Biomedical subjects

Jean-Marc Lanchy

Publications and source records attributed to Jean-Marc Lanchy.

7 recordsLinked to original sources

PubMed2025

Interstrain Recombinants of Human Cytomegalovirus Reveal Complex Genetic Correlates and Epistasis Influencing Glycoprotein Display, Virion Infectivity and Spread Characteristics.

Most of the nucleotide diversity in the human cytomegalovirus (HCMV) genome is due to approximately 17 genes with 2-14 alleles each. These allelic genes are interspersed among longer stretches of highly conserved sequences with signatures of extensive recombination that would shuffle the allelic genes into a vast number of allelic haplotypes. Bacterial artificial chromosome clones derived from 3 independent clinical isolates (TB40/e (TB), TR and Merlin (ME)) display dramatic differences in the abundance of entry-mediating glycoproteins gH/gL/gO and gH/gL/UL128-131, virion infectivity and efficiency of cell-free and cell-to-cell modes of spread. Of these, TB and ME are the most phenotypically different and share only 2 of the 17 allelic genes. A set of recombinant HCMV was generated by coinfecting cells with TB and ME and restriction fragment length polymorphism (RFLP) analyses demonstrated complex crossover patterns. Most recombinants were either "TB-like" with much more gH/gL/gO than gH/gL/UL128-131, or "ME-like" with much more gH/gL/UL128-131. This correlated with a TB or ME UL128 sequence, consistent with a G/T polymorphism affecting UL128 pre-mRNA splicing. One recombinant had a gH/gL/gO:gH/gL/UL128-131 ratio of 0.8, suggesting genetic determinants beyond UL128. Virion infectivity correlated with TB versus ME-like glycoprotein display, but intragroup variability indicated additional factors and variability in spread efficiency and the contribution of cell-free and cell-to-cell spread modes indicated an influence of characteristics beyond virion infectivity. Results suggest that the relationships among these three phenotypes are not strictly causal and that all three phenotypes are genetically complex and influenced by epistasis among polymorphic loci across the genome.

Journal Article↗

PubMed2007

An extended stem-loop 1 is necessary for human immunodeficiency virus type 2 replication and affects genomic RNA encapsidation.

Genomic RNA encapsidation in lentiviruses is a highly selective and regulated process. The unspliced RNA molecules are selected for encapsidation from a pool of many different viral and cellular RNA species. Moreover, two molecules are encapsidated per viral particle, where they are found associated as a dimer. In this study, we demonstrate that a 10-nucleotide palindromic sequence (pal) located at the 3' end of the psi encapsidation signal is critical for human immunodeficiency virus type 2 (HIV-2) replication and affects genomic RNA encapsidation. We used short-term and long-term culture of pal-mutated viruses in permissive C8166 cells and their phenotypic reversion to show the existence of a structurally extended SL1 during HIV-2 replication, formed by the interaction of the 3' end of the pal within psi with a motif located downstream of SL1. The stem extending HIV-2 SL1 is structurally similar to stem B described for HIV-1 SL1. Despite the high degree of phylogenetic conservation, these results show that mutant viruses are viable when the autocomplementary nature of the pal sequence is disrupted, but not without a stable stem B. Our observations show that formation of the extended SL1 is necessary during viral replication and positively affects HIV-2 genomic RNA encapsidation. Sequestration of part of the packaging signal into SL1 may be a means of regulating its presentation during the replication cycle.

Animals↗

PubMed2004

Splicing affects presentation of RNA dimerization signals in HIV-2 in vitro.

During retroviral replication, full-length viral RNAs are encapsidated into new virus particles, while spliced RNAs are excluded. The Retroviridae are unique among viruses in that infectious viral particles contain a dimer of two identical genomic RNA strands. A variety of experimental data has suggested that dimerization and encapsidation of full-length viral RNAs are linked processes, although whether dimerization is a prerequisite for encapsidation, or conversely, dimerization follows encapsidation, has not been firmly established. If dimerization was the sole determinant for encapsidation, then spliced viral RNAs might be expected to display a reduced capacity for dimerization, resulting in their exclusion from the dimerization pool. Here, we studied the in vitro dimerization properties of unspliced and spliced HIV-2 RNA. We find that the rate and yield of dimerization of Nef, Rev and Tat spliced RNAs exceeded those of unspliced RNA. Although these data do not support a simple correlation between dimerization efficiency and the presence of introns, they establish that splicing affects the presentation of dimerization signal(s), which we corroborate with structure probing. This change in RNA conformation likely affects the RNA's suitability for packaging. Furthermore, the presence of upstream and downstream elements that affect the conformation of the packaging signal represents a potentially efficient viral strategy for correctly sorting spliced versus unspliced RNAs.

Base Sequence↗

PubMed2003

Elements located upstream and downstream of the major splice donor site influence the ability of HIV-2 leader RNA to dimerize in vitro.

An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during budding and maturation of the viral particle. In HIV-1, a 5' leader region site termed stem-loop 1 (SL1) promotes RNA dimerization in vitro and influences dimerization in vivo. In HIV-2, two sequences promote dimerization of RNA fragments in vitro: the 5'-end of the primer-binding site (PBS) and a stem-loop region homologous to the HIV-1 SL1 sequence. Because HIV-2 RNA constructs of different lengths use these two dimerization signals disproportionately, we hypothesized that other sequences could modulate their relative utilization. Here, we characterized the influence of sequences upstream and downstream of the major splice donor site on the formation of HIV-2 RNA dimers in vitro using a variety of RNA constructs and dimerization and electrophoresis protocols. We first assayed the formation of loose or tight dimers for 1-444 and 1-561 model RNAs. Although both RNAs could form PBS-dependent loose dimers, the 1-561 RNA was unable to make SL1-dependent tight dimers. Using RNAs truncated at their 5'- and/or 3'-ends and by making compensatory base substitutions, we found that two elements interfere with the formation of SL1-dependent tight dimers. The cores of these elements are located at nucleotides 189-196 and 543-550. Our results suggest that base pairing between these sequences prevents the formation of SL1-dependent tight dimers, probably by sequestering SL1 in a stable intramolecular arrangement. Moreover, we found that nucleotides downstream of SL1 decreased the rate of tight dimerization. Interestingly, dimerization at 37 degrees C in the presence of nucleocapsid protein increased the yield of SL1-mediated tight dimerization in vitro, even in the presence of the two interfering elements, suggesting a relationship between the nucleocapsid protein and activation of the SL1 dimerization signal in vivo.

3' Untranslated Regions↗

PubMed2003

Elucidation and characterization of oligonucleotide-accessible sites on HIV-2 leader region RNA.

The retroviruses, including the human pathogens HIV-1 and HIV-2, are diploid inasmuch as they encapsidate two copies of their RNA genome. Prior to or during encapsidation, two copies of full-length genomic RNA recognize and stably bind each other in a process called dimerization. RNA structures within the viral genome promote dimerization in both HIV-1 and HIV-2 and are located in the 5'-untranslated leader region. Inhibition of dimerization by mutation of these RNA signals has been demonstrated to drastically reduce viral infectivity and replication kinetics and, thus, represents a potential target for antiretroviral therapy. In this study, we identified sites in HIV-2 leader region RNA that are functionally accessible to hybridization with oligonucleotides (ODNs) by reverse transcription with random ODN libraries (RT-ROL). We then tested specific ODNs directed against these regions for their efficacy in inhibiting RNA dimerization in vitro. We determined that of several hybridization-competent ODNs, only two were very effective in inhibiting RNA dimerization. Both of these ODNs were complementary to viral RNA at the primer binding site (PBS). These results identify regions with high accessibility to ODN binding on HIV-2 RNA and help to map the region(s) essential for dimerization within the viral RNA.

5' Untranslated Regions↗

PubMed2003

A structural linkage between the dimerization and encapsidation signals in HIV-2 leader RNA.

The 5' untranslated leader region of retroviral RNAs contains noncoding information that is essential for viral replication, including signals for transcriptional transactivation, splicing, primer binding for reverse transcription, dimerization of the genomic RNA, and encapsidation of the viral RNA into virions. These RNA motifs have considerable structural and functional overlap. In this study, we investigate the conformational dynamics associated with the use and silencing of a sequence in HIV-2 RNA that is involved in genomic RNA dimerization called stem-loop 1 (SL1) and its relationship with a flanking sequence that is known to be important for encapsidation of viral RNAs. We demonstrate that a long-distance intramolecular interaction between nucleotides located upstream of the primer-binding site domain and nucleotides encompassing the Gag translation start codon functionally silences SL1 as a dimerization element. This silencing can be relieved by mutation or by hybridization of an oligonucleotide that disrupts the long-distance interaction. Furthermore, we identify a palindrome within the packaging/encapsidation signal Psi (just 5' of SL1) that can either serve as an efficient dimerization signal itself, or can mediate SL1 silencing through base pairing with SL1. These results provide a tangible link between the functions of genomic RNA dimerization and encapsidation, which are known to be related, but whose physical relationship has been unclear. A model is proposed that accounts for observations of dimerization, packaging, and translation of viral RNAs during different phases of the viral replication cycle.

Base Sequence↗

PubMed2002

Alternate usage of two dimerization initiation sites in HIV-2 viral RNA in vitro.

An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during encapsidation and budding. In HIV-1, a stem-loop structure in the genomic RNA called the dimerization initiation site, or DIS, has been well characterized. However, the identification of the structure(s) necessary for dimerization of HIV-2 genomic RNA has been less straightforward, as reflected by recent conflicting reports. Here, using a variety of mutant and wild-type RNA constructs and a systematic analysis of experimental conditions, we demonstrate that two dimerization sites in HIV-2 RNA are clearly discernible under different experimental conditions. A short sequence overlapping the primer binding site acts as the default dimerization site for wild-type viral RNA transcripts of several lengths provided that dimerization incubation conditions do not include a high heat step (>50 degrees C), and electrophoresis is carried out under mild conditions that do not deplete the RNA of magnesium. However, some RNA constructs are able to dimerize through stem-loop 1 (SL1), which is the structure homologous to the HIV-1 DIS, under certain experimental conditions. Interestingly, deletion or mutation of the default PBS dimerization site leads to efficient usage of the SL1 dimerization site. This study defines conditions under which each site may be used for dimerization and demonstrates, furthermore, the facility with which the two sites can substitute for each other. This is suggestive of a switching mechanism that may be used in the viral replication cycle.

Base Sequence↗