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

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

Evidence for Gene Expression by Unintegrated Human Immunodeficiency Virus Type 1 DNA Species

The integrated form of human immunodeficiency virus type 1 (HIV-1) DNA is classically considered to be the sole template for viral gene expression. However, several studies have suggested that unintegrated viral DNA species could also support transcription. To determine the contribution of the different species of HIV-1 DNA to viral expression, we first monitored intracellular levels of various HIV-1 DNA and RNA species in a single-round infection assay. We observed that, in comparison to the precocity of HIV-1 DNA synthesis, viral expression was delayed, suggesting that only the HIV-1 DNA species that persist for a sufficient period of time would be transcribed efficiently. We next evaluated the transcriptional activity of the circular forms of HIV-1 DNA bearing two long terminal repeats, since these episomes were reported to exhibit an intrinsic molecular stability. Our results support the notion that these circular species of HIV-1 DNA are naturally transcribed during HIV-1 infection, thereby participating in virus replication

Vaccination contre le virus de l'immunodéficience féline

PARIS-AgroParisTech Centre Paris (751052302) / SudocSudocFranceF

Vaccination contre le virus de l'immunodéficience féline (VIF)

PARIS-AgroParisTech Centre Paris (751052302) / SudocSudocFranceF

Analysis of Early Human Immunodeficiency Virus Type 1 DNA Synthesis by Use of a New Sensitive Assay for Quantifying Integrated Provirus

A novel Alu-long terminal repeat (LTR)-based real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type 1 (HIV-1) DNA in infected cells with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents). Parallel assays for total HIV-1 DNA and two-LTR HIV-1 DNA circles allowed the synthesis and fate of the different HIV-1 DNA species to be monitored upon a single round of viral replication

Rôle des différentes formes d'ADN viral dans la réplication et la persistance du Virus de l'Immunodéficience Humaine de type 1

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Dossier Interdisciplinarité. Une ouverture interdisciplinaire pour la biologie : l'école de Berder

Ce texte discute les enjeux des approches interdisciplinaires, par opposition à une spécialisation accrue, face aux grandes questions de la biologie actuelle et aux défis que posent l'analyse et l'interprétation de données expérimentales de plus en plus riches et extensives. Pour favoriser l'établissement de collaborations interdisciplinaires efficaces et durables, nous organisons depuis 2002 une école-chercheurs annuelle, dans l'île de Berder (Morbihan), soutenue par les formations permanentes du CNRS et de l'Inra. Nous décrivons les spécificités de cette école et comment elle répond aux attentes de formation et d'échanges de ses participants : un grand nombre d'orateurs, un équilibre entre les différentes disciplines, des exposés synthétiques et pédagogiques, une grande place laissée aux questions, débats, interactions et animations de terrain, pendant une semaine intense et coupée des préoccupations immédiates