Gladys Mbemba

Inhibition of Early Steps of HIV-1 Replication by SNF5/Ini1

To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection.

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.

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.

Synthesis and antiviral properties of some polyphenols related to Salvia genus

An efficient synthesis of the acid part of salvianolic acid E 2 is described. Compound 2 was obtained from vanillin in 10 steps and 21% overall yield. During the synthesis of 2 an unexpected 5-oxo-4b,9b-dihydroindano[1,2-b]benzofuran rac-12 was isolated. Both compounds together with the acid part of salvianolic acid D were active as HIV-1 integrase inhibitors at the submicromolar level. But they did not inhibit the replication of the virus on MT-4 cells.

Synthesis, biological evaluation and molecular modeling studies of quinolonyl diketo acid derivatives: New structural insight into the HIV-1 integrase inhibition

New quinolonyl diketo acid compounds bearing various substituents at position 6 of the quinolone scaffold were designed and synthesized as potential HIV-1 integrase inhibitors. These new compounds were evaluated for their antiviral and anti-integrase activity and showed inhibitory potency similar to that of 6-bromide analog 2. Molecular modeling and docking studies were performed to rationalize these data and to provide a detailed understanding of the mechanism of inhibition for this class of compounds.

New 2-arylnaphthalenediols and triol inhibitors of HIV-1 integrase—Discovery of a new polyhydroxylated antiviral agent

A series of 13 hydroxylated 2-arylnaphthalenes have been synthesized and evaluated as HIV-1 integrase inhibitors. 7-(3,4,5-trihydroxyphenyl)naphthalene-1,2,3-triol 1c revealed chemical instability upon storage, leading to the isolation of a dimer 5c which was also tested. In the 2-arylnaphthalene series, all compounds were active against HIV-1 IN with IC50s within the 1-10 microM range, except for 1c and 5c which displayed submicromolar activity. Antiviral activity against HIV-1 replication was measured on 1b-c and 5c. Amongst the tested molecules, only 5c was found to present antiviral properties with a low cytotoxicity on two different cell lines.

Structural and theoretical studies of [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl)]-4-hydroxy-2-oxo-3-butenoïc acid as HIV-1 integrase inhibitor.

Ethyl [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl]-4-hydroxy-2-oxo-3-butenoate 1 and [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl)]-4-hydroxy-2-oxo-3-butenoïc acid 2 were synthesized as potential HIV-1 integrase inhibitors and evaluated for their enzymatic and antiviral activity, acidic compound 2 being more potent than ester compound 1. X-ray diffraction analyses and theoretical calculations show that the diketoacid chain of compound 2 is preferentially coplanar with the quinolinone ring (dihedral angle of 0-30 degrees ). Docking studies suggest binding modes in agreement with structure-activity relationships.

The total synthesis of fukiic acid, an HIV-1 integrase inhibitor.

A successful synthesis of fukiic acid is described in 7% overall yield (6 steps from veratraldehyde). rac-Fukiic acid was found to be a potent inhibitor of HIV-1 integrase but did not reveal any antiviral activity in the MT-4 cells assay.