Lentiviral vectors derived from simian immunodeficiency virus.
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Animals↗
Biomedical subjects
J L Darlix
Publications and source records attributed to J L Darlix.
Assembling the human immunodeficiency virus type 1.
Retroviral assembly proceeds through a series of concerted events that lead to the formation and release of infectious virion particles from the infected cell. Upon translation, structural proteins are targeted to the plasma membrane where they accumulate. There, the nascent particle forces the plasma membrane to form a bud, which pinches off releasing the virion particle from the cell. In this review we describe the molecular mechanisms now known to be behind the process of virion assembly. In particular, we focus on the human immunodeficiency virus type 1, the prototype member of the lentivirus subfamily of the Retroviridae.
Cell Membrane↗
Efficient gene transfer in mouse neural precursors with a bicistronic retroviral vector.
Gene transfer into neural precursors is a powerful approach to study the function of specific gene products during nervous system development. Here we describe a retrovirus-based methodology to transduce foreign genes into mouse neural precursors. We used a high-titer bicistronic retroviral vector that encodes a marker gene, placental alkaline phosphatase (plap), and a selection gene, neomycin phosphotransferase II (neoR), under the translational control of two retroviral internal ribosome entry segments. Transduction efficiency even without selection was up to 95% for multipotential neurospheres derived from embryonic striata and grown with basic fibroblast growth factor 2. Expression of plap and neoR was sustained with time in culture and upon differentiation into neurons, astrocytes, and oligodendrocytes, as shown by double immunofluorescence labeling with cell type-specific markers, Western blotting, and neomycin resistance. However, levels of plap were decreased in differentiated oligodendrocytes. Transduction with the same vector of neonatal oligodendrocyte precursors grown in oligospheres consistently resulted in a lower proportion of plap-immunoreactive cells and enhanced cell death in the absence of neomycin. However, plap expression was maintained in some differentiated oligodendrocytes expressing galactocerebroside or myelin basic protein. In that neurospheres can be easily expanded in vitro and factors enabling their differentiation into the three main central nervous system cell types are being elucidated, this methodology could be used in the future to produce large number of transduced, differentiated neural cells.
Alkaline Phosphatase↗
The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein.
The transmissible spongiform encephalopathies are fatal neurodegenerative diseases that are associated with the accumulation of a protease-resistant form of the cellular prion protein (PrP). Although PrP is highly conserved and widely expressed in vertebrates, its function remains a matter of speculation. Indeed PrP null mice develop normally and are healthy. Recent results show that PrP binds to nucleic acids in vitro and is found associated with retroviral particles. Furthermore, in mice the scrapie infectious process appears to be accelerated by MuLV replication. These observations prompted us to further investigate the interaction between PrP and nucleic acids, and compare it with that of the retroviral nucleocapsid protein (NC). As the major nucleic acid-binding protein of the retroviral particle, NC protein is tightly associated with the genomic RNA in the virion nucleocapsid, where it chaperones proviral DNA synthesis by reverse transcriptase. Our results show that the human prion protein (huPrP) functionally resembles NCp7 of HIV-1. Both proteins form large nucleoprotein complexes upon binding to DNA. They accelerate the hybridization of complementary DNA strands and chaperone viral DNA synthesis during the minus and plus DNA strand transfers necessary to generate the long terminal repeats. The DNA-binding and strand transfer properties of huPrP appear to map to the N-terminal fragment comprising residues 23 to 144, whereas the C-terminal domain is inactive. These findings suggest that PrP could be involved in nucleic acid metabolism in vivo.
Capsid↗
Role of distal zinc finger of nucleocapsid protein in genomic RNA dimerization of human immunodeficiency virus type 1; no role for the palindrome crowning the R-U5 hairpin.
Genomic RNA isolated from HIV-1 variously mutated in nucleocapsid protein (NC) was characterized by nondenaturing gel electrophoresis. Mutations in the C-terminal, the N-terminal, and the linker regions had no effect on genomic RNA dimerization [they are R7R10K11S, P31L, R32G, S3(32-34), and K59L], while a C36S/C39S mutation in the distal zinc knuckle (Cys-His box or zinc finger) inhibited genome dimerization as much as disrupting the kissing-loop domain. The four mutations which inhibited tRNA(Lys3) genomic placement (i.e., the in vivo placement of tRNA(Lys3) on the primer binding site) had no effect on genome dimerization. Among five mutations which inhibited genome packaging, four had no effect on genome dimerization. Thus the N-terminal and linker regions of NC control genome packaging/tRNA(Lys3) placement (two processes which do not require mature NC) but have little influence on genome dimerization and 2-base extension of tRNA(Lys3) (two processes which are likely to require mature NC). It has been suggested, based on electron microscopy, that the AAGCUU82 palindrome crowning the R-U5 hairpin stimulates genomic RNA dimerization. To test this hypothesis, we deleted AGCU81 from wild-type viruses and from viruses bearing a disrupted kissing-loop hairpin or kissing-loop domain; in another mutant, we duplicated AGCU81. The loss of AGCU81 reduced dimerization by 2.5 +/- 4%; its duplication increased it by 3 +/- 6%. Dissociation temperature was left unchanged. We reach two conclusions. First, the palindrome crowning the R-U5 hairpin has no impact on HIV-1 genome dimerization. Second, genomic RNA dimerization is differentially influenced by NC sequence: it is Zn finger dependent but independent of the basic nature of the N-terminal and linker subdomains. We propose that the NC regions implicated in 2-base extension of tRNA(Lys3) are required for a second (maturation) step of tRNA placement. Genome dimerization and mature tRNA placement would then become two RNA-RNA interactions sharing similar NC sequence requirements.
Amino Acid Sequence↗
The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1.
Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP). Although PrP is conserved in vertebrates, its function remains to be identified. In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP. Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome. NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes. This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT. The NC-like properties of huPrP map to the N-terminal region of huPrP. Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells. These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.
5' Untranslated Regions↗
New ways of initiating translation in eukaryotes.
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Animals↗
Characterization of loose and tight dimer forms of avian leukosis virus RNA.
Retroviral genomes consist of two identical RNA molecules joined non-covalently near their 5'-ends. Recently, we showed that an imperfect autocomplementary sequence, located in the L3 domain, plays an essential role in avian sarcoma-leukosis virus (ASLV) RNA dimerization in vitro. This sequence can adopt a stem-loop structure and is involved in ASLV replication. Here, we found that in the absence of nucleocapsid protein, RNA transcripts of avian leukosis virus (ALV) were able to form two types of dimers in vitro that differ in their stability: a loose dimer, formed at a physiological temperature, and a tight dimer, formed at a high temperature. A mutational analysis was performed to define the features of these dimers. The results of this analysis unambiguously confirm that the two L3 stem-loops interact directly in both types of dimers. A loop-loop interaction is the main linkage in the loose dimer. In contrast, in the tight dimer, the stem and the loop of the L3 hairpin form an extended duplex. Surprisingly, we also found that the dimerization properties defined for our ALV strain (type SR-A) differ from those found in other ASLV strains.
Animals↗
The GAG-like protein of the yeast Ty1 retrotransposon contains a nucleic acid chaperone domain analogous to retroviral nucleocapsid proteins.
The reverse transcription process for retroviruses and retrotransposons takes place in a nucleocore structure in the virus or virus-like particle. In retroviruses the major protein of the nucleocore is the nucleocapsid protein (NC protein), which derives from the C-terminal region of GAG. Retroviral NC proteins are formed of either one or two CCHC zinc finger(s) flanked by basic residues and have nucleic acid chaperone and match-maker properties essential for virus replication. Interestingly, the GAG protein of a number of retroelements including Spumaviruses does not possess the hallmarks of retroviral GAGs and in particular lacks a canonical NC protein. In an attempt to search for a nucleic acid chaperone activity in this class of retroelements we used the yeast Ty1 retrotransposon as a model system. Results shows that the C-terminal region of Ty1 GAG contains a nucleic acid chaperone domain capable of promoting the annealing of primer tRNA(i)(Met) to the multipartite primer binding site, Ty1 RNA dimerization and initiation of reverse transcription. Moreover Ty1 RNA dimerization, in a manner similar to Ty3 but unlike retroviral RNAs, appears to be mediated by tRNA(i)(Met). These findings suggest that nucleic acid chaperone proteins probably are general co-factors for reverse transcriptases.
Dimerization↗
An internal ribosome entry segment promotes translation of the simian immunodeficiency virus genomic RNA.
The retroviral genomic RNA is the messenger for the synthesis of the group-specific antigen (gag) and polymerase precursors of the major structural proteins and enzymes of the virion. The 5'-untranslated leader of the simian immunodeficiency virus (SIV) genomic RNA is formed of highly structured domains involved in key steps of the viral life cycle. Thus, the presence of stable RNA structures between the 5'-cap and the gag start codon are thought to strongly inhibit scanning of a 43 S preinitiation ribosomal complex. This prompted us to look for an alternative to the canonical ribosome scanning. By using a standard bicistronic assay in the rabbit reticulocyte lysate, we show that the SIVmac 5'-leader contains an internal ribosome entry segment (IRES) and that gene expression driven by this IRES is stimulated upon cleavage of eukaryotic initiation factor 4G. Deletion analysis revealed that the sequence between the major splice donor and the gag AUG codon is required for IRES activity. DNA transfection and viral transduction experiments in both NIH-3T3 and COS-7 cells confirmed that translation driven by the SIV leader is IRES-dependent and thus insensitive to the immunosuppressant rapamycin. Identification of an IRES in SIV is of particular interest for the understanding of lentivirus replication and also for the design of novel lentiviral vectors suitable for gene transfer.
3T3 Cells↗
Nucleocapsid protein of human immunodeficiency virus as a model protein with chaperoning functions and as a target for antiviral drugs.
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Amino Acid Sequence↗
Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells.
We describe the generation and the characterization of new lentiviral vectors derived from SIVmac251, a simian immunodeficiency virus (SIV). A methodical approach was used to engineer both efficient and safe packaging constructs allowing the production of SIV viral core proteins. SIV-vectors encoding GFP (green fluorescent protein) were generated as VSV-G-pseudotyped particles upon transient expression of the vector construct and helper functions in 293 cells. The SIV vectors were able to transduce efficiently various target cell types at low multiplicity of infection, including monocyte-differentiated human dendritic cells (DCs) which retained their capacity to differentiate into mature DCs after gene transfer. Transduction of the DCs by the SIV vectors was prevented when infections were performed in the presence of AZT, a reverse-transcriptase inhibitor. After gene transfer, expression of the GFP in the target cells remained constant after several weeks, indicating that the vectors had been stably integrated into the genome of the host cells. Preparations of SIV vectors were systematically checked for the absence of replication-competent and recombinant retroviruses but remained negative, suggesting the innocuousness of these novel gene delivery vectors. Side-to-side comparisons with vectors derived from HIV-1 (human immunodeficiency virus) indicated that the SIV vectors were equally potent in transducing proliferating target cells. Finally, we have determined the infectivity of SIV vectors pseudotyped with surface glycoproteins of several membrane-enveloped viruses.
Animals↗
Development of minimal lentivirus vectors derived from simian immunodeficiency virus (SIVmac251) and their use for gene transfer into human dendritic cells.
Lentivirus-derived vectors are very promising gene delivery systems since they are able to transduce nonproliferating differentiated cells, while murine leukemia virus-based vectors can only transduce cycling cells. Here we report the construction and characterization of highly efficient minimal vectors derived from simian immunodeficiency virus (SIVmac251). High-fidelity PCR amplification of DNA fragments was used to generate a minimal SIV vector formed from a 5' cytomegalovirus early promoter, the 5' viral sequences up to the 5' end of gag required for reverse transcription and packaging, the Rev-responsive element, a gene-expressing cassette, and the 3' long terminal repeat (LTR). Production of SIV vector particles was achieved by transfecting 293T cells with the vector DNA and helper constructs coding for the viral genes and the vesicular stomatitis virus glycoprotein G envelope. These SIV vectors were found to have transducing titers reaching 10(7) transducing units/ml on HeLa cells and to deliver a gene without transfer of helper functions to target cells. The central polypurine tract can be included in the minimal vector, resulting in a two- to threefold increase in the transduction titers on dividing or growth-arrested cells. Based on this minimal SIV vector, a sin vector was designed by deleting 151 nucleotides in the 3' LTR U3 region, and this SIV sin vector retained high transduction titers. Furthermore, the minimal SIV vector was efficient at transducing terminally differentiated human CD34(+) cell-derived or monocyte-derived dendritic cells (DCs). Results show that up to 40% of human primary DCs can be transduced by the SIV vectors. This opens a new perspective in the field of immunotherapy.
Cell Line↗
Characterization of an internal ribosomal entry segment in the 5' leader of murine leukemia virus env RNA.
The 5' untranslated region, also called the leader, of oncoretroviruses and lentiviruses is long and is formed of several structured domains critically important in virus replication. The 5' leader of murine leukemia virus (MLV) RNA contains an internal ribosomal entry segment (IRES) which promotes synthesis of Gag and glyco-Gag polyprotein precursors. In the present study we investigated the translational features of the 5' leader of MLV subgenomic RNA (env RNA) encoding the Env polyprotein precursor. When the env leader was inserted between two genes, such as lacZ and the neomycin resistance cassette, in a dicistronic vector, it allowed IRES-dependent translation of the 3' cistron in the rabbit reticulocyte lysate system and in murine cells. The drug rapamycin and the foot-and-mouth disease virus L protease, known to inhibit cap-dependent translation, caused an enhancement of the translation driven by the env leader sequence, consistent with an IRES activity promoting Env expression. Analysis of several deletion mutants led us to localize the minimal env IRES between the splice junction and the env AUG start codon.
3T3 Cells↗
Rous sarcoma virus translation revisited: characterization of an internal ribosome entry segment in the 5' leader of the genomic RNA.
The 5' leader of Rous sarcoma virus (RSV) genomic RNA and of retroviruses in general is long and contains stable secondary structures that are critical in the early and late steps of virus replication such as RNA dimerization and packaging and in the process of reverse transcription. The initiation of RSV Gag translation has been reported to be 5' cap dependent and controlled by three short open reading frames located in the 380-nucleotide leader upstream of the Gag start codon. Translation of RSV Gag would thus differ from that prevailing in other retroviruses such as murine leukemia virus, reticuloendotheliosis virus type A, and simian immunodeficiency virus, in which an internal ribosome entry segment (IRES) in the 5' end of the genomic RNA directs efficient Gag expression despite stable 5' secondary structures. This prompted us to investigate whether RSV Gag translation might be controlled by an IRES-dependent mechanism. The results show that the 5' leaders of RSV and v-Src RNA exhibit IRES properties, since these viral elements can promote efficient translation of monocistronic RNAs in conditions inhibiting 5' cap-dependent translation. When inserted between two cistrons in a canonical bicistronic construct, both the RSV and v-Src leaders promote expression of the 3' cistron. A genetic analysis of the RSV leader allowed the identification of two nonoverlapping 5' and 3' leader domains with IRES activity. In addition, the v-Src leader was found to contain unique 3' sequences promoting an efficient reinitiation of translation. Taken together, these data lead us to propose a new model for RSV translation.
Animals↗
Canine large granular lymphocyte leukemia and its derived cell line produce infectious retroviral particles.
We describe a case of large granular lymphocyte (LGL) leukemia in a dog that we followed over a period of 2 years. Analysis of a hematological profile revealed lymphocytosis (19,500 lymphocytes per microliter; reference values, 1,000-4,800 lymphocytes per microliter), with a majority of LGL on the blood smear. LGL is defined as a lymphoid subset comprising 10% of peripheral blood mononuclear cells and corresponding to either CD3- CD8- NK cells or CD3+ CD8+ T cells. The cells are characterized by abundant basophilic cytoplasm containing distinct granules of variable size and number. The characteristic phenotype of our leukemic LGL is of a cytotoxic T cell, CD3+ and CD8+. A new cell line, DLC 02, was established from the peripheral lymphocytes of the leukemic dog. Particles with type C retroviral morphology were found in ultrathin sections of DLC 02 cell pellets. These particles were found to have a sucrose gradient density of 1.17 g/liter and a reverse transcriptase activity with an Mn2+ preference, suggesting that they correspond to a mammalian type C oncovirus.
Animals↗
Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 in vitro.
Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5' RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5'-3' bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5' PBS (). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5'-3' PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.
Amino Acid Sequence↗
Retroviral vectors for the expression of two genes in human multipotent neural precursors and their differentiated neuronal and glial progeny.
Retroviral vectors allow stable integration of exogenous DNA into genomic DNA and therefore gene transmission to progeny. Multipotent neural precursors and immortal cell lines prepared from them have been demonstrated to integrate into either adult or developing brain in a nontumorigenic, functional manner, without interfering with normal neurobiological processes. These cells thus appear to provide a Trojan horse ideally adapted to directing the expression of transgenes appropriately in a host brain. Here we investigated and optimized the transduction capacity of MuLV-based retroviral vectors in which internal ribosomal entry segments (IRESs) drive coexpression of two heterologous gene products from a single bicistronic mRNA in a human multipotent neural precursor cell line, "Dev," which was prepared from a medulloblastoma. For this, two vectors containing two different combinations of three viral IRESs were used and the capacity of different pseudotyped vectors to permit an efficient and stable transduction of Dev cells was compared. Our data show that (1) the best recombinant vectors for Dev cell transduction are those pseudotyped with the 10A1 MuLV envelope (40% of transduction) and (2) the initial coexpression of neo and plap, observed in transduced undifferentiated Dev cells, is maintained in differentiated Dev cells with a neuronal or glial phenotype. Therefore, these double-IRES vectors may provide an efficient means of transducing the coexpression of two proteins in undifferentiated human neural precursors that is maintained in their differentiated progeny. These data suggest that the double-IRES strategy is well adapted to potential therapeutic situations when coexpression of two different transgenes may be required in the same cell.
Animals↗