Intranuclear Positions of HIV-1 Proviruses Are Dynamic and Do Not Correlate with Transcriptional Activity

HIV-1 integrates its genomic DNA into the chromosomes of the infected cell, but how it selects the site of integration and the impact of their location in the 3-dimensional nuclear space is not well understood. Here, we examined the nuclear locations of proviruses 1 and 5 days after infection and found that integration sites are first located near the nuclear envelope but become randomly distributed throughout the nucleus after a few cell divisions, indicating that the locations of the chromosomal sites of integration that harbor transcriptionally active proviruses are dynamic.

Analysis of HIV quasispecies and virological outcome of an HIV D+/R+ kidney–liver transplantation

Introduction Transplantation among HIV positive patients may be a valuable therapeutic intervention. This study involves an HIV D+/R+ kidney–liver transplantation, where PBMC-associated HIV quasispecies were analyzed in donor and transplant recipients (TR) prior to transplantation and thereafter, together with standard viral monitoring. Methods The donor was a 54 year of age HIV infected woman: kidney and liver recipients were two HIV infected men, aged 49 and 61. HIV quasispecies in PBMC was analyzed by ultra-deep sequencing of V3 env region. During TR follow-up, plasma HIV-1 RNA, HIV-1 DNA in PBMC, analysis of proviral integration sites and drug-resistance genotyping were performed. Other virological and immunological monitoring included CMV and EBV DNA quantification in blood and CD4 T cell counts. Results Donor and TR were all ART-HIV suppressed at transplantation. Thereafter, TR maintained a nearly suppressed HIV-1 viremia, but HIV-1 RNA blips and the increase of proviral integration sites in PBMC attested some residual HIV replication. A transient peak in HIV-1 DNA occurred in the liver recipient. No major changes of drug-resistance genotype were detected after transplantation. CMV and EBV transient reactivations were observed only in the kidney recipient, but did not require specific treatment. CD4 counts remained stable. No intermixed quasispecies between donor and TR was observed at transplantation or thereafter. Despite signs of viral evolution in TR, HIV genetic heterogeneity did not increase over the course of the months of follow up. Conclusions No evidence of HIV superinfection was observed in the donor nor in the recipients. The immunosuppressive treatment administrated to TR did not result in clinical relevant viral reactivations.

Heterogeneity and genomic loci of ubiquitous Cre reporter transgenes in zebrafish

The most-common strategy for zebrafish Cre/lox-mediated lineage labeling experiments combines ubiquitously expressed, lox-based Switch reporter transgenes with tissue-specific Cre or 4-OH-Tamoxifen-inducible CreERT2 driver lines. Although numerous Cre driver lines have been produced, only a few broadly expressed Switch reporters exist in zebrafish and their generation by random transgene integration has been challenging due to position-effect sensitivity of the lox-flanked recombination cassettes. Here, we compare commonly used Switch reporter lines for their recombination efficiency and reporter expression pattern during zebrafish development. Using different experimental setups, we show that ubi:Switch and hsp70l:Switch outperform current generations of two additional Switch reporters due to favorable transgene integration sites. Our comparisons also document preferential Cre-dependent recombination of ubi:Switch and hsp70l:Switch in distinct zebrafish tissues at early developmental stages. To investigate what genomic features may influence Cre accessibility and lox recombination efficiency in highly functional Switch lines, we mapped these transgenes and charted chromatin dynamics at their integration sites. Our data documents the heterogeneity among lox-based Switch transgenes towards informing suitable transgene selection for lineage labeling experiments. Our work further proposes that ubi:Switch and hsp70l:Switch define genomic integration sites suitable for universal transgene or switch reporter knock-in in zebrafish.

Recurrent integration of human papillomavirus genomes at transcriptional regulatory hubs

Oncogenic human papillomavirus (HPV) genomes are often integrated into host chromosomes in HPV-associated cancers. HPV genomes are integrated either as a single copy or as tandem repeats of viral DNA interspersed with, or without, host DNA. Integration occurs frequently in common fragile sites susceptible to tandem repeat formation and the flanking or interspersed host DNA often contains transcriptional enhancer elements. When co-amplified with the viral genome, these enhancers can form super-enhancer-like elements that drive high viral oncogene expression. Here we compiled highly curated datasets of HPV integration sites in cervical (CESC) and head and neck squamous cell carcinoma (HNSCC) cancers, and assessed the number of breakpoints, viral transcriptional activity, and host genome copy number at each insertion site. Tumors frequently contained multiple distinct HPV integration sites but often only one “driver” site that expressed viral RNA. As common fragile sites and active enhancer elements are cell-type-specific, we mapped these regions in cervical cell lines using FANCD2 and Brd4/H3K27ac ChIP-seq, respectively. Large enhancer clusters, or super-enhancers, were also defined using the Brd4/H3K27ac ChIP-seq dataset. HPV integration breakpoints were enriched at both FANCD2-associated fragile sites and enhancer-rich regions, and frequently showed adjacent focal DNA amplification in CESC samples. We identified recurrent integration “hotspots” that were enriched for super-enhancers, some of which function as regulatory hubs for cell-identity genes. We propose that during persistent infection, extrachromosomal HPV minichromosomes associate with these transcriptional epicenters and accidental integration could promote viral oncogene expression and carcinogenesis.

Fanconi Anemia Pathway Genes Advance Cervical Cancer via Immune Regulation and Cell Adhesion

Fanconi anemia (FA) pathway is a typical and multienzyme-regulated DNA damage repairer that influences the occurrence and development of disease including cancers. Few comprehensive analyses were reported about the role of FA-related genes (FARGs) and their prognostic values in cancers. In this study, a comprehensive pan-cancer analysis on 79 FARGs was performed. According to the correlation analyses between HPV integration sites and FARGs, we found that FARGs played specific and critical roles in HPV-related cancers, especially in cervical cancer (CC). Based on this, a FARGs-associated prognostic risk score (FPS) model was constructed, and subsequently a nomogram model containing the FPS was developed with a good accuracy for CC overall survival (OS) and recurrence-free survival (RFS) outcome prediction. We also used the similar expression pattern of FARGs by consensus clustering analysis to separate the patients into three subgroups that exhibited significant differential OS but not RFS. Moreover, differential expressed genes (DEGs) between the two risk groups or three clusters were identified and immune pathways as well as cell adhesion processes were determined by functional enrichment analysis. Results indicated that FARGs might promote occurrence and development of CC by regulating the immune cells’ infiltration and cell adhesion. In addition, through the machine learning models containing decision tree, random forest, naïve bayes, and support vector machine models, screening of important variables on CC prognosis, we finally determined that ZBTB32 and CENPS were the main elements affecting CC OS, while PALB2 and BRCA2 were for RFS. Kaplan-Meier analysis revealed that bivariate prediction of CC outcome was reliable. Our study systematically analyzed the prognostic prediction values of FARGs and demonstrated their potential mechanism in CC aggressiveness. Results provided perspective in FA pathway-associated modification and theoretical basis for CC clinical treatments.

Selective clonal persistence of human retroviruses in vivo: radial chromatin organization, integration site and host transcription

The human retroviruses HTLV-1 and HIV-1 persist in vivo, despite the host immune response and antiretroviral therapy, as a reservoir of latently infected T-cell clones. It is poorly understood what determines which clones survive in the reservoir and which are lost. We compared >160,000 HTLV-1 integration sites from T-cells isolated ex vivo from naturally-infected subjects with >230,000 integration sites from in vitro infection, to identify the genomic features that determine selective clonal survival. Three factors explained >40% of the observed variance in clone survival of HTLV-1 in vivo: the radial intranuclear position of the provirus, its absolute genomic distance from the centromere, and the intensity of host genome transcription flanking the provirus. The radial intranuclear position of the provirus and its distance from the centromere also explained ~7% of clonal persistence of HIV-1 in vivo. Selection for transcriptionally repressive nuclear compartments favours clonal persistence of human retroviruses in vivo.

Detection of HPV and Human Chromosome Sites by Dual-Color Fluorescence In Situ Hybridization Reveals Recurrent HPV Integration Sites and Heterogeneity in Cervical Cancer

Human papillomavirus (HPV) integration in the human genome is suggested to be an important cause of cervical cancer. With the development of sequencing technologies, an increasing number of integration “hotspots” have been identified. However, this HPV integration information was derived from analysis of whole cervical cancer tissue, and we know very little about the integration in different cancer cell subgroups or individual cancer cells. This study optimized the preparation of probes and provided a dual-color fluorescence in situ hybridization (FISH) method to detect HPV integration sites in paraffin-embedded cervical cancer samples. We used both HPV probes and site-specific probes: 3p14 (FHIT), 8q24 (MYC), 13q22 (KLF5/KLF12), 3q28 (TP63), and 5p15 (TERT). We detected HPV signals in 75 of the 96 cases of cervical cancer; 62 cases showed punctate signals, and 13 cases showed diffuse punctate signals. We identified 3p14 as a high-frequency HPV integration site in 4 cervical cancer cases. HPV integration at 8p14 occurred in 2 cases of cervical cancer. In the same cervical cancer tissue of sample No.1321, two distinct subgroups of cells were observed based on the HPV probe but showed no difference in cell and nucleus morphology. Our study provides a new method to investigate the frequent HPV integration sites in cervical cancer and reports the heterogeneity within cervical cancer from the perspective of HPV integration.

Comparative analysis of integrative and conjugative mobile genetic elements in the genus Mesorhizobium

Members of the Mesorhizobium genus are soil bacteria that often form nitrogen-fixing symbioses with legumes. Most characterised Mesorhizobium spp. genomes are ~8 Mb in size and harbour extensive pangenomes including large integrative and conjugative elements (ICEs) carrying genes required for symbiosis (ICESyms). Here, we document and compare the conjugative mobilome of 41 complete Mesorhizobium genomes. We delineated 56 ICEs and 24 integrative and mobilizable elements (IMEs) collectively occupying 16 distinct integration sites, along with 24 plasmids. We also demonstrated horizontal transfer of the largest (853,775 bp) documented ICE, the tripartite ICEMspSymAA22. The conjugation systems of all identified ICEs and several plasmids were related to those of the paradigm ICESym ICEMlSymR7A, with each carrying conserved genes for conjugative pilus formation (trb), excision (rdfS), DNA transfer (rlxS) and regulation (fseA). ICESyms have likely evolved from a common ancestor, despite occupying a variety of distinct integration sites and specifying symbiosis with diverse legumes. We found extensive evidence for recombination between ICEs and particularly ICESyms, which all uniquely lack the conjugation entry-exclusion factor gene trbK. Frequent duplication, replacement and pseudogenization of genes for quorum-sensing-mediated activation and antiactivation of ICE transfer suggests ICE transfer regulation is constantly evolving. Pangenome-wide association analysis of the ICE identified genes potentially involved in symbiosis, rhizosphere colonisation and/or adaptation to distinct legume hosts. In summary, the Mesorhizobium genus has accumulated a large and dynamic pangenome that evolves through ongoing horizontal gene transfer of large conjugative elements related to ICEMlSymR7A.

ViMIC: a database of human disease-related virus mutations, integration sites and cis-effects

Molecular mechanisms of virus-related diseases involve multiple factors, including viral mutation accumulation and integration of a viral genome into the host DNA. With increasing attention being paid to virus-mediated pathogenesis and the development of many useful technologies to identify virus mutations (VMs) and viral integration sites (VISs), much research on these topics is available in PubMed. However, knowledge of VMs and VISs is widely scattered in numerous published papers which lack standardization, integration and curation. To address these challenges, we built a pilot database of human disease-related Virus Mutations, Integration sites and Cis-effects (ViMIC), which specializes in three features: virus mutation sites, viral integration sites and target genes. In total, the ViMIC provides information on 31 712 VMs entries, 105 624 VISs, 16 310 viral target genes and 1 110 015 virus sequences of eight viruses in 77 human diseases obtained from the public domain. Furthermore, in ViMIC users are allowed to explore the cis-effects of virus-host interactions by surveying 78 histone modifications, binding of 1358 transcription regulators and chromatin accessibility on these VISs. We believe ViMIC will become a valuable resource for the virus research community. The database is available at http://bmtongji.cn/ViMIC/index.php.