes ph 7, no b is required for high strand transfer activity. Structural basis of secondgeneration hiv integrase inhibitor action. The development of hiv integrase in strand transfer inhibitors. Intasome assembly and catalysis proceeds through a multistep process that involves several distinct intermediates fig. Retroviral intasome assembly and inhibition of dna strand transfer stephen hare1, saumya shree gupta1,, eugene valkov1, alan engelman2, and peter cherepanov1 1. Structures of intasomes are needed to understand inhibitor. During viral integration, integrase binds the ends of viral dna, forming a higherorder structure called the intasome. Recently, we reported a crystal structure of the functional retroviral intasome containing full. E under conditions of optimal activity, in remains active in the presence of 200 mm nacl. Superposition of infab2 structure with dnabound structures e. Many dna transposases and retroviral integrases belong to a large superfamily of polynucleotide transferases 3. Retroviral genomes encode for functionally conserved gag, pro, pol, and env regions, as well as integrase in, which is required for retroviral integration. Inhibition of human immunodeficiency virus1 integrase by.
Hence, targeting integration is not only expected to block hiv. Structurebased modeling of the functional hiv1 intasome. The process of integration, governed by integrase in, is highly conserved across the retroviridae family. Assembly and purification of prototype foamy virus intasomes randi m. Integrase in is required for lentivirus replication and is a proven drug target for the prevention of aids in hiv1infected patients. Retroviral intasomes arising spiral imperial college london. The establishment of dna oligonucleotidebased hiv1 in 3. With larger size viral dna substrates 1 kb in complexes capable of concerted integration, the length of viral dna protected by hiv1 in varies from 16 to 32 base pairs, depending on the conditions of assembly, the step of dna processing, and the presence of strand transfer inhibitors. Xray crystal structures of functional pfv intasomes have yielded unprecedented details on the mechanism of retroviral dna integration, 18, 26. In has multiple roles in the retrovirus life cycle.
A schematic of indna complexes observed in in vitro systems. Retroviral integrase proteins and hiv1 dna integration journal of. Recent advances in the development of integrase inhibitors. The structure could not have been predicted based on the structure of the hiv1 integrase protein domains alone because the viral dna acts as a glue that holds the intasome. Retroviral intasome assembly and inhibition of dna strand transfer article pdf available in nature 4647286. Retroviral integrase in recognizes and acts on the termini of the linear doublestranded dna molecule that is produced by reverse transcription 1,2. Retrovirus integrasedna structure elucidates concerted. The binding of strandtransfer inhibitors displaces the reactive viral dna end from the active site, disarming the viral nucleoprotein complex. Request pdf structural basis for strand transfer inhibitor binding to hiv intasomes the hiv intasome is a large nucleoprotein assembly that mediates the integration of a dna copy of the viral. Retroviral integration intermediates and detection of 3. Xray crystal structures of functional spumaviral intasomes were previously revealed to harbor a homotetramer of integrase, and it was generally believed that integrase tetramers catalyzed the integration of other retroviruses. Structural basis for strand transfer inhibitor binding to. All tested compounds caused significant inhibition of in vitro strand transfer at nanomolar. The intasome is the basic recombination unit of retroviral integra tion, comprising.