16 research outputs found
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Investigation of the sequences and structural elements required for HIV-2 infectivity and genome dimerisation
A unique feature of retroviruses is that two copies of the genomic RNA are packaged in each particle. The selective encapsidation of viral genomes is ensured by the binding of the Nucleocapsid to a specific motif on the RNA genome, the packaging signal (Psi). The Psi regions of many retroviruses overlap with sequences that promote the dimerisation of the genome, the dimerisation initiation site (DIS), and it has been suggested that the two mechanisms are closely linked.
The aim of the research presented herein was to identify the sequences and structural elements required for the dimerisation of HIV-2 genomic RNA and to investigate the relationship between HIV-2 genome dimerisation and encapsidation, infectivity and particle morphogenesis.
Mutations of two palindromic sequences, introduced in an infectious molecular clone of the HIV-2rod isolate, revealed that a palindrome within HIV-2 Psi was important for genome dimerisation. In contrast with previous studies, the palindrome termed DIS is not required for genome dimerisation and viral replication.
Viruses bearing mutations within the Psi region failed to dimerise and to replicate in T-cells, a defect that could not be rescued by targeting more genomes to the cells. Psi-deleted viruses also displayed a defect in particle morphogenesis. A reduced packaging efficiency, combined with the presence of RNA monomers or unstable dimers in these virions, resulted in the production of fewer mature particles. However an increase in the number of particles containing two cores was observed.
Further characterisation of the sequences and structural elements required for RNA dimerisation, packaging and viral replication showed that the formation of stem B is not critical for viral replication. However, a GGAG purine-rich motif at position 392-395 of the HIV-2rod genome is absolutely essential for genome dimerisation and viral infectivity, and a correlation was observed between dimer formation and viral replication
Identification of a GGAG motif in the HIV-2 5' leader RNA that is critical for genome dimerisation and viral replication
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Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity.
BACKGROUND: Retroviruses selectively encapsidate two copies of their genomic RNA, the Gag protein binding a specific RNA motif in the 5' UTR of the genome. In human immunodeficiency virus type 2 (HIV-2), the principal packaging signal (Psi) is upstream of the major splice donor and hence is present on all the viral RNA species. Cotranslational capture of the full length genome ensures specificity. HIV-2 RNA dimerisation is thought to occur at the dimer initiation site (DIS) located in stem-loop 1 (SL-1), downstream of the main packaging determinant. However, the HIV-2 packaging signal also contains a palindromic sequence (pal) involved in dimerisation. In this study, we analysed the role of the HIV-2 packaging signal in genomic RNA dimerisation in vivo and its implication in viral replication. RESULTS: Using a series of deletion and substitution mutants in SL-1 and the Psi region, we show that in fully infectious HIV-2, genomic RNA dimerisation is mediated by the palindrome pal. Mutation of the DIS had no effect on dimerisation or viral infectivity, while mutations in the packaging signal severely reduce both processes as well as RNA encapsidation. Electron micrographs of the Psi-deleted virions revealed a significant reduction in the proportion of mature particles and an increase in that of particles containing multiple cores. CONCLUSION: In addition to its role in RNA encapsidation, the HIV-2 packaging signal contains a palindromic sequence that is critical for genomic RNA dimerisation. Encapsidation of a dimeric genome seems required for the production of infectious mature particles, and provides a promising therapeutic target.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are
Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity
<p>Abstract</p> <p>Background</p> <p>Retroviruses selectively encapsidate two copies of their genomic RNA, the Gag protein binding a specific RNA motif in the 5' UTR of the genome. In human immunodeficiency virus type 2 (HIV-2), the principal packaging signal (Psi) is upstream of the major splice donor and hence is present on all the viral RNA species. Cotranslational capture of the full length genome ensures specificity. HIV-2 RNA dimerisation is thought to occur at the dimer initiation site (DIS) located in stem-loop 1 (SL-1), downstream of the main packaging determinant. However, the HIV-2 packaging signal also contains a palindromic sequence (pal) involved in dimerisation. In this study, we analysed the role of the HIV-2 packaging signal in genomic RNA dimerisation <it>in vivo </it>and its implication in viral replication.</p> <p>Results</p> <p>Using a series of deletion and substitution mutants in SL-1 and the Psi region, we show that in fully infectious HIV-2, genomic RNA dimerisation is mediated by the palindrome pal. Mutation of the DIS had no effect on dimerisation or viral infectivity, while mutations in the packaging signal severely reduce both processes as well as RNA encapsidation. Electron micrographs of the Psi-deleted virions revealed a significant reduction in the proportion of mature particles and an increase in that of particles containing multiple cores.</p> <p>Conclusion</p> <p>In addition to its role in RNA encapsidation, the HIV-2 packaging signal contains a palindromic sequence that is critical for genomic RNA dimerisation. Encapsidation of a dimeric genome seems required for the production of infectious mature particles, and provides a promising therapeutic target.</p
Widespread disruption of host transcription termination in HSV-1 infection.
Herpes simplex virus 1 (HSV-1) is an important human pathogen and a paradigm for virus-induced host shut-off. Here we show that global changes in transcription and RNA processing and their impact on translation can be analysed in a single experimental setting by applying 4sU-tagging of newly transcribed RNA and ribosome profiling to lytic HSV-1 infection. Unexpectedly, we find that HSV-1 triggers the disruption of transcription termination of cellular, but not viral, genes. This results in extensive transcription for tens of thousands of nucleotides beyond poly(A) sites and into downstream genes, leading to novel intergenic splicing between exons of neighbouring cellular genes. As a consequence, hundreds of cellular genes seem to be transcriptionally induced but are not translated. In contrast to previous reports, we show that HSV-1 does not inhibit co-transcriptional splicing. Our approach thus substantially advances our understanding of HSV-1 biology and establishes HSV-1 as a model system for studying transcription termination.This work was supported by MRC Fellowship grant G1002523 and NHSBT grant WP11-05 to LD, and DFG grant FR2938/1–2 to C.C.F. We thank Viv Connor for excellent technical assistance and Professor Rozanne Sandri-Goldin (University of California) for the ΔICP27 mutant and complementing cell line. The support of the Cluster of Excellence (Nucleotide lab) to P.R. is acknowledged.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/ncomms812
Improved Ribo-seq enables identification of cryptic translation events.
Ribosome profiling has been used to predict thousands of short open reading frames (sORFs) in eukaryotic cells, but it suffers from substantial levels of noise. PRICE (https://github.com/erhard-lab/price) is a computational method that models experimental noise to enable researchers to accurately resolve overlapping sORFs and noncanonical translation initiation. We experimentally validated translation using major histocompatibility complex class I (MHC I) peptidomics and observed that sORF-derived peptides efficiently enter the MHC I presentation pathway and thus constitute a substantial fraction of the antigen repertoire
Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity-1
<p><b>Copyright information:</b></p><p>Taken from "Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity"</p><p>http://www.retrovirology.com/content/4/1/90</p><p>Retrovirology 2007;4():90-90.</p><p>Published online 13 Dec 2007</p><p>PMCID:PMC2222663.</p><p></p>. RNA inputs were normalised on RT activity and an equivalent to 2.5 × 10cpm was used. WT, wild type; DM, Psi deletion mutant; SM1, DIS mutant; SM2, Psi pal mutant; mock, mock transfection; MM, Millennium RNA Markers (Ambion); M, monomer; D, dimer. (B) Bar chart representing the percentage of dimer present in each virion RNA sample. Data from at least three independent experiments are shown, error bars correspond to the SD. *, Student t test p value < 0.05
Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity-0
<p><b>Copyright information:</b></p><p>Taken from "Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity"</p><p>http://www.retrovirology.com/content/4/1/90</p><p>Retrovirology 2007;4():90-90.</p><p>Published online 13 Dec 2007</p><p>PMCID:PMC2222663.</p><p></p>viously [8]. In SM1 the first three bases in the DIS palindrome at position 420 of the HIV-2 RNA genome are substituted. In SM2 the first four bases of the Psi palindrome at position 392 of the HIV-2 RNA genome are substituted. (B) Structure of the SL-1 region predicted by previously published biochemical analyses [20, 22, 55, 56] and computer modelling [57, 58. The position of the stem proposed to extend SL-1 is indicated (stem B) [14]
Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity-2
<p><b>Copyright information:</b></p><p>Taken from "Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity"</p><p>http://www.retrovirology.com/content/4/1/90</p><p>Retrovirology 2007;4():90-90.</p><p>Published online 13 Dec 2007</p><p>PMCID:PMC2222663.</p><p></p>otection assay (RPA). Protected mutant genomic RNAs vary in size between 329 and 355 nt due to the position of the mutations. (B) Representative RPA where 2 μg of cytoplasmic RNA and an equivalent of 2.5 × 10cpm of virion RNA was probed with 1 × 10cpm of KS2ΨKE riboprobe. Samples were also probed with 1 × 10cpm of KS2ΨEP and GAPDH riboprobes to detect plasmid DNA contamination and control for the loading, respectively. WT, wild type; DM, Psi deletion mutant; SM1, DIS mutant; SM2, pal mutant; mock, mock transfection; Y, yeast RNA + RNase; I, yeast RNA – RNAse (diluted 1:10); M, Century Plus RNA Markers (Ambion). (C) Packaging efficiencies of mutant HIV-2 relative to WT virus. Data from 3 independent experiments are shown, error bars correspond to the SD. *, Student t test p value < 0.005
Two murine cytomegalovirus microRNAs target the major viral immediate early 3 gene
Human cytomegalovirus is responsible for morbidity and mortality in immune compromised patients and is the leading viral cause of congenital infection. Virus-encoded microRNAs (miRNAs) represent interesting targets for novel antiviral agents. While many cellular targets that augment productive infection have been identified in recent years, regulation of viral genes such as the major viral immediate early protein 72 (IE72) by hcmv-miR-UL112-1 may contribute to both the establishment and the maintenance of latent infection. We employed photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking (PAR-iCLIP) to identify murine cytomegalovirus (MCMV) miRNA targets during lytic infection. While the PAR-iCLIP data were of insufficient quality to obtain a comprehensive list of cellular and viral miRNA targets, the most prominent PAR-iCLIP peak in the MCMV genome mapped to the 3′ untranslated region of the major viral immediate early 3 (
) transcript. We show that this results from two closely positioned binding sites for the abundant MCMV miRNAs miR-M23-2-3p and miR-m01-2-3p. Their pre-expression significantly impaired viral plaque formation. However, mutation of the respective binding sites did not alter viral fitness during acute or subacute infection
. Furthermore, no differences in the induction of virus-specific CD8
T cells were observed. Future studies will probably need to go beyond studying immunocompetent laboratory mice housed in pathogen-free conditions to reveal the functional relevance of viral miRNA-mediated regulation of key viral immediate early genes