Porcine endogenous retrovirus-A/C: biochemical properties of its integrase and susceptibility to raltegravir.

Porcine endogenous retroviruses (PERVs) are present in the genomes of pig cells. The PERV-A/C recombinant virus can infect human cells and is a major risk of zoonotic disease in the case of xenotransplantation of pig organs to humans. Raltegravir (RAL) is a viral integrase (IN) inhibitor used in highly active antiretroviral treatment. In the present study, we explored the potential use of RAL against PERV-A/C. We report (i) a three-dimensional model of the PERV-A/C intasome complexed with RAL, (ii) the sensitivity of PERV-A/C IN to RAL in vitro and (iii) the sensitivity of a PERV-A/C-IRES-GFP recombinant virus to RAL in cellulo. We demonstrated that RAL is a potent inhibitor against PERV-A/C IN and PERV-A/C replication with IC50s in the nanomolar range. To date, the use of retroviral inhibitors remains the only way to control the risk of zoonotic PERV infection during pig-to-human xenotransplantation.

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.

Special Issue "Viral and Host Factors Driving the Emergence and the Evolution of the SARS-CoV-2 and Other Coronaviruses".

Two and half years ago, humanity was facing the emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causal agent of the COVID-19 pandemics that significantly impact public health, society and the global economy [...].

A single lentivector DNA based immunization contains a late heterologous SIVmac251 mucosal challenge infection.

Variety of conventional vaccine strategies tested against HIV-1 have failed to induce protection against HIV acquisition or durable control of viremia. Therefore, innovative strategies that can induce long lasting protective immunity against HIV chronic infection are needed. Recently, we developed an integration-defective HIV lentiDNA vaccine that undergoes a single cycle of replication in target cells in which most viral antigens are produced. A single immunization with such lentiDNA induced long-lasting T-cell and modest antibody responses in cynomolgus macaques. Here eighteen months after this single immunization, all animals were subjected to repeated low dose intra-rectal challenges with a heterologous pathogenic SIVmac251 isolate. Although the viral set point in SIVmac-infected cynomolgus is commonly lower than that seen in Indian rhesus macaques, the vaccinated group of macaques displayed a two log reduction of peak of viremia followed by a progressive and sustained control of virus replication relative to control animals. This antiviral control correlated with antigen-specific CD4+ and CD8+ T cells with high capacity of recall responses comprising effector and central memory T cells but also memory T cell precursors. This is the first description of SIV control in NHP model infected at 18 months following a single immunization with a non-integrative single cycle lentiDNA HIV vaccine. While not delivering sterilizing immunity, our single immunization strategy with a single-cycle lentivector DNA vaccine appears to provide an interesting and safe vaccine platform that warrants further exploration.

In vitro, in cellulo and structural characterizations of the interaction between the integrase of Porcine Endogenous Retrovirus A/C and proteins of the BET family.

Retroviral integrase (IN) proteins catalyze the permanent integration of the viral genome into host DNA. They can productively recruit cellular proteins, and the human Bromodomain and Extra-Terminal domain (hBET) proteins have been shown to be co-factors for integration of gamma-retroviruses such as Murine Leukemia Virus (MLV) into human cells. By using two-hybrid, co-immunoprecipitation and in vitro interaction assays, we showed that IN of the gamma- Porcine Endogenous Retrovirus-A/C (PERV IN) interacts through its C-terminal domain (CTD) with hBET proteins. We observed that PERV IN interacts with the BRD2, BRD3 and BRD4 proteins in vitro and that the BRD2 protein specifically binds and co-localizes with PERV IN protein in the nucleus of cells. We further mapped the interaction sites to the conserved Extra-Terminal (ET) domain of the hBET proteins and to several amino acids of the of the C-terminal tail of the PERV IN CTD. Finally, we determined the first experimental structure of an IN CTD - BET ET complex from small-angle X-ray scattering data (SAXS). We showed that the two factors assemble as two distinct modules linked by a short loop which confers partial flexibility. The SAXS-restrained model is structurally compatible with the binding of the PERV intasome to BRD2. Altogether, these data confirm the important role of host BET proteins in the gamma-retroviruses' targeting site and efficiency of integration.

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.

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.

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.

Transfer of the Cystic Fibrosis Transmembrane Conductance Regulator to Human Cystic Fibrosis Cells Mediated by Extracellular Vesicles.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in a deficiency in chloride channel activity. In this study, extracellular vesicles (EVs), microvesicles, and exosomes were used as vehicles to deliver exogenous CFTR glycoprotein and its encoding mRNA (mRNA(GFP-CFTR)) to CF cells to correct the CFTR chloride channel function. We isolated microvesicles and exosomes from the culture medium of CFTR-positive Calu-3 cells, or from A549 cells transduced with an adenoviral vector overexpressing a GFP-tagged CFTR (GFP-CFTR). Both microvesicles and exosomes had the capacity to package and deliver the GFP-CFTR glycoprotein and mRNA(GFP-CFTR) to target cells in a dose-dependent manner. Homologous versus heterologous EV-to-cell transfer was studied, and it appeared that the cellular uptake of EVs was significantly more efficient in homologous transfer. The incubation of CF15 cells, a nasal epithelial cell line homozygous for the ΔF508 CFTR mutation, with microvesicles or exosomes loaded with GFP-CFTR resulted in the correction of the CFTR function in CF cells in a dose-dependent manner. A time-course analysis of EV-transduced CF cells suggested that CFTR transferred as mature glycoprotein was responsible for the CFTR-associated channel activity detected at early times posttransduction, whereas GFP-CFTR translated from exogenous mRNA(GFP-CFTR) was responsible for the CFTR function at later times. Collectively, this study showed the potential application of microvesicles and exosomes as vectors for CFTR transfer and functional correction of the genetic defect in human CF cells.

Persistent production of an integrase-deleted HIV-1 variant with no resistance mutation and wild-type proviral DNA in a treated patient.

An HIV-infected patient presenting an unexpected viral escape under combined antiretroviral treatment is described. The virus isolated from plasma contained a large deletion in the HIV-1 integrase gene but no known resistance mutation. Nested polymerase chain reactions (PCRs) with patient virus integrase-specific primers and probes were developed and used to detect the mutant from plasma, blood, rectal biopsies, and sperm. The variant progressively emerged during a period of therapy-induced virosuppression, and persisted at a low but detectable level for at least 5 years. Surprisingly, proviral DNA from lymphocytes, rectal cells, and sperm cells was, and remained, mainly wild type. Cellular HIV RNA with the deletion was detected only once from the rectum. The origin and mechanisms underlying this so far not described production at a detectable level are largely hypothetical. This observation raised concern about the ability of defective viruses to spread.

In vitro functional analyses of the human immunodeficiency virus type 1 (HIV-1) integrase mutants give new insights into the intasome assembly.

A functional study of mutants of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) was conducted with the support of a recently proposed HIV-1 intasome model. Firstly, we investigated the predicted position of the C-terminal domain (CTD) and the flexibility of the alpha-6 helix by mutating the residue Ile-203. This had no impact on the 3'-processing reaction but reduced the strand transfer reaction and the formation of tetramers. Secondly, the residues Ile-141 of the catalytic loop and Glu-246 of the CTD are predicted to bind the Td-3 base of the viral DNA maintaining it in a "flipped out" position and stabilizing the catalytic core domain (CCD)-CTD interface. Our data showed that the Ile-141/Td-3 interaction was important for the strand transfer activity and the oligomerization of IN. Interestingly, mutating the Glu-246 residue by an alanine enhanced half- and full-site integrations, suggesting that this residue may not be optimized for integration.

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 (ß2/ß3 turn-ß3 strands) following binding to viral DNA and highlight the dynamic nature of IN.

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.

Characterization of two distinct RNA domains that regulate translation of the Drosophila gypsy retroelement.

The genomic RNA of the gypsy retroelement from Drosophila melanogaster exhibits features similar to other retroviral RNAs because its 5' untranslated (5' UTR) region is unusually long (846 nucleotides) and potentially highly structured. Our initial aim was to search for an internal ribosome entry site (IRES) element in the 5' UTR of the gypsy genomic RNA by using various monocistronic and bicistronic RNAs in the rabbit reticulocyte lysate (RRL) system and in cultured cells. Results reported here show that two functionally distinct and independent RNA domains control the production of gypsy encoded proteins. The first domain corresponds to the 5' UTR of the env subgenomic RNA and exhibits features of an efficient IRES (IRES(E)) both in the reticulocyte lysate and in cells. The second RNA domain that encompasses the gypsy insulator can function as an IRES in the rabbit reticulocyte lysate but strongly represses translation in cultured cells. Taken together, these results suggest that expression of the gypsy encoded proteins from the genomic and subgenomic RNAs can be regulated at the level of translation.

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.