HIV-Tocky system to visualize proviral expression dynamics.

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.

Assessing the effects of antiretroviral therapy-latency-reversing agent combination therapy on eradicating replication-competent HIV provirus in a Jurkat cell culture model.

Eradication of HIV-1 latently infected cells is an important issue in HIV treatment. However, there are limited models available to assess therapeutic efficacy in vitro. Here, we present a protocol for establishing a variety of HIV-infected Jurkat cells, including productive and latent status, evaluating the efficacy of antiviral agents, followed by PCR/sequencing-based detection of replication competent HIV provirus. This protocol is useful for optimization of treatment of HIV-1 and provides insights into the mechanisms of clonal selection of heterogeneous HIV-1-infected cells. For complete details on the use and execution of this protocol, please refer to Matsuda et al. (2021).1.

Clone Dynamics and Its Application for the Diagnosis of Enzootic Bovine Leukosis.

Bovine leukemia virus (BLV) infection results in polyclonal expansion of infected B lymphocytes, and ~5% of infected cattle develop enzootic bovine leukosis (EBL). Since BLV is a retrovirus, each individual clone can be identified by using viral integration sites. To investigate the distribution of tumor cells in EBL cattle, we performed viral integration site analysis by using a viral DNA capture-sequencing method. We found that the same tumor clones existed in peripheral blood, with a dominance similar to that in lymphoma tissue. Additionally, we observed that multiple tumor tissues from different sites harbored the identical clones, indicating that tumor cells can circulate and distribute systematically in EBL cattle. To investigate clonal expansion of BLV-infected cells during a long latent period, we collected peripheral blood samples from asymptomatic cattle every 2 years, among which several cattle developed EBL. We found that no detectable EBL clone existed before the diagnosis of EBL in some cases; in the other cases, clones that were later detected as malignant clones at the EBL stage were present several months or even years before the disease onset. To establish a feasible clonality-based method for the diagnosis of EBL, we simplified a quick and cost-effective method, namely, rapid amplification of integration sites for BLV infection (BLV-RAIS). We found that the clonality values (Cvs) were well correlated between the BLV-RAIS and viral DNA capture-sequencing methods. Furthermore, receiver operating characteristic (ROC) curve analysis identified an optimal Cv cutoff value of 0.4 for EBL diagnosis, with excellent diagnostic sensitivity (94%) and specificity (100%). These results indicated that the RAIS method efficiently and reliably detected expanded clones not only in lymphoma tissue but also in peripheral blood. Overall, our findings elucidated the clonal dynamics of BLV- infected cells during EBL development. In addition, Cvs of BLV-infected cells in blood can be used to establish a valid and noninvasive diagnostic test for potential EBL onset. IMPORTANCE 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. However, there are no effective drugs or vaccines to control BLV infection and related diseases. The strategy of eradication of infected cattle is not practical due to the high endemicity of BLV. Furthermore, how BLV-infected B cell clones proliferate during oncogenesis and their distribution in EBL cattle have yet to be elucidated. Here, we provided evidence that tumor cells are circulating in the blood of diseased cattle. Thus, the Cv of virus-infected cells in blood is useful information for the evaluation of the disease status. The BLV-RAIS method provides quantitative and accurate clonality information and therefore is a promising method for the diagnosis of EBL.

Single-Cell Transcriptome Analysis of Treg.

Regulatory T-cells (Treg) are considerably heterogeneous. Thymically derived Treg (tTreg) are those, which differentiate in the thymus, while peripherally derived Treg (pTreg) differentiate from peripheral mature CD4+ T-cells. These two populations are often identified using markers such as neuropilin-1 and Helios (for tTreg) and ROR-γt (for pTreg) in intestines (Tanoue et al., Nat Rev Immunol 16:295-309, 2016).

Identification and characterization of a novel enhancer in the HTLV-1 proviral genome.

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.

Multiomics Investigation Revealing the Characteristics of HIV-1-Infected Cells In Vivo.

For eradication of HIV-1 infection, it is important to elucidate the detailed features and heterogeneity of HIV-1-infected cells in vivo. To reveal multiple characteristics of HIV-1-producing cells in vivo, we use a hematopoietic-stem-cell-transplanted humanized mouse model infected with GFP-encoding replication-competent HIV-1. We perform multiomics experiments using recently developed technology to identify the features of HIV-1-infected cells. Genome-wide HIV-1 integration-site analysis reveals that productive HIV-1 infection tends to occur in cells with viral integration into transcriptionally active genomic regions. Bulk transcriptome analysis reveals that a high level of viral mRNA is transcribed in HIV-1-infected cells. Moreover, single-cell transcriptome analysis shows the heterogeneity of HIV-1-infected cells, including CXCL13high cells and a subpopulation with low expression of interferon-stimulated genes, which can contribute to efficient viral spread in vivo. Our findings describe multiple characteristics of HIV-1-producing cells in vivo, which could provide clues for the development of an HIV-1 cure.

The Nature of the HTLV-1 Provirus in Naturally Infected Individuals Analyzed by the Viral DNA-Capture-Seq Approach.

The retrovirus human T-cell leukemia virus type 1 (HTLV-1) integrates into the host DNA, achieves persistent infection, and induces human diseases. Here, we demonstrate that viral DNA-capture sequencing (DNA-capture-seq) is useful to characterize HTLV-1 proviruses in naturally virus-infected individuals, providing comprehensive information about the proviral structure and the viral integration site. We analyzed peripheral blood from 98 naturally HTLV-1-infected individuals and found that defective proviruses were present not only in patients with leukemia, but also in those with other clinical entities. We further demonstrated that clones with defective-type proviruses exhibited a higher degree of clonal abundance than those with full-length proviruses. The frequency of defective-type proviruses in HTLV-1-infected humanized mice was lower than that in infected individuals, indicating that defective proviruses were rare at the initial phase of infection but preferentially selected during persistent infection. These results demonstrate the robustness of viral DNA-capture-seq for HTLV-1 infection and suggest potential applications for other virus-associated cancers in humans.

Genome Editing: A Comparative Study on the Efficiency of CRISPR/Cas9 Nuclease Versus Nickase Using HIV as a Model System

@#Introduction: CRISPR/Cas9 nuclease has gained popularity as a genome editing tool due to its straight-forward mechanism. However, there are concerns that CRISPR nuclease would cause off-target and toxicity. The CRISPR/ Cas9 D10A nickase was designed to enhance genome editing. Nevertheless, this raised the question of whether the efficiency of nickase is compromised compared to CRISPR/Cas9 nuclease. Targeting HIV genes, we investigated if CRISPR nuclease performed better than the nickase in efficacy and safety. Methods: CRISPR nucleases and nickases were designed to target Gag, Pol, Rev, Vif, Tat and LTR. HIV latently infected cell line, ACH-2, was transfected with the nucleases and nickases. Changes to viral load after CRISPR treatment was measured using p24 ELISA. Safety of nuclease and nickase was monitored using GFP expression with fluorescence microscopy and flow cytometry. Targeting two sites within the same gene, and targeting multiple genes concurrently were also studied to determine efficacy of CRISPR in reducing viral load. Results: A 44.9 to 68.1% and a 34.4 to 49.7% decrease in viral load was seen in CRISPR nuclease and nickase respectively. Microscopy and flow cytometry results showed that the nickase system was slightly toxic with a 0.31 to 0.7-fold cell death. There was a 34% decrease in viral load when two sites were targeted within a gene, and the largest decrease was seen when all the nucleases were combined, giving a 75.4% decrease in viral load at day 5. Conclusion: The knowledge gained from this study will be employed to improve genome editing in other disease models.