Abstract The stably integrated pool of HIV-1 proviruses in the host genome stands against curative strategies. This reservoir is extremely heterogeneous with respect to host cell type, anatomical location, integration site, and replication fitness. During the initial phase of infection, only a few infected cells can resist host immune clearance or cytopathic effect and establish this resistant pool. The mechanisms underlying HIV latency initiation are not fully resolved yet. In the current study, we propose and validate a new reporter model for monitoring HIV-1 provirus silencing and reactivation using Timer of cell kinetics and activity (Tocky). HIV-Tocky system uses a fluorescent Timer protein whose emission spectrum spontaneously shifts from blue to red to reveal HIV-1 provirus dynamics. We dissected provirus transcriptional phases into early, persistent, recently silenced, and latent. To our knowledge, this is the first report to distinguish two latent subsets: a directly non-expressing population and a recently silenced after brief expression. In-depth integration site analysis suggested that the distribution of proviruses in directly latent cells was similar to that in actively transcribing cell population, whereas recently silenced cells tended to harbor proviruses integrated into heterochromatin. Furthermore, we established a library of various single integration clones at which we utilized to demonstrate the efficiency of the block-and-lock strategy by capturing the fast dynamics of silencing that were overlooked in previous models. In summary, we propose HIV-Tocky system to serve as a time-sensitive model that can capture the dynamics of provirus expression, making it a useful tool for HIV latency research. Significance Statement Determinants of HIV-1 latency establishment are yet to be elucidated. This reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allowed; for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies. Classification Biological Sciences, Microbiology.
Background: The incidence of non-B non-C hepatocellular carcinoma (NBNC-HCC), which is negative for hepatitis B surface antigen and hepatitis C virus antibodies, is on the rise. Relatively high numbers of NBNC-HCC patients are hepatitis B core antibody (HBcAb) positive, suggesting that previous HBV infection may play a role in NBNC-HCC development, though the exact mechanisms are unclear. This study aimed to investigate whether HBV genomes are integrated into the host genome of HBcAb-positive NBNC-HCC cases and how these integrations may contribute to cancer development and progression. Methods: HBV detection PCR using HBV-specific primers on DNA extracted from HBcAb-positive NBNC-HCC tissue samples was performed. Positive samples were further examined for HBV integration sites using viral DNA-capture sequencing. Additionally, hepatitis B core-related antigen (HBcrAg) serum levels were measured to assess whether they could be predictive for HBV detection PCR results. Results: Among 90 HBcAb-positive NBNC-HCC samples, HBV genome amplification was detected in 18 samples, and elevated HBcrAg levels were associated with the HBV detection PCR results. Seventeen of these samples exhibited HBV integration. The HBV genome was integrated near the TERT gene in 7 samples, resulting in significantly increased TERT mRNA levels; in the KMT2B gene (2 samples); and downstream of LOC441666 (2 samples). Conclusion: The integration sites we identified in our samples have been previously reported in HBV-related HCC, suggesting that HBV integration may also contribute to hepatocarcinogenesis in HBcAb-positive NBNC-HCC. Furthermore, HBcrAg could serve as a potential, noninvasive marker for detecting HBV integration in these cases.
Virus-related cancers are malignancies caused by specific viruses, such as human papillomavirus (HPV), hepatitis B virus, and human T-cell leukemia virus, contributing significantly to the global cancer burden through persistent infection and oncogenic transformation. The current study aimed to develop a robust HPV-16 detection method for formalin-fixed cancer specimens. To prevent false negatives resulting from DNA fragmentation, a DNA quality check step was added. Additionally, this study used multiplex polymerase chain reaction (PCR) covering the entire HPV-16 genome to mitigate effects caused by viral sequence variation. To prove this concept, we analyzed genomic DNA extracted from oropharyngeal cancer tissues known as HPV-16-positive. Subsequently, the protocol was tested on oral squamous cell carcinoma (OSCC) samples in our cohort. Given the wide variation in HPV-16 positivity in previous studies, it remains elusive how frequently HPV-16 is positive in OSCC. The results showed faint bands or smears in the multiplex PCR of 7 out of 112 cases. Droplet digital PCR confirmed variable positivity levels of HPV-16, suggesting two scenarios of HPV-16 positivity in cancer tissue: cancer cells derived from infected cells or only a portion being HPV-16-positive. Finally, we comprehensively analyzed the case and identified the integration of a deleted HPV-16 genome into the intronic region of the host gene TMEM94 on chromosome 17. To the best of our knowledge, this is the first evidence showing the integration of HPV-16 in OSCC cells and providing its complete viral sequence. The established protocol should be applicable to various cancer tissues for analyzing the association with HPV-16 infection.
Abstract Determinants of HIV-1 latency establishment are yet to be elucidated. HIV reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model; HIV-Timer of cell kinetics and activity (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allowed; for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies.
Determinants of HIV-1 latency establishment are yet to be elucidated. HIV reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model, HIV-Timer of cell kinetics and activity (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allows, for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies.
ABSTRACT Bovine leukemia virus (BLV), a retrovirus, causes Enzootic Bovine Leukosis (EBL) in cattle following a latent infection period. The BLV infection results in polyclonal expansion of infected B-lymphocytes and ∼5% of infected cattle develop monoclonal leukosis. Since the clonal expansion of virus-infected cell is a key in the pathogenesis of EBL, assessing the clonality of malignant cells is crucial for both understanding viral pathogenesis, which might be useful for EBL diagnosis. For the investigation of clonality of BLV-infected cells in non-EBL and EBL cattle, two methods were used to evaluate the status of EBL; BLV-DNA-capture-seq method with high sensitivity and specificity and simple and cost-effective Rapid Amplification of Integration Site for BLV (BLV-RAIS) method. We found that the RAIS method efficiently detect expanded clone in EBL tissue sample as BLV-DNA-capture-seq method. Taking advantage of high frequency of BLV-infected cells in blood, we simplified RAIS method and showed that similar to BLV-DNA-capture-seq, this method could reliably provide quantitative value about clonal abundance of BLV-infected cells. Next, we aimed to establish a diagnostic blood test for EBL by using the clonality information. First, we compared clonality of BLV-infected cells in blood with that in tumor tissue in EBL cattle. There was a remarkably similar clonality between blood and tissue in each animal. Furthermore, BLV integration site information clearly showed that the same clone was the most expanded in both blood and tumor tissue, indicating that tumor cells were circulating in blood in the disease cattle. We also analyzed tumor tissue at two independent anatomical regions and found the same clones was most expanded in both regions, supporting the idea that tumor cells are systemically circulating in the diseased cattle. Finally, we compared clonality value between non-EBL and EBL cattle by using BLV-RAIS method and found that there was clear difference between non-EBL and EBL. More importantly, we found that clonality value was low in asymptomatic phase but high in EBL phase in the longitudinal cohort study. These findings have demonstrated that BLV integration site and clonality value are is a useful information to establish diagnostic blood test for EBL. That would contribute to reduction of economic damage caused by EBL and improvement of productivity in cattle industry.
Although BLV has been eradicated in some European countries, BLV is still endemic in other countries, including Japan and the United States. EBL causes huge economic damage to the cattle industry.
Abstract High transmissible viruses including SARS-CoV-2 frequently accumulate novel mutations that are detected via high-throughput sequencing. However, there is a need to develop an alternative rapid and non-expensive approach. Here we developed a novel multiplex DNA detection method Intelli-OVI for analysing existing and novel mutations of SARS-CoV-2. Intelli-OVI uses the micro-disc-based method IntelliPlex and computational algorithms of objective variant identification (OVI). IntelliPlex uses micro-discs printed with a unique pictorial pattern as a labelling conjugate for DNA probes, and OVI allows simultaneous identification of several variants using multidimensional data obtained by the IntelliPlex method. Importantly, de novo mutations can be identified by decreased signals, which prompts the need to design additional primers and probes. Thus, Intelli-OVI can be upgraded to keep up with rapidly evolving viruses. We believe that Intelli-OVI will be useful in establishing a world better equipped to tackle emerging novel pathogens.
Highly transmissible viruses including SARS-CoV-2 frequently accumulate novel mutations that are detected via high-throughput sequencing. However, there is a need to develop an alternative rapid and non-expensive approach. Here we developed a novel multiplex DNA detection method Intelli-OVI for analysing existing and novel mutations of SARS-CoV-2.
Abstract Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL), a cancer of infected CD4 + T-cells. There is both sense and antisense transcription from the integrated provirus. Sense transcription tends to be suppressed, but antisense transcription is constitutively active. Various efforts have been made to elucidate the regulatory mechanism of HTLV-1 provirus for several decades; however, it remains unknown how HTLV-1 antisense transcription is maintained. Here, using proviral DNA-capture sequencing, we found a previously unidentified viral enhancer in the middle of the HTLV-1 provirus. The transcription factors, SRF and ELK-1, play a pivotal role in the activity of this enhancer. Aberrant transcription of genes in the proximity of integration sites was observed in freshly isolated ATL cells. This finding resolves certain long-standing questions concerning HTLV-1 persistence and pathogenesis. We anticipate that the DNA-capture-seq approach can be applied to analyze the regulatory mechanisms of other oncogenic viruses integrated into the host cellular genome.
