19 research outputs found
Heterogeneous loss of HIV transcription and proviral DNA from 8E5/LAV lymphoblastic leukemia cells revealed by RNA FISH:FLOW analyses
8E5/LAV cells harbor a single HIV provirus, and are used frequently to generate standards for HIV genome quantification. Using flow cytometry-based in situ mRNA hybridization validated by qPCR, we find that different batches of 8E5 cells contain varying numbers of cells lacking viral mRNA and/or viral genomes. These findings raise concerns for studies employing 8E5 cells for quantitation, and highlight the value of mRNA FISH and flow cytometry in the detection and enumeration of HIV-positive cells
The Left-Right Pitx2 Pathway Drives Organ-Specific Arterial and Lymphatic Development in the Intestine
SummaryThe dorsal mesentery (DM) is the major conduit for blood and lymphatic vessels in the gut. The mechanisms underlying their morphogenesis are challenging to study and remain unknown. Here we show that arteriogenesis in the DM begins during gut rotation and proceeds strictly on the left side, dependent on the Pitx2 target gene Cxcl12. Although competent Cxcr4-positive angioblasts are present on the right, they fail to form vessels and progressively emigrate. Surprisingly, gut lymphatics also initiate in the left DM and arise only after—and dependent on—arteriogenesis, implicating arteries as drivers of gut lymphangiogenesis. Our data begin to unravel the origin of two distinct vascular systems and demonstrate how early left-right molecular asymmetries are translated into organ-specific vascular patterns. We propose a dual origin of gut lymphangiogenesis in which prior arterial growth is required to initiate local lymphatics that only subsequently connect to the vascular system
TZM-gfp cells: a tractable fluorescent tool for analysis of rare and early HIV-1 infection
Here we describe TZM-gfp, a novel HIV-1 reporter cell derived from the same parental clone JC.53, used previously to generate the widely-utilized indicator cell line TZM-bl. We re-engineered JC.53 cells to express GFP under regulation of HIV Tat and Rev. We characterize the new reporter cell line to show that TZM-gfp cells are equally susceptible to HIV infection, exhibit minimal background signal, and can report HIV infection in rare cells from a bulk population of experimentally-infected human monocyte-derived macrophages. We demonstrate the utility and sensitivity of the cells in detection of even a single HIV-positive macrophage by fluorescence-assisted correlative electron microscopy, using the GFP signal to guide imaging of HIV virions in primary co-culture. Finally, we used TZM-gfp cells for viral capture during co-culture with human peripheral blood mononuclear cells, showing that TZM-gfp can support outgrowth and analyses of patient-derived primary HIV-1 isolates
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A novel, sensitive dual-indicator cell line for detection and quantification of inducible, replication-competent latent HIV-1 from reservoir cells
Funder: DH | National Institute for Health Research (NIHR); doi: https://doi.org/10.13039/501100000272Funder: U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)Abstract: Understanding the mechanisms involved in HIV infection and latency, and development of a cure, rely on the availability of sensitive research tools such as indicator cells, which allow rigorous quantification of viral activity. Here we describe the construction and validation of a novel dual-indicator cell line, Sup-GGR, which offers two different readouts to quantify viral replication. A construct expressing both Gaussia luciferase and hrGFP in a Tat- and Rev-dependent manner was engineered into SupT1-CCR5 to create Sup-GGR cells. This cell line supports the replication of both X4 and R5-tropic HIV as efficiently as its parental cell line, SupT1-CCR5, and allows repeated sampling without the need to terminate the culture. Sup-GGR demonstrates comparable sensitivity and similar kinetics in virus outgrowth assays (VOA) to SupT1-CCR5 using clinical samples. However the Gaussia luciferase reporter is significantly less labor-intensive and allows earlier detection of reactivated latent viruses compared to the conventional HIV p24 ELISA assay. The Sup-GGR cell line constitutes a versatile new tool for HIV research and clinical trials
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A novel, sensitive dual-indicator cell line for detection and quantification of inducible, replication-competent latent HIV-1 from reservoir cells
Funder: DH | National Institute for Health Research (NIHR); doi: https://doi.org/10.13039/501100000272Funder: U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)Abstract: Understanding the mechanisms involved in HIV infection and latency, and development of a cure, rely on the availability of sensitive research tools such as indicator cells, which allow rigorous quantification of viral activity. Here we describe the construction and validation of a novel dual-indicator cell line, Sup-GGR, which offers two different readouts to quantify viral replication. A construct expressing both Gaussia luciferase and hrGFP in a Tat- and Rev-dependent manner was engineered into SupT1-CCR5 to create Sup-GGR cells. This cell line supports the replication of both X4 and R5-tropic HIV as efficiently as its parental cell line, SupT1-CCR5, and allows repeated sampling without the need to terminate the culture. Sup-GGR demonstrates comparable sensitivity and similar kinetics in virus outgrowth assays (VOA) to SupT1-CCR5 using clinical samples. However the Gaussia luciferase reporter is significantly less labor-intensive and allows earlier detection of reactivated latent viruses compared to the conventional HIV p24 ELISA assay. The Sup-GGR cell line constitutes a versatile new tool for HIV research and clinical trials
Heterogeneous loss of HIV transcription and proviral DNA from 8E5/LAV lymphoblastic leukemia cells revealed by RNA FISH:FLOW analyses
EVOLVED PLATFORMS FOR VIRAL OUTGROWTH AND MOLECULAR CHARACTERIZATION OF HIV WITHIN TISSUE RESERVOIRS
HIV persists during long-term, effective antiretroviral therapy within reservoir cells. While CD4-positive lymphocytes are accepted as bona fide reservoirs, the contribution of other HIV-susceptible cell types to viral persistence remains unknown. ART therapy is a lifelong commitment for those with HIV, as treatment interruption induces swift rebound of peripheral viremia and progression toward AIDS. This demonstrates that persistent HIV reservoir cells are not eliminated by the virus or cleared by the immune system. However, while the memory T-cell HIV reservoir was discovered twenty years ago, strategies to specifically target these cells for destruction remain elusive. Furthermore, we know far less about HIV- susceptible cells within tissues than those in the blood, including subsets of myeloid cells such as tissue macrophages. Indeed, challenges in the study and isolation of tissue macrophages and the sheer number of organ-specific myeloid subsets has complicated unified efforts to learn whether these cells harbor HIV during treatment. Here I present a series of experimental platforms for the study of HIV tissue reservoirs, with a focus on pulmonary alveolar macrophages of the human lung. To learn whether these cells are HIV-infected in vivo, I describe a flow-cytometry assay using fluorescence RNA in situ hybridization (FISH) that reveals individual HIV- infected primary cells in a cell suspension. These assays are amenable to multiplex HIV antigen staining and sorting of HIV-expressing cells for RNAseq analyses. Using this FISH:FLOW tool I demonstrate a universal caveat in the industry standard cell line 8E5, used routinely as a calibration standard for clinical measurements of the HIV reservoir. Secondly, I engineered several reporter cell platforms for the outgrowth of HIV from tissue reservoirs, and use these tools to develop novel paradigms in the study of HIV replication and cellular transmission in myeloid cells. Finally, I deploy some of these cell lines in a combined HIV outgrowth pipeline to capture and characterize Clade C HIV in the blood and lungs of adults in Blantyre, Malawi. Together, these contributions will accelerate our progress toward therapies that specifically target HIV reservoir cells in the tissues, the key obstacle to an HIV cure
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A novel, sensitive dual-indicator cell line for detection and quantification of inducible, replication-competent latent HIV-1 from reservoir cells.
Understanding the mechanisms involved in HIV infection and latency, and development of a cure, rely on the availability of sensitive research tools such as indicator cells, which allow rigorous quantification of viral activity. Here we describe the construction and validation of a novel dual-indicator cell line, Sup-GGR, which offers two different readouts to quantify viral replication. A construct expressing both Gaussia luciferase and hrGFP in a Tat- and Rev-dependent manner was engineered into SupT1-CCR5 to create Sup-GGR cells. This cell line supports the replication of both X4 and R5-tropic HIV as efficiently as its parental cell line, SupT1-CCR5, and allows repeated sampling without the need to terminate the culture. Sup-GGR demonstrates comparable sensitivity and similar kinetics in virus outgrowth assays (VOA) to SupT1-CCR5 using clinical samples. However the Gaussia luciferase reporter is significantly less labor-intensive and allows earlier detection of reactivated latent viruses compared to the conventional HIV p24 ELISA assay. The Sup-GGR cell line constitutes a versatile new tool for HIV research and clinical trials.National Institutes for Health Research, UK (NIHR, Cambridge Biomedical Research Centres), the Cambridge Clinical Academic Reserve and the Medical Research Council, UK (MR/N02043X/1) . This work was also supported by the National Institute of Health (AI118582 and AI36097)
Recommended from our members
A novel, sensitive dual-indicator cell line for detection and quantification of inducible, replication-competent latent HIV-1 from reservoir cells.
Understanding the mechanisms involved in HIV infection and latency, and development of a cure, rely on the availability of sensitive research tools such as indicator cells, which allow rigorous quantification of viral activity. Here we describe the construction and validation of a novel dual-indicator cell line, Sup-GGR, which offers two different readouts to quantify viral replication. A construct expressing both Gaussia luciferase and hrGFP in a Tat- and Rev-dependent manner was engineered into SupT1-CCR5 to create Sup-GGR cells. This cell line supports the replication of both X4 and R5-tropic HIV as efficiently as its parental cell line, SupT1-CCR5, and allows repeated sampling without the need to terminate the culture. Sup-GGR demonstrates comparable sensitivity and similar kinetics in virus outgrowth assays (VOA) to SupT1-CCR5 using clinical samples. However the Gaussia luciferase reporter is significantly less labor-intensive and allows earlier detection of reactivated latent viruses compared to the conventional HIV p24 ELISA assay. The Sup-GGR cell line constitutes a versatile new tool for HIV research and clinical trials
Inhibition of the lncRNA SAF drives activation of apoptotic effector caspases in HIV-1-infected human macrophages.
Long noncoding RNAs (lncRNAs) impart significant regulatory functions in a diverse array of biological pathways and manipulation of these RNAs provides an important avenue to modulate such pathways, particularly in disease. Our knowledge about lncRNAs' role in determination of cellular fate during HIV-1 infection remains sparse. Here, we have identified the impact of the lncRNA SAF in regulating apoptotic effector caspases in macrophages, a long-lived cellular reservoir of HIV-1, that are largely immune to virus-induced cell death. Expression of SAF is significantly up-regulated in HIV-1-infected human monocyte-derived macrophages (MDM) compared with bystander and virus-nonexposed cells. A similar enhancement in SAF RNA expression is also detected in the HIV-1-infected airway macrophages obtained by bronchoalveolar lavage of HIV-1-infected individuals. Down-regulation of SAF with siRNA treatment increases caspase-3/7 activity levels in virus-infected MDMs. This induction of apoptotic caspases occurs exclusively in HIV-1-infected macrophages and not in bystander cells, leading to a significant reduction in HIV-1 replication and overall viral burden in the macrophage culture. This study identifies targeting of the lncRNA SAF as a potential means to specifically induce cell death in HIV-1-infected macrophages