Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes malignant and inflammatory diseases in ∼10% of infected people. A typical host has between 10 4 and 10 5 clones of HTLV-1-infected T lymphocytes, each clone distinguished by the genomic integration site of the single-copy HTLV-1 provirus. The HTLV-1 bZIP (HBZ) factor gene is constitutively expressed from the minus strand of the provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is suppressed or intermittent in vivo, allowing escape from host immune surveillance. It remains unknown what regulates this pattern of proviral transcription and latency. Here, we show that CTCF, a key regulator of chromatin structure and function, binds to the provirus at a sharp border in epigenetic modifications in the pX region of the HTLV-1 provirus in T cells naturally infected with HTLV-1. CTCF is a zinc-finger protein that binds to an insulator region in genomic DNA and plays a fundamental role in controlling higher order chromatin structure and gene expression in vertebrate cells. We show that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates HTLV-1 mRNA splicing, and forms long-distance interactions with flanking host chromatin. CTCF-binding sites (CTCF-BSs) have been propagated throughout the genome by transposons in certain primate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic CTCF-BS introduced by the retrovirus in tens of thousands of genomic locations has the potential to cause widespread abnormalities in host cell chromatin structure and gene expression.retrovirus | latency | epigenetics | HTLV-1 | CTCF
Combination anti-retroviral therapy (cART) has drastically improved the clinical outcome of HIV-1 infection. Nonetheless, despite effective cART, HIV-1 persists indefinitely in infected individuals. Clonal expansion of HIV-1-infected cells in peripheral blood has been reported recently. cART is effective in stopping the retroviral replication cycle, but not in inhibiting clonal expansion of the infected host cells. Thus, the proliferation of HIV-1-infected cells may play a role in viral persistence, but little is known about the kinetics of the generation, the tissue distribution or the underlying mechanism of clonal expansion in vivo. Here we analyzed the clonality of HIV-1-infected cells using high-throughput integration site analysis in a hematopoietic stem cell-transplanted humanized mouse model. Clonally expanded, HIV-1-infected cells were detectable at two weeks post infection, their abundance increased with time, and certain clones were present in multiple organs. Expansion of HIV-1-infected clones was significantly more frequent when the provirus was integrated near host genes in specific gene ontological classes, including cell activation and chromatin regulation. These results identify potential drivers of clonal expansion of HIV-1-infected cells in vivo.
The recent development and advancement of next-generation sequencing (NGS) technologies have enabled the characterization of the human genome at extremely high resolution. In the retrovirology field, NGS technologies have been applied to integration-site analysis and deep sequencing of viral genomes in combination with PCR amplification using virus-specific primers. However, virus-specific primers are not available for some epigenetic analyses, like chromatin immunoprecipitation sequencing (ChIP-seq) assays. Viral sequences are poorly detected without specific PCR amplification because proviral DNA is very scarce compared to human genomic DNA. Here, we have developed and evaluated the use of biotinylated DNA probes for the capture of viral genetic fragments from a library prepared for NGS. Our results demonstrated that viral sequence detection was hundreds or thousands of times more sensitive after enrichment, enabling us to reduce the economic burden that arises when attempting to analyze the epigenetic landscape of proviruses by NGS. In addition, the method is versatile enough to analyze proviruses that have mismatches compared to the DNA probes. Taken together, we propose that this approach is a powerful tool to clarify the mechanisms of transcriptional and epigenetic regulation of retroviral proviruses that have, until now, remained elusive.
The Bioinformation and DDBJ Center (DDBJ Center, https://www.ddbj.nig.ac.jp) provides databases that capture, preserve and disseminate diverse biological data to support research in the life sciences. This center collects nucleotide sequences with annotations, raw sequencing data, and alignment information from high-throughput sequencing platforms, and study and sample information, in collaboration with the National Center for Biotechnology Information (NCBI) and the European Bioinformatics Institute (EBI). This collaborative framework is known as the International Nucleotide Sequence Database Collaboration (INSDC). In collaboration with the National Bioscience Database Center (NBDC), the DDBJ Center also provides a controlled-access database, the Japanese Genotype–phenotype Archive (JGA), which archives and distributes human genotype and phenotype data, requiring authorized access. The NBDC formulates guidelines and policies for sharing human data and reviews data submission and use applications. To streamline all of the processes at NBDC and JGA, we have integrated the two systems by introducing a unified login platform with a group structure in September 2020. In addition to the public databases, the DDBJ Center provides a computer resource, the NIG supercomputer, for domestic researchers to analyze large-scale genomic data. This report describes updates to the services of the DDBJ Center, focusing on the NBDC and JGA system enhancements.
Our data identified some effects of the long-term treatment on total HIV DNA levels and highlighted the partial influence of comorbidities and coinfections. Total HIV DNA monitoring contributed to therapy response estimates and HIV reservoir quantification. The results suggest that HIV DNA monitoring during ART might be useful as a persistence marker in both HIV-monoinfected patients and those with comorbidities and coinfections.
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