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.

Small ruminant lentivirus Tat protein induces apoptosis in caprine cells in vitro by the intrinsic pathway.

The small ruminant lentiviruses, caprine arthritis-encephalitis virus (CAEV) and maedi visna virus (MVV) naturally cause inflammatory disease in goats and sheep, provoking chronic lesions in several different organs. We have previously demonstrated that in vitro infection of caprine cells by CAEV induces apoptosis through the intrinsic pathway (Rea-Boutrois, A., Pontini, G., Greenland, T., Mehlen, P., Chebloune, Y., Verdier, G. and Legras-Lachuer, C. 2008). In the present study, we used Tat deleted viruses and SLRV Tat-expression vectors to show that the SRLV Tat proteins are responsible for this apoptosis. We have also studied the activation of caspases-3, -8 and -9 by fluorescent assays in caprine cells expressing SRLV Tat proteins, and the effects of transfected dominant negative variants of these caspases, to show that Tat-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of the mitochondrial pathway.

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.

A novel self-deleting retroviral vector carrying an additional sequence recognized by the viral integrase (IN).

During retroviral integration, the viral integrase recognizes the attachment (att) sequence (formed by juxtaposition of two LTRs ends) as the substrate of integration. We have developed a self-deleting Avian Leukosis and Sarcoma Viruses (ALSVs)-based retroviral vector carrying an additional copy of the att sequence, between neo and puro genes. We observed that: (i) the resulting NP3Catt vector was produced at neo and puro titers respectively smaller and higher than that of the parental vector devoid of the att sequence; (ii) 61% of NP3Catt proviruses were flanked by LTRs; most of them were deleted of internal sequences, probably during the reverse transcription step; (iii) 31% of clones were deleted of the whole 5' part of their genome and were flanked, in 5', by the additional att sequence and, in 3', by an LTR. Integration of these last proviruses was often imprecise with respect to the viral ends. At total, 77% of proviruses had lost the packaging signal and were not mobilizable by a replication-competent virus and 92% had lost the selectable gene in a single round of replication. Although still to improve, the att vector could be considered as an interesting new safe retroviral vector for gene transfer experiments.

Caprine arthritis-encephalitis virus induces apoptosis in infected cells in vitro through the intrinsic pathway.

Caprine arthritis-encephalitis virus (CAEV) is a lentivirus that causes natural inflammatory disease in goats, with chronic lesions in several different organs. CAEV infection of in vitro cultured cells is accompanied by apoptosis, but the involvement of the intrinsic and extrinsic pathways has not previously been elucidated. We have studied the activation of caspases-3, -8 and -9 by fluorescent assays in various goat cells infected in vitro by CAEV, and the effects of transfected dominant negative variants of theses caspases, to show that CAEV-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of mitochondrial pathway.

Gene transfer into mammalian cells using a self-deleting avian leukemia and sarcoma virus-based vector.

OBJECTIVES: We have previously described an avian leukemia and sarcoma virus-based vector containing an additional att sequence in an internal position that is capable of self-deleting most of its 5' viral sequences during one cycle of replication in avian cells [Virus Res 2008;135:72-82; Arch Virol 2008;153:2233-2243]. Herein, our aim was to test the infectivity and self-deleting properties of this avian retroviral vector in human cells. METHODS: Human Hela cells transiently expressing the cellular receptor for avian leukemia and sarcoma viruses (tva) were infected with the avian vector. Molecular analyses of thirteen clones were performed. RESULTS: Data showed that more than 77% of proviruses had lost the 5' part of their genome including the selectable gene. At least 61% of these proviruses were flanked on the left by the additional att sequence and on the right by the LTR. None of the thirteen proviruses was able to express a full-length genomic RNA. CONCLUSION: This study demonstrates that the self-deleting properties of the avian vector in avian cells may be also applicable to human cells.

Mutational analyses of the core domain of Avian Leukemia and Sarcoma Viruses integrase: critical residues for concerted integration and multimerization.

During replicative cycle of retroviruses, the reverse-transcribed viral DNA is integrated into the cell DNA by the viral integrase (IN) enzyme. The central core domain of IN contains the catalytic site of the enzyme and is involved in binding viral ends and cell DNA as well as dimerization. We previously performed single amino acid substitutions in the core domain of an Avian Leukemia and Sarcoma Virus (ALSV) IN [Arch. Virol. 147 (2002) 1761]. Here, we modeled the resulting IN mutants and analyzed the ability of these mutants to mediate concerted DNA integration in an in vitro assay, and to form dimers by protein-protein cross-linking and size exclusion chromatography. The N197C mutation resulted in the inability of the mutant to perform concerted integration that was concomitant with a loss of IN dimerization. Surprisingly, mutations Q102G and A106V at the dimer interface resulted in mutants with higher efficiencies than the wild-type IN in performing two-ended concerted integration of viral DNA ends. The G139D and A195V mutants had a trend to perform one-ended DNA integration of viral ends instead of two-ended integration. More drastically, the I88L and L135G mutants preferentially mediated nonconcerted DNA integration although the proteins form dimers. Therefore, these mutations may alter the formation of IN complexes of higher molecular size than a dimer that would be required for concerted integration. This study points to the important role of core domain residues in the concerted integration of viral DNA ends as well as in the oligomerization of the enzyme.

Mutations in the C-terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process in vitro.

Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. The C-terminal domain of IN is involved in DNA binding and enzyme multimerization. We previously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma viruses (ALSV) IN. Here, we modelled these IN mutants and analysed their ability to mediate concerted DNA integration (in an in vitro assay) as well as to form dimers (by size exclusion chromatography and protein-protein cross-linking). Mutations of residues located at the dimer interface (V239, L240, Y246, V257 and K266) have the greatest effects on the activity of the IN. Among them: (a) the L240A mutation resulted in a decrease of integration efficiency that was concomitant with a decrease of IN dimerization; (b) the V239A, V249A and K266A mutants preferentially mediated non-concerted DNA integration rather than concerted DNA integration although they were found as dimers. Other mutations (V260E and Y246W/DeltaC25) highlight the role of the C-terminal domain in the general folding of the enzyme and, hence, on its activity. This study points to the important role of residues at the IN C-terminal domain in the folding and dimerization of the enzyme as well as in the concerted DNA integration of viral DNA ends.

Maedi-visna virus and caprine arthritis encephalitis virus genomes encode a Vpr-like but no Tat protein.

A small open reading frame (ORF) in maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) was initially named "tat" by analogy with a similarly placed ORF in the primate lentiviruses. The encoded "Tat" protein was ascribed the function of up regulation of the viral transcription from the long terminal repeat (LTR) promoter, but we have recently reported that MVV and CAEV Tat proteins lack trans-activation function activity under physiological conditions (S. Villet, C. Faure, B. Bouzar, G. Verdien, Y. Chebloune, and C. Legras, Virology 307:317-327, 2003). In the present work, we show that MVV Tat localizes to the nucleus of transfected cells, probably through the action of a nuclear localization signal in its C-terminal portion. We also show that, unlike the human immunodeficiency virus (HIV) Tat protein, MVV Tat was not secreted into the medium by transfected human or caprine cells in the absence of cell lysis but that, like the primate accessory protein Vpr, MVV and CAEV Tat proteins were incorporated into viral particles. In addition, analysis of the primary protein structures showed that small-ruminant lentivirus (SRLV) Tat proteins are more similar to the HIV type 1 (HIV-1) Vpr protein than to HIV-1 Tat. We also demonstrate a functional similarity between the SRLV Tat proteins and the HIV-1 Vpr product in the induction of a specific G(2) arrest of the cell cycle in MVV Tat-transfected cells, which increases the G(2)/G(1) ratio 2.8-fold. Together, these data strongly suggest that the tat ORF in the SRLV genomes does not code for a regulatory transactivator of the LTR but, rather, for a Vpr-like accessory protein.

Lack of trans-activation function for Maedi Visna virus and Caprine arthritis encephalitis virus Tat proteins.

All lentiviruses contain an open reading frame located shortly upstream or inside of the env gene and encoding a small protein which has been designated Tat. This designation was mainly with respect to the positional analogy with the first exon of the trans-activator protein of the well studied human immunodeficiency virus type 1 (HIV-1). In this work we comparatively studied the trans- activation activity induced by Tat proteins of the small ruminant Maedi Visna virus (MVV) of sheep and Caprine arthritis encephalitis virus (CAEV) of goats on MVV and CAEV LTRs with that induced by the human lentivirus HIV-1 on its own LTR. The HIV-1 LTR alone weakly expresses the reporter GFP gene except when the HIV-1 Tat protein is coexpressed, the GFP expression is increased 60-fold. In similar conditions only minimal trans-activation increasing two- to three-fold the MVV and CAEV LTR activity was found with MVV Tat protein, and no trans-activation activity was detected in any used cell type or with any virus strain when CAEV Tat was tested. These results indicate that the small ruminant lentiviruses (SRLV) differ from the primate lentiviruses in their control of expression from the viral LTRs and put into question the biological role of the encoded protein named "Tat."