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

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

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

Dynamic localization of SPE-9 in sperm: a protein required for sperm-oocyte interactions in Caenorhabditis elegans

BACKGROUND: Fertilization in Caenorhabditis elegans requires functional SPE-9 protein in sperm. SPE-9 is a transmembrane protein with a predicted extracellular domain that contains ten epidermal growth factor (EGF)-like motifs. The presence of these EGF-like motifs suggests that SPE-9 is likely to function in gamete adhesive and/or ligand-receptor interactions. RESULTS: We obtained specific antisera directed against different regions of SPE-9 in order to determine its subcellular localization. SPE-9 is segregated to spermatids with a pattern that is consistent with localization to the plasma membrane. During spermiogenesis, SPE-9 becomes localized to spiky projections that coalesce to form a pseudopod. This leads to an accumulation of SPE-9 on the pseudopod of mature sperm. CONCLUSIONS: The wild type localization patterns of SPE-9 provide further evidence that like the sperm of other species, C. elegans sperm have molecularly mosaic and dynamic regions. SPE-9 is redistributed by what is likely to be a novel mechanism that is very fast (~5 minutes) and is coincident with dramatic rearrangements in the major sperm protein cytoskeleton. We conclude that SPE-9 ends up in a location on mature sperm where it can function during fertilization and this localization defines the sperm region required for these interactions

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

A Novel Sperm-Delivered Toxin Causes Late-Stage Embryo Lethality and Transmission Ratio Distortion in C. elegans

The evolutionary fate of an allele ordinarily depends on its contribution to host fitness. Occasionally, however, genetic elements arise that are able to gain a transmission advantage while simultaneously imposing a fitness cost on their hosts. We previously discovered one such element in C. elegans that gains a transmission advantage through a combination of paternal-effect killing and zygotic self-rescue. Here we demonstrate that this element is composed of a sperm-delivered toxin, peel-1, and an embryo-expressed antidote, zeel-1. peel-1 and zeel-1 are located adjacent to one another in the genome and co-occur in an insertion/deletion polymorphism. peel-1 encodes a novel four-pass transmembrane protein that is expressed in sperm and delivered to the embryo via specialized, sperm-specific vesicles. In the absence of zeel-1, sperm-delivered PEEL-1 causes lethal defects in muscle and epidermal tissue at the 2-fold stage of embryogenesis. zeel-1 is expressed transiently in the embryo and encodes a novel six-pass transmembrane domain fused to a domain with sequence similarity to zyg-11, a substrate-recognition subunit of an E3 ubiquitin ligase. zeel-1 appears to have arisen recently, during an expansion of the zyg-11 family, and the transmembrane domain of zeel-1 is required and partially sufficient for antidote activity. Although PEEL-1 and ZEEL-1 normally function in embryos, these proteins can act at other stages as well. When expressed ectopically in adults, PEEL-1 kills a variety of cell types, and ectopic expression of ZEEL-1 rescues these effects. Our results demonstrate that the tight physical linkage between two novel transmembrane proteins has facilitated their co-evolution into an element capable of promoting its own transmission to the detriment of organisms carrying it

The ubiquitin conjugation system is involved in the disassembly of cilia and flagella

The disassembly of cilia and flagella is linked to the cell cycle and environmental cues. We have found that ubiquitination of flagellar proteins is an integral part of flagellar disassembly. Free ubiquitin and the ubiquitin-conjugating enzyme CrUbc13 are detected in flagella, and several proteins are ubiquitinated in isolated flagella when exogenous ubiquitin and adenosine triphosphatase are added, suggesting that the ubiquitin conjugation system operates in flagella. Levels of ubiquitinated flagellar proteins increase during flagellar resorption, especially in intraflagellar transport (IFT) mutants, suggesting that disassembly products are labeled with ubiquitin and transported to the cell body by IFT. Substrates of the ubiquitin conjugation system include α-tubulin (but not β-tubulin), a dynein subunit (IC2), two signaling proteins involved in the mating process, cyclic guanosine monophosphate–dependent kinase, and the cation channel polycystic kidney disease 2. Ubiquitination of flagellar proteins is enhanced early in mating, suggesting that ubiquitination also plays an active role in regulating signaling pathways in flagella

Acetyltransferases and tumour suppression

The acetyltransferase p300 was first identified associated with the adenoviral transforming protein E1A, suggesting a potential role for p300 in the regulation of cell proliferation. Direct evidence demonstrating a role for p300 in human tumours was lacking until the recentl publication by Gayther et al, which strongly supports a role for p300 as a tumour suppressor. The authors identify truncating mutations associated with the loss or mutation of the second allele in both tumour samples and cell lines, suggesting that loss of p300 may play a role in the development of a subset of human cancers

SPE-44 Implements Sperm Cell Fate

The sperm/oocyte decision in the hermaphrodite germline of Caenorhabditis elegans provides a powerful model for the characterization of stem cell fate specification and differentiation. The germline sex determination program that governs gamete fate has been well studied, but direct mediators of cell-type-specific transcription are largely unknown. We report the identification of spe-44 as a critical regulator of sperm gene expression. Deletion of spe-44 causes sperm-specific defects in cytokinesis, cell cycle progression, and organelle assembly resulting in sterility. Expression of spe-44 correlates precisely with spermatogenesis and is regulated by the germline sex determination pathway. spe-44 is required for the appropriate expression of several hundred sperm-enriched genes. The SPE-44 protein is restricted to the sperm-producing germline, where it localizes to the autosomes (which contain sperm genes) but is excluded from the transcriptionally silent X chromosome (which does not). The orthologous gene in other Caenorhabditis species is similarly expressed in a sex-biased manner, and the protein likewise exhibits autosome-specific localization in developing sperm, strongly suggestive of an evolutionarily conserved role in sperm gene expression. Our analysis represents the first identification of a transcriptional regulator whose primary function is the control of gamete-type-specific transcription in this system