Integration of retroviral DNA into host cell DNA is a defining feature of retroviral replication. HIV integration is known to be favored in active transcription units, which promotes efficient ...transcription of the viral genes, but the molecular mechanisms responsible for targeting are not fully clarified. Here we used pyrosequencing to map 40,569 unique sites of HIV integration. Computational prediction of nucleosome positions in target DNA indicated that integration sites are periodically distributed on the nucleosome surface, consistent with favored integration into outward-facing DNA major grooves in chromatin. Analysis of integration site positions in the densely annotated ENCODE regions revealed a wealth of new associations between integration frequency and genomic features. Integration was particularly favored near transcription-associated histone modifications, including H3 acetylation, H4 acetylation, and H3 K4 methylation, but was disfavored in regions rich in transcription-inhibiting modifications, which include H3 K27 trimethylation and DNA CpG methylation. Statistical modeling indicated that effects of histone modification on HIV integration were partially independent of other genomic features influencing integration. The pyrosequencing and bioinformatic methods described here should be useful for investigating many aspects of retroviral DNA integration.
Unravelling HIV-1 Latency, One Cell at a Time Kok, Yik Lim; Ciuffi, Angela; Metzner, Karin J.
Trends in microbiology (Regular ed.),
November 2017, 2017-11-00, 20171101, Letnik:
25, Številka:
11
Journal Article
Recenzirano
A single virus is capable of infecting and replicating in a single cell. Recent advances across single-cell omics technologies – genomics, epigenomics, transcriptomics, epitranscriptomics, ...proteomics, and metabolomics – will offer unprecedented opportunities to gain more insights into the various aspects of the life cycle of viruses and their impact on the host cell. Here, using the human immunodeficiency virus type 1 (HIV-1) as an example, we summarize the current knowledge and the future potential of single-cell omics in the investigation of an important aspect of the life cycle of HIV-1 that represents a major hurdle in achieving viral eradication, HIV-1 latency.
HIV-1 latency-associated determinants show certain tendencies at the cell population level.
The examination of HIV-1 latency-associated determinants at single-cell resolution is emerging owing to the recent identification of HIV-1 latency biomarkers and development of single-cell omics technologies.
Single-cell omics studies could define specific signature interactions between biomolecules (DNA, RNA, and/or protein) that govern HIV-1 latency, contributing towards curative efforts.
Rift Valley fever virus (RVFV) (family
) can cause severe disease, and outbreaks of this mosquito-borne pathogen pose a significant threat to public and animal health. Yet many molecular aspects of ...RVFV pathogenesis remain incompletely understood. Natural RVFV infections are acute, characterized by a rapid onset of peak viremia during the first days post-infection, followed by a rapid decline. Although
studies identified a major role of interferon (IFN) responses in counteracting the infection, a comprehensive overview of the specific host factors that play a role in RVFV pathogenesis
is still lacking. Here, the host
transcriptional profiles in the liver and spleen tissues of lambs exposed to RVFV are studied using RNA sequencing (RNA-seq) technology. We validate that IFN-mediated pathways are robustly activated in response to infection. We also link the observed hepatocellular necrosis with severely compromised organ function, which is reflected as a marked downregulation of multiple metabolic enzymes essential for homeostasis. Furthermore, we associate the elevated basal expression of
in the liver with RVFV tissue tropism. Collectively, the results of this study deepen the knowledge of the
host response during RVFV infection and reveal new insights into the gene regulation networks underlying pathogenesis in a natural host.
Rift Valley fever virus (RVFV) is a mosquito-transmitted pathogen capable of causing severe disease in animals and humans. Outbreaks of RVFV pose a significant threat to public health and can result in substantial economic losses. Little is known about the molecular basis of RVFV pathogenesis
, particularly in its natural hosts. We employed RNA-seq technology to investigate genome-wide host responses in the liver and spleen of lambs during acute RVFV infection. We show that RVFV infection drastically decreases the expression of metabolic enzymes, which impairs normal liver function. Moreover, we highlight that basal expression levels of the host factor
may be a determinant of RVFV tissue tropism. This study links the typical pathological phenotype induced by RVFV infection with tissue-specific gene expression profiles, thereby improving our understanding of RVFV pathogenesis.
The study of RNA modifications, today known as epitranscriptomics, is of growing interest. The N6-methyladenosine (m
6
A) and 5-methylcytosine (m
5
C) RNA modifications are abundantly present on mRNA ...molecules, and impact RNA interactions with other proteins or molecules, thereby affecting cellular processes, such as RNA splicing, export, stability, and translation. Recently m
6
A and m
5
C marks were found to be present on human immunodeficiency (HIV) transcripts as well and affect viral replication. Therefore, the discovery of RNA methylation provides a new layer of regulation of HIV expression and replication, and thus offers novel array of opportunities to inhibit replication. However, no study has been performed to date to investigate the impact of HIV replication on the transcript methylation level in the infected cell. We used a productive HIV infection model, consisting of the CD4+ SupT1 T cell line infected with a VSV-G pseudotyped HIVeGFP-based vector, to explore the temporal landscape of m
6
A and m
5
C epitranscriptomic marks upon HIV infection, and to compare it to mock-treated cells. Cells were collected at 12, 24, and 36 h post-infection for mRNA extraction and FACS analysis. M
6
A RNA modifications were investigated by methylated RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-Seq). M
5
C RNA modifications were investigated using a bisulfite conversion approach followed by high-throughput sequencing (BS-Seq). Our data suggest that HIV infection impacted the methylation landscape of HIV-infected cells, inducing mostly increased methylation of cellular transcripts upon infection. Indeed, differential methylation (DM) analysis identified 59 m
6
A hypermethylated and only 2 hypomethylated transcripts and 14 m
5
C hypermethylated transcripts and 7 hypomethylated ones. All data and analyses are also freely accessible on an interactive web resource (
http://sib-pc17.unil.ch/HIVmain.html
). Furthermore, both m
6
A and m
5
C methylations were detected on viral transcripts and viral particle RNA genomes, as previously described, but additional patterns were identified. This work used differential epitranscriptomic analysis to identify novel players involved in HIV life cycle, thereby providing innovative opportunities for HIV regulation.
One of the seven key scientific priorities identified in the road map on HIV cure research is to 'determine the host mechanisms that control HIV replication in the absence of therapy'. This review ...summarizes the recent work in genomics and in epigenetic control of viral replication that is relevant for this mission.
New technologies allow the joint analysis of host and viral transcripts. They identify the patterns of antisense transcription of the viral genome and its role in gene regulation. High-throughput studies facilitate the assessment of integration at the genome scale. Integration site, orientation and host genomic context modulate the transcription and should also be assessed at the level of single cells. The various models of latency in primary cells can be followed using dynamic study designs to acquire transcriptome and proteome data of the process of entry, maintenance and reactivation of latency. Dynamic studies can be applied to the study of transcription factors and chromatin modifications in latency and upon reactivation.
The convergence of primary cell models of latency, new high-throughput quantitative technologies applied to the study of time series and the identification of compounds that reactivate viral transcription bring unprecedented precision to the study of viral latency.
To replicate, a retrovirus must integrate a DNA copy of its RNA genome into a chromosome of the host cell. Integration is not random in the host genome but favors particular regions, and preferences ...differ among retroviruses. Several mechanisms might play a part in this favored integration targeting: (i) open chromatin might be preferentially accessible for viral DNA integration; (ii) DNA replication during cell division might facilitate access of integration complexes to favored sites; and (iii) cellular proteins bound to the host chromosome might tether integration complexes to favored regions. This review summarizes recent advances in understanding the mechanisms of retroviral integration, focusing on LEDGF/p75 – the first cellular protein shown to have a role in directing HIV DNA integration. Studies on LEDGF/p75 indicate that it directs HIV integration site selection by a tethering interaction, whereas the chromatin accessibility or cell cycle models are less well supported. Understanding viral integration will help improve the safety of retrovirus-based vectors used in gene therapy.
Studying the effects of HIV infection on the host transcriptome has typically focused on protein-coding genes. However, recent advances in the field of RNA sequencing revealed that long non-coding ...RNAs (lncRNAs) add an extensive additional layer to the cell's molecular network. Here, we performed transcriptome profiling throughout a primary HIV infection in vitro to investigate lncRNA expression at the different HIV replication cycle processes (reverse transcription, integration and particle production). Subsequently, guilt-by-association, transcription factor and co-expression analysis were performed to infer biological roles for the lncRNAs identified in the HIV-host interplay. Many lncRNAs were suggested to play a role in mechanisms relying on proteasomal and ubiquitination pathways, apoptosis, DNA damage responses and cell cycle regulation. Through transcription factor binding analysis, we found that lncRNAs display a distinct transcriptional regulation profile as compared to protein coding mRNAs, suggesting that mRNAs and lncRNAs are independently modulated. In addition, we identified five differentially expressed lncRNA-mRNA pairs with mRNA involvement in HIV pathogenesis with possible cis regulatory lncRNAs that control nearby mRNA expression and function. Altogether, the present study demonstrates that lncRNAs add a new dimension to the HIV-host interplay and should be further investigated as they may represent targets for controlling HIV replication.
In this scoping review, we offer a comprehensive understanding of the current and recent epidemiology, challenges, and emerging issues related to bacterial sexually transmitted infections (STIs) in ...the WHO European Region. We endeavour in collating data from both EU/EEA and non- EU/EEA countries, thereby giving a complete picture of the region which highlights the higher notification rates in Northern and Western countries than other regions, likely due to differences in testing, access to testing, and surveillance capacity. We provide an up-to-date review on the current knowledge of determinants and persistent inequities in key populations as well as the use of molecular epidemiology for identifying transmission networks in gonorrhoea and syphilis, and detecting chlamydia mutations that evade molecular diagnosis. Finally, we explore the emerging STIs in the region and the evolving transmission routes of food and waterborne diseases into sexual transmission. Our findings call for harmonized STI surveillance systems, proactive strategies, and policies to address social factors, and staying vigilant for emerging STIs.
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