Karen Moreau

The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1

ABSTRACT Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4+ cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.

Analysis of conserved and non-conserved amino acids critical for ALSV (Avian leukemia and sarcoma viruses) integrase functions in vitro

Summary. Retroviral integrase (IN) is the viral enzyme responsible for the integration of viral DNA into host cellular DNA. In vitro, recombinant IN protein is able to catalyze the 3′-processing, strand transfer and disintegration activities. In order to analyze the importance of specific residues of ALSV (Avian leukemia and sarcoma viruses) IN protein, we introduced 31 amino acid substitutions either in residues previously shown by others to be involved in IN oligomerization or in selected conserved and non-conserved residues through the IN sequence. We tested, in vitro, the three catalytic activities of these mutants as well as their capacity to bind DNA. We found that (i) 88% of the substitutions occurring on well-conserved residues have an effect on IN activities (ii) two mutants (S85T in the central catalytic domain and N197C in the C-terminal domain) present a reduced efficiency of DNA binding compared to the wild type protein. Moreover, all mutations made on the dimer interface of C-terminal domain present reduced activities, suggesting an important role of this part of the protein. Finally, for some mutations, we observed differences between the ALSV and HIV (Human immunodeficiency virus) IN corresponding residues.

A Crystal Structure of the Catalytic Core Domain of an Avian Sarcoma and Leukemia Virus Integrase Suggests an Alternate Dimeric Assembly

Integrase (IN) is an important therapeutic target in the search for anti-Human Immunodeficiency Virus (HIV) inhibitors. This enzyme is composed of three domains and is hard to crystallize in its full form. First structural results on IN were obtained on the catalytic core domain (CCD) of the avian Rous and Sarcoma Virus strain Schmidt-Ruppin A (RSV-A) and on the CCD of HIV-1 IN. A ribonuclease-H like motif was revealed as well as a dimeric interface stabilized by two pairs of α-helices (α1/α5, α5/α1). These structural features have been validated in other structures of IN CCDs. We have determined the crystal structure of the Rous-associated virus type-1 (RAV-1) IN CCD to 1.8 Å resolution. RAV-1 IN shows a standard activity for integration and its CCD differs in sequence from that of RSV-A by a single accessible residue in position 182 (substitution A182T). Surprisingly, the CCD of RAV-1 IN associates itself with an unexpected dimeric interface characterized by three pairs of α-helices (α3/α5, α1/α1, α5/α3). A182 is not involved in this novel interface, which results from a rigid body rearrangement of the protein at its α1, α3, α5 surface. A new basic groove that is suitable for single-stranded nucleic acid binding is observed at the surface of the dimer. We have subsequently determined the structure of the mutant A182T of RAV-1 IN CCD and obtained a RSV-A IN CCD-like structure with two pairs of buried α-helices at the interface. Our results suggest that the CCD of avian INs can dimerize in more than one state. Such flexibility can further explain the multifunctionality of retroviral INs, which beside integration of dsDNA are implicated in different steps of the retroviral cycle in presence of viral ssRNA.

Avian Sarcoma and Leukemia Virus (ASLV) integration in vitro: Mutation or deletion of integrase (IN) recognition sequences does not prevent but only reduces the efficiency and accuracy of DNA integration

Integrase (IN) is the enzyme responsible for provirus integration of retroviruses into the host cell genome. We used an Avian Sarcoma and Leukemia Viruses (ASLV) integration assay to investigate the way in which IN integrates substrates mutated or devoid of one or both IN recognition sequences. We found that replacing U5 by non-viral sequences (U5del) or U3 by a mutated sequence (pseudoU3) resulted in two and three fold reduction of two-ended integration (integration of the two ends from a donor DNA) respectively, but had a slight effect on concerted integration (integration of both ends at the same site of target DNA). Further, IN was still able to integrate the viral ends of the double mutant (pseudoU3/U5del) in a two-ended and concerted integration reaction. However, efficiency and accuracy (i.e. fidelity of size duplication and of end cleavage) of integration were reduced.

Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity

A human immunodeficiency virus type (HIV-1)-based lentiviral vector pseudotyped with the vesicular stomatitis virus envelope glycoprotein and encoding the GFP reporter gene was used to evaluate different methods of lentiviral vector titration. GFP expression, viral DNA quantification and the efficiency of vector DNA integration were assayed after infection of conventional HIV-1-permissive cell lines and human primary adult fibroblasts with the vector. We found that vector titers based on GFP expression determined by flow cytometry may vary by more than 50-fold depending on the cell type and the promoter-cell combination used. Interestingly, we observed that the viral integration process in primary HDFa cells was significantly more efficient compared to that in SupT1 or 293T cells. We propose that determination of the amount of integrated viral DNA by quantitative PCR be used in combination with the reporter gene expression assay.

Functional analyses of mutants of the central core domain of an Avian Sarcoma/Leukemia Virus integrase.

Integrase (IN) is the enzyme responsible for the integration of the retroviral genome into the host cell DNA. Herein, three mutants of conserved residues (V79, S85 and I146) of the central core domain (CCD) of an Avian Sarcoma/Leukemia Virus IN were analyzed in vitro. Our data revealed (i) the inability of S85T mutant to form dimers and tetramers in the absence of DNA and (ii) a slightly reduced ability of V79A IN in tetramers formation. Surprisingly, both mutants were still able to efficiently achieve concerted DNA integration. This could be explained by the ability of the two mutants to form complexes in the presence of DNA. These data suggest a strong structural role of the region encompassing V79 and S85 residues (β2/β3 turn-β3 strands) following binding to viral DNA and highlight the dynamic nature of IN.

An improved self-deleting retroviral vector derived from avian leukemia and sarcoma virus

We have previously developed a self-deleting avian leukosis and sarcoma virus (ALSV)- based retroviral vector carrying an additional attachment (att) sequence. Resulting proviruses underwent deletion of viral sequences and were flanked either by two LTRs (LTRs proviruses) or by the additional att sequence and the 3′ LTR (att proviruses). Herein, we have tried to increase (1) the self-deleting properties of this vector, either by raising the selection pressure applied on target cells or by optimizing the size of the internal att sequence, (2) the titer of the vector by deleting or inverting some viral sequences. Moreover, a new type of provirus flanked by att sequences at each end was isolated. Finally, under specific conditions, 100% of proviruses had internal sequences deleted, and as many as 92–100% of proviruses were no longer mobilizable by a replication-competent virus. The inactivation procedure achieved here might improve the biosafety of retroviral vectors.