[Show abstract][Hide abstract] ABSTRACT: The Pr55 Gag of HIV-1 orchestrates viral particle assembly in producer cells, which requires the genomic RNA and a lipid membrane as scaffolding platforms. The nucleocapsid (NC) domain with its two invariant CCHC zinc fingers flanked by unfolded basic sequences is thought to direct genomic RNA selection, dimerization and packaging during virus assembly. To further investigate the role of NC domain, we analyzed the assembly of Gag with deletions in the NC domain in parallel with that of wild-type Gag using fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) in HeLa cells. We found that upon binding to nucleic acids, the NC domain promotes the formation of compact Gag oligomers in the cytoplasm. Moreover, the intracellular distribution of the population of oligomers further suggests that oligomers progressively assemble during their trafficking towards the plasma membrane (PM), but with no dramatic changes in their compact arrangement. This ultimately results in the accumulation at the PM of closely packed Gag oligomers that likely arrange in hexameric lattices, as revealed by the perfect match between the experimental FRET value with the one calculated from the structural model of Gag in immature viruses. The distal finger and flanking basic sequences, but not the proximal finger appear to be essential for Gag oligomer compaction and membrane binding. Moreover, the full NC domain was found to be instrumental in the kinetics of Gag oligomerization and intracellular trafficking. These findings further highlight the key roles played by the NC domain in virus assembly.
[Show abstract][Hide abstract] ABSTRACT: During reverse transcription, the HIV-1 RNA is converted by the reverse transcriptase (RT) into proviral DNA. RT is
assisted by the HIV-1 nucleocapsid (NCp7) protein that notably increases the ability of RT to synthesize DNA through
pause sites. Using single molecule FRET, we monitored the NCp7 effect on the binding of RT to nucleic acid sequences
corresponding to two different pause sites. NCp7 was found to modify the distribution of RT orientations on the
oligonucleotides and decrease the residence time of RT on one of the pause sites. These results give direct insight into
the NCp7 molecular mechanism in reverse transcription.
Proceedings of SPIE - The International Society for Optical Engineering 02/2013; DOI:10.1117/12.2003478 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the rush to develop anti-viral drugs against the human immunodeficiency virus type I (HIV-1), all the steps of the viral life cycle are potential targets of therapeutic intervention. In this review, we will explore the recent advances on strategies that aim at obstructing the formation, the release and the infectivity of newly formed virion particles from HIV-1 infected cells.
Current Pharmaceutical Design 02/2004; 10(30):3725-39. DOI:10.2174/1381612043382701 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Cellular and Molecular Life Sciences CMLS 08/2002; 59(7):1166-84. DOI:10.1007/s00018-002-8495-6 · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is conclusive evidence that the host gene encoding the prion precursor protein (PrPc) is implicated in the development and propagation of transmissible spongiform encephalopathies collectively known as prion diseases. Nevertheless, the normal cellular function of this widely expressed and highly conserved gene product remains elusive. Here we review evidence implicating PrPc in a number of diverse phenomena including the transportation and metabolism of metal ions associated with protection against oxidative stress; behavior as a membrane receptor or ligand, or a receptor-bound molecule implicated in signal transduction; and as a nucleic acid-binding protein with the functional properties of a nucleic acid chaperone protein. A complex picture is emerging of PrPc as a multifunctional protein. (c) 2002 Prous Science. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Neuroscience Research 09/2001; 65(3):208-19. DOI:10.1002/jnr.1144 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 01/2001; 74(24):11581-8. DOI:10.1128/JVI.74.24.11581-11588.2000 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 10/2000; 74(18):8307-15. DOI:10.1128/JVI.74.18.8307-8315.2000 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.