Jean-Luc Darlix

Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1.

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it is in tight association with genomic RNA and the tRNA primer. NC protein is necessary for the dimerization and encapsidation of genomic RNA, the annealing of the tRNA primer to the primer binding site (PBS) and the initial strand transfer event. Due to the general nature of NC protein-promoted annealing, its use to improve nucleic acid interactions in various reactions can be envisioned. Parameters affecting NC-promoted nucleic acid annealing of NCp7 from HIV-1 have been analyzed. The promotion of RNA:RNA and RNA:DNA annealing by NCp7 is more sensitive to the concentration of MgCl2 than the promotion of DNA:DNA hybridization. Stimulation of complex formation for all three complexes was efficient at 0-90 mM NaCl, between 23 and 55 degrees C and at pH values between 6.5 and 9.5, inclusive. Parameters affecting NCp7-promoted hybridization of tRNA(Lys,3) to the PBS, which appears to be specific for NC protein, will be discussed. Results implicate the basic regions of NCp7, but not the zinc fingers, in promoting the annealing of complementary nucleic acid sequences. Finally, NCp7 strand transfer activity aids the formation of the most stable nucleic acid complex.

Development of Minimal Lentivirus Vectors Derived from Simian Immunodeficiency Virus (SIVmac251) and Their Use for Gene Transfer into Human Dendritic Cells

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 gagrequired 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 107 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, asin 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.

Intracellular Trafficking of Gag and Env Proteins and Their Interactions Modulate Pseudotyping of Retroviruses

ABSTRACT Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses.

Analysis of hepatitis C virus RNA dimerization and core–RNA interactions

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

ABSTRACT HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.

Composite structure of the chloroplast 23 S ribosomal RNA genes of Chlamydomonas reinhardii. Evolutionary and functional implications.

Abstract The chloroplast ribosomal unit of Chlamydomonas reinhardii displays two features which are not shared by other chloroplast ribosomal units. These include the presence of an intron in the 23 S ribosomal RNA gene and of two small genes coding for 3 S and 7 S rRNA in the spacer between the 16 S and 23 S rRNA genes (Rochaix & Malnoe, 1978). Sequencing of the 7 S and 3 S rRNAs as well as their genes and neighbouring regions has shown that: (1) the 7 S and 3 S rRNA genes are 282 and 47 base-pairs long, respectively, and are separated by a 23 base-pair A + T-rich spacer. (2) A sequence microheterogeneity exists within the 3 S RNA genes. (3) The sequences of the 7 S and 3 S rRNAs are homologous to the 5′ termini of prokaryotic and other chloroplast 23 S rRNAs, indicating that the C. reinhardii counterparts of 23 S rRNA have a composite structure. (4) The sequences of the 7 S and 3 S rRNAs are related to that of cytoplasmic 5.8 S rRNA, suggesting that these RNAs may perform similar functions in the ribosome. (5) Partial nucleotide sequence complementarity is observed between the 5′ ends of the 7 S and 3 S RNAs on one hand and the 23 S rRNA sequences which flank the ribosomal intron on the other. These data are compatible with the idea that these small rRNAs may play a role in the processing of the 23 S rRNA precursor.

It is Rous Sarcoma virus protein P12 and not P19 that binds tightly to Rous Sarcoma virus RNA

The interactions between Rous Sarcoma virus (RSV) RNA and the viral proteins in the virus have been analysed by Sen & Todaro (1977) using ultraviolet light irradiation; they showed that the major protein ultraviolet light cross-linked to the viral RNA was P19 as identified by polyacrylamide gel electrophoresis. We report here that it is not viral protein P19 but P12 that binds tightly to RSV RNA upon ultraviolet light irradiation of the virus. Therefore, the binding sites of the viral protein along RSV RNA that we have characterized previously should be correctly attributed now to P12 and not P19.

Characterization of an Internal Ribosomal Entry Segment in the 5′ Leader of Murine Leukemia Virus env RNA

ABSTRACT 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 theenv leader was inserted between two genes, such aslacZ 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.

Rous Sarcoma Virus Translation Revisited: Characterization of an Internal Ribosome Entry Segment in the 5′ Leader of the Genomic RNA

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.

Binding sites of viral protein P19 onto Rous sarcoma virus RNA and possible controls of viral functions

Abstract Protein P19, a constituent of the core of Rous sarcoma virus, was shown to bind tightly to the virus RNA (Sen & Todaro, 1977) and more precisely to structured regions of the RNA (Leis et al., 1978). We report here that specific RNA sequences are recognized by viral protein P19, and that such sequences are close to or within structured RNA regions which can themselves interact with each other. Among P19 binding sites two are located in the middle of the P19 coding sequence which is also the putative dimer linkage sequence where the two 35 S RNA subunits are associated to form viral 70 S RNA. In addition, protein-protein cross-linking studies indicate that P19 molecules bound to Rous sarcoma virus RNA are closely associated. In vitro, viral protein P19 bound to the viral RNA inhibits both translation and reverse transcription. These results are discussed with respect to the role that P19 may play in controlling the various functions of Rous sarcoma virus RNA in vivo.