Integrase and integration: biochemical activities of HIV-1 integrase

Integration of retroviral DNA is an obligatory step of retrovirus replication because proviral DNA is the template for productive infection. Integrase, a retroviral enzyme, catalyses integration. The process of integration can be divided into two sequential reactions. The first one, named 3'-processing, corresponds to a specific endonucleolytic reaction which prepares the viral DNA extremities to be competent for the subsequent covalent insertion, named strand transfer, into the host cell genome by a trans-esterification reaction. Recently, a novel specific activity of the full length integrase was reported, in vitro, by our group for two retroviral integrases (HIV-1 and PFV-1). This activity of internal cleavage occurs at a specific palindromic sequence mimicking the LTR-LTR junction described into the 2-LTR circles which are peculiar viral DNA forms found during viral infection. Moreover, recent studies demonstrated the existence of a weak palindromic consensus found at the integration sites. Taken together, these data underline the propensity of retroviral integrases for binding symmetrical sequences and give perspectives for targeting specific sequences used for gene therapy

A novel function for spumaretrovirus integrase: an early requirement for integrase-mediated cleavage of 2 LTR circles

Retroviral integration is central to viral persistence and pathogenesis, cancer as well as host genome evolution. However, it is unclear why integration appears essential for retrovirus production, especially given the abundance and transcriptional potential of non-integrated viral genomes. The involvement of retroviral endonuclease, also called integrase (IN), in replication steps apart from integration has been proposed, but is usually considered to be accessory. We observe here that integration of a retrovirus from the spumavirus family depends mainly on the quantity of viral DNA produced. Moreover, we found that IN directly participates to linear DNA production from 2-LTR circles by specifically cleaving the conserved palindromic sequence found at LTR-LTR junctions. These results challenge the prevailing view that integrase essential function is to catalyze retroviral DNA integration. Integrase activity upstream of this step, by controlling linear DNA production, is sufficient to explain the absolute requirement for this enzyme. The novel role of IN over 2-LTR circle junctions accounts for the pleiotropic effects observed in cells infected with IN mutants. It may explain why 1) 2-LTR circles accumulate in vivo in mutants carrying a defective IN while their linear and integrated DNA pools decrease; 2) why both LTRs are processed in a concerted manner. It also resolves the original puzzle concerning the integration of spumaretroviruses. More generally, it suggests to reassess 2-LTR circles as functional intermediates in the retrovirus cycle and to reconsider the idea that formation of the integrated provirus is an essential step of retrovirus production

Branched oligonucleotide-intercalator conjugate forming a parallel stranded structure inhibits HIV-1 integrase

AbstractIntegration of a DNA copy of the HIV-1 genome into chromosomal DNA of infected cells is a key step of viral replication. Integration is carried out by integrase, a viral protein which binds to both ends of viral DNA and catalyses reactions of the 3′-end processing and strand transfer. A 3′-3′ branched oligonucleotide functionalised by the intercalator oxazolopyridocarbazole at each 5′-end was found to inhibit integration in vitro. We show that both a specific (G,A) sequence and the OPC intercalating agent contribute to the capability of the branched oligonucleotide to form a parallel stranded structure responsible for the inhibition

In Silico

Most antiretroviral medical treatments were developed and tested principally on HIV-1 B nonrecombinant strain, which represents less than 10% of the worldwide HIV-1-infected population. HIV-1 circulating recombinant form CRF02_AG is prevalent in West Africa and is becoming more frequent in other countries. Previous studies suggested that the HIV-1 polymorphisms might be associated to variable susceptibility to antiretrovirals. This study is pointed to compare the susceptibility to integrase (IN) inhibitors of HIV-1 subtype CRF02_AG IN respectively to HIV-1 B. Structural models of B and CRF02_AG HIV-1 INs as unbound enzymes and in complex with the DNA substrate were built by homology modeling. IN inhibitors—raltegravir (RAL), elvitegravir (ELV) and L731,988—were docked onto the models, and their binding affinity for both HIV-1 B and CRF02_AG INs was compared. CRF02_AG INs were cloned and expressed from plasma of integrase strand transfer inhibitor (INSTI)-naïve infected patients. Our in silico and in vitro studies showed that the sequence variations between the INs of CRF02_AG and B strains did not lead to any notable difference in the structural features of the enzyme and did not impact the susceptibility to the IN inhibitors. The binding modes and affinities of INSTI inhibitors to B and CRF02_AG INs were found to be similar. Although previous studies suggested that several naturally occurring variations of CRF02_AG IN might alter either IN/vDNA interactions or INSTIs binding, our study demonstrate that these variations do affect neither IN activity nor its susceptibility to INSTIs

Efficient and Specific Internal Cleavage of a Retroviral Palindromic DNA Sequence by Tetrameric HIV-1 Integrase

BACKGROUND: HIV-1 integrase (IN) catalyses the retroviral integration process, removing two nucleotides from each long terminal repeat and inserting the processed viral DNA into the target DNA. It is widely assumed that the strand transfer step has no sequence specificity. However, recently, it has been reported by several groups that integration sites display a preference for palindromic sequences, suggesting that a symmetry in the target DNA may stabilise the tetrameric organisation of IN in the synaptic complex. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the ability of several palindrome-containing sequences to organise tetrameric IN and investigated the ability of IN to catalyse DNA cleavage at internal positions. Only one palindromic sequence was successfully cleaved by IN. Interestingly, this symmetrical sequence corresponded to the 2-LTR junction of retroviral DNA circles-a palindrome similar but not identical to the consensus sequence found at integration sites. This reaction depended strictly on the cognate retroviral sequence of IN and required a full-length wild-type IN. Furthermore, the oligomeric state of IN responsible for this cleavage differed from that involved in the 3'-processing reaction. Palindromic cleavage strictly required the tetrameric form, whereas 3'-processing was efficiently catalysed by a dimer. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the restriction-like cleavage of palindromic sequences may be a general physiological activity of retroviral INs and that IN tetramerisation is strongly favoured by DNA symmetry, either at the target site for the concerted integration or when the DNA contains the 2-LTR junction in the case of the palindromic internal cleavage

Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection

Contact with HIV-1 envelope protein elicits release of ATP through pannexin-1 channels on target cells; by activating purinergic receptors and Pyk2 kinase in target cells, this extracellular ATP boosts HIV-1 infectivity

La détermination de la susceptibilité et de la résistance des intégrases (INs) du virus de l immunodéficience humaine aux inhibiteurs de transfert de brins de l'IN et le développement d anticorps simple-chaîne ciblant l IN

Ce mémoire de thèse présente mes travaux sur la détermination de la susceptibilité et de la résistance des intégrases (INs) du virus de l immunodéficience humaine (VIH) aux inhibiteurs de transfert de brins de l'IN (INSTIs) ainsi que le développement de fragments d anticorps simple-chaîne (sdAbs) ciblant l IN du VIH. Tout d abord, car les études antérieures ont suggéré que les variations significatives de l IN de souche CRF02_AG pourrait avoir des effets consécutifs sur l'interaction entre l'inhibiteur et l IN, la susceptibilité de l IN de souche CRF02_AG du VIH-1 aux dernières INSTIs a été déterminée. Accord avec l'étude in silico, nous avons mis en évidence que l activité de 3 -processing et de transfert de brin des INs de souche B et de souche CRF02_AG sont comparables. La susceptibilité des INs recombinantes de souche CRF02_AG aux INSTIs utilisés (Raltégravir-RAL, Elevitégravir-EVG et L-731, 988) est similaire à celle de l IN de souche B, malgré les variations naturelles qui se produisent dans les INs de souche CRF02_AG. Le polymorphisme de l IN de CRF02_AG n a pas d effet significatif sur la susceptibilité aux INSTIs. Dans un second temps, la résistance de l IN du VIH-2 au RAL, l unique INSTI approuvé, a été confirmée in vitro avec des enzymes mutées portant des mutations de résistance. Les mutations aux positions 155 et 148 jouent un rôle similaire pour les VIH-1 et VIH-2, en rendant l'IN résistante au RAL. La mutation G140S confère peu de résistance, mais compense le défaut catalytique dû à la mutation Q148R. À l'inverse, Y143C seule ne confère pas de résistance au RAL excepté si la mutation E92Q est également présente. De plus, l'introduction de la mutation Y143C dans le mutant résistant N155H baisse le niveau de résistance de l enzyme contenant la mutation N155H, ce qui pourrait expliquer l'absence de détection de ces deux mutations ensemble dans un seul génome. Enfin, des anti-VIH sdAbs avec nombreuses propriétés intéressantes ont été sélectionnés pour développer des agents antirétroviraux. Après la sélection de sdAb ciblant l IN du VIH, nous avons obtenu des qui sdAbs qui reconnaissent spécifiquement une vaste gamme d INs in vitro, y compris le mutant G140S/Q148R résistant aux INSTIs. Néanmoins, l'activité inhibitrice des sdAbs n'a pas été observée. Les sdAbs ciblant l IN du VIH peuvent être utilisés pour d'autres applications, telles que des réactifs ciblant des nanocapteurs. À l'avenir, en raison des avantages uniques des sdAbs, le développement de sdAbs anti-IN du VIH qui bloquent la réplication du VIH reste attractive pour l'obtenir des inhibiteurs efficaces de l IN.This thesis presents the determination of susceptibility and resistance of HIV integrases (INs) to IN strand transfer inhibitors (INSTIs) and the development of single domain antibody (sdAb) targeting HIV IN. Firstly, the susceptibility of HIV-1 subtype CRF02_AG INs to the latest INSTIs was determined, since previous studies suggested that the significant variations of CRF02_AG IN may have consequential effects on the interaction between the inhibitor and IN. Consistent with in silico study, we found that 3 -processing and strand transfer activity of both HIV-1 subtype B IN and subtype CRF02_AG IN are comparable. The susceptibility of recombinant CRF02_AG INs to employed INSTIs (Raltegravir-RAL, Elevitegravir-EVG and L-731, 988) is similar to that of HIV-1 B IN. Hence, the polymorphism of CRF02_AG IN cannot significantly effect on the susceptibility to INSTIs. Secondly, the resistance of HIV-2 IN to RAL, the unique approved INSTI, has been confirmed in vitro with mutated enzymes harboring resistance mutations. Mutations at positions 155 and 148 played a similar role in HIV-1 and HIV-2, rendering the IN resistant to RAL. The G140S mutation conferred little resistance, but compensated for the catalytic defect due to the Q148R mutation. Conversely, Y143C alone did not confer resistance to RAL unless E92Q is also present. Furthermore, the introduction of the Y143C mutation into the N155H resistant background decreased the resistance level of enzyme containing the N155H mutation, possibly accounting for the lack of detection of these two mutations together in a single genome. Finally, anti-HIV IN sdAb that is endowed with many attractive properties was selected for developing antiretroviral agents. After the selections, we have obtained some sdAbs that specifically recognize a broad range of INs in vitro, including INSTI-resistance mutant G140S/Q148R. However, the inhibition activity of anti-HIV IN sdAbs has not been observed yet. Anti-HIV IN sdAbs can be applied for other application, such as targeting reagents for nanosensor. In future, development of anti-HIV IN sdAbs which are able to block HIV replication remains attractive for obtaining efficient inhibitor of IN.CACHAN-ENS (940162301) / SudocSudocFranceF

Chemistry and structure-activity relationship of the styrylquinoline-type HIV integrase inhibitors

molecule