Project description:Pseudomonas virus PA5oct has a large, linear, double-stranded DNA genome (286,783 bp) and is related to Escherichia phages 121Q/PBECO 4, Klebsiella phage vB_KleM-RaK2, Klebsiella phage K64-1, and Cronobacter phage vB_CsaM_GAP32. A protein-sharing network analysis highlights the conserved core genes within this clade. Combining hybrid genome sequencing, RNA-Seq and mass spectrometry analyses of its virion proteins allowed us to accurately identify genes and elucidate regulatory elements for this phage (ncRNAs, tRNAs and promoter elements). In total PA5oct encodes 449 CDS of which 93, have been identified as virion-associated based on ESI-MS/MS. The RNA-Seq-based temporal genome organization suggests a gradual take-over by viral transcripts from 21%, 69%, and 93% at 5, 15 and 25 min after infection, respectively . Like many large phages, PA5oct is not organized into contiguous regions of temporal transcription. However, although the temporal regulation of the PA5oct genome expression reveals specific genome clusters expressed in early and late infection, many genes encoding experimentally observed structural proteins surprisingly appear to remain almost untranscribed throughout the infection cycle. Within the host, operons associated with elements of a cryptic Pf1-like prophage are upregulated, as are operons responsible for Psl exopolysaccharide (pslE-J) and periplasmic nitrate reductase (napA-F) production. The characterization described here represents a crucial step towards understanding the genomic complexity as well as molecular diversity of jumbo viruses.
2020-06-04 | GSE130190 | GEO
Project description:Ultra-deep sequencing of large double-stranded DNA viruses
| PRJNA592818 | ENA
Project description:Insect transposable elements in large double-stranded DNA viruses
Project description:ϕXacN1 is a novel jumbo myovirus infecting the causative agent of Asian citrus canker, Xanthomonas citri. Its linear 384,670 bp double-stranded DNA genome encodes 592 predicted protein coding genes and shows 65,875 bp direct terminal repeats (DTRs), so far the longest DTRs among sequence phage genomes. The DTRs harbor 56 tRNA genes, corresponding to all 20 amino acids. This is the highest number of tRNA genes reported in a phage genome. Codon usage analyses revealed a propensity that the phage encoded tRNAs target codons that are highly used by the phage but less frequently by its host. The existence of these tRNA genes, additional seven translation-related genes as well as a chaperonin gene found in the ϕXacN1 genome suggests an increased level of independence of phage replication on host molecular machinery and a wide host range. Consistently, ϕXacN1 showed a wider host range than other X. citri phages in an infection test against a panel of X. citri strains. Phylogenetic analyses revealed a clade of phages composed of ϕXacN1 and ten other jumbo phages showing an evolutionary stability in their large genome sizes.
Project description:Survey of single and double stranded DNA non-enveloped viruses on the skin of WHIM patients, healthy controls and other immune compromised patients
Project description:Viral infections involve packaging of the viral genome and proteins into virions. Knowing virion composition and structure is critical to understand viral pathogenesis. However, an integrated picture of virion proteome organization of large viruses, such as Herpesviruses, is lacking. Here we use cross-linking mass spectrometry to derive a spatially resolved structural interactome of intact human cytomegalovirus virions. We capture interactions of 82 host and 33 viral proteins, allowing us to de novo allocate them into distinct virion-layers and identify several host proteins as constitutive virion components recruited via specific protein interactions. The abundant viral protein pp150 forms domain-specific interactions with all virion layers and scaffolds numerous viral and host proteins such as PP1 phosphatase and 14-3-3 proteins, which are incorporated via nearby short linear motifs in pp150’s C-terminus. PP1 recruitment antagonizes 14-3-3 proteins and is pivotal during early and late viral replication steps. Collectively, this study gives a spatial and quantitative inventory of the system-wide organization and functional relevance of protein interactions inside native herpesvirus particles.
Project description:Retroviral integration is mediated by a unique enzymatic process shared by all retroviruses and retrotransposons. During integration, double-stranded linear viral DNA is inserted into the host genome in a process catalyzed by viral-encoded integrase. However, host cell defenses against HIV-1 integration are not clear. This study identifies -catenin-like protein 1 (CTNNBL1) as a potent inhibitor of HIV-1 integration via association with viral IN and its cofactor, lens epithelium-derived growth factor/p75. CTNNBL1 overexpression blocks HIV-1 integration and inhibits viral replication, whereas CTNNBL1 depletion significantly upregulates HIV-1 integration into the genome of various target cells. Further, CTNNBL1 expression is downregulated in CD4+ T cells by activation, and CTNNBL1 depletion also facilitates HIV-1 integration in resting CD4+ T cells. Thus, host cells may employ CTNNBL1 to inhibit HIV-1 integration into the genome. This finding suggests a strategy for the treatment of HIV infections.
Project description:The RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double-stranded) engage in a high-efficiency HDR mechanism that requires only ~35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity-sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand-annealing (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.
Project description:Telomeres are the ends of linear chromosomes and consist of repetitive double- and single-stranded DNA sequences. Telomeres are bound by dedicated protein complexes, such as shelterin in mammals. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to screen for proteins binding to C. elegans telomeres, and identified TEBP-1 and TEBP-2, two paralogs that associate to telomeres in vitro and in vivo. TEBP-1 and TEBP-2 form a telomeric complex with the known single-stranded telomere-binding proteins POT-1, POT-2, and MRT-1. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp 2 mutants display shorter telomeres and a mortal germline, a phenotype characterized by transgenerational germline deterioration. Notably, tebp 1;tebp-2 double mutant animals are synthetic sterile, with germlines showing signs of severe mitotic and meiotic arrest. These results define the first telomere-binding complex of C. elegans, including TEBP-1 and TEBP-2, two double-stranded telomere binders required for fertility.