The kinetics, mechanisms, and consequences of HTLV-1 plus-strand expression in naturally-infected T-cell clones

HTLV-1 replication requires the expression of plus-strand-encoded transcriptional transactivator protein Tax. However, Tax protein, a surrogate for HTLV-1 plus-strand expression is seldom detected in freshly isolated infected blood. The kinetics and consequences of plus-strand expression remain poorly understood. I used two fluorescent protein-based Tax reporter systems to study the dynamics and consequences of plus-strand expression and the changes to the host gene expression during plus-strand expression in naturally HTLV-1-infected, non-malignant T-cell clones. Time-lapse live-cell imaging followed by single-cell analysis of two T-cell clones stably transduced with a short-lived enhanced green fluorescent protein Tax reporter system identified five patterns of Tax expression in both clones and the distribution of these patterns was different between the two clones. Mathematical modelling of the experimental data revealed that the mean duration of Tax expression differed between the two clones – 94 and 417 hours, respectively. Host cell transcriptome analysis during successive stages of plus-strand strand expression using a fluorescent timer protein-based Tax reporter system in naturally-infected T-cell clones identified dysregulation in the expression of genes related to multiple cellular processes, including cell cycle, DNA damage response, and apoptosis at the initiation of the plus-strand transcriptional burst. The plus-strand expression showed immediate but transient adverse effects, including reduced proliferation, increased apoptosis, upregulation of a DNA damage marker, and impaired cell cycle progression. In the longer term, the immediate negative consequences of Tax expression were offset by reduced apoptosis and increased proliferation as cells terminated plus-strand expression. Plus-strand expression was also associated with cell-to-cell adhesion and reduced motility. These findings show within and between clone variability in the patterns and duration of HTLV-1 plus-strand expression, changes to the host gene expression during successive stages of the plus-strand expression, and the balance between the beneficial and adverse effects on the host cell associated with the plus-strand expression.Open Acces

Expression and reactivation of HIV in a chemokine induced model of HIV latency in primary resting CD4+ T cells

<p>Abstract</p> <p>Background</p> <p>We recently described that HIV latent infection can be established <it>in vitro </it>following incubation of resting CD4+ T-cells with chemokines that bind to CCR7. The main aim of this study was to fully define the post-integration blocks to virus replication in this model of CCL19-induced HIV latency.</p> <p>Results</p> <p>High levels of integrated HIV DNA but low production of reverse transcriptase (RT) was found in CCL19-treated CD4+ T-cells infected with either wild type (WT) NL4.3 or single round envelope deleted NL4.3 pseudotyped virus (NL4.3- Δenv). Supernatants from CCL19-treated cells infected with either WT NL4.3 or NL4.3- Δenv did not induce luciferase expression in TZM-bl cells, and there was no expression of intracellular p24. Following infection of CCL19-treated CD4+ T-cells with NL4.3 with enhanced green fluorescent protein (EGFP) inserted into the <it>nef </it>open reading frame (NL4.3- Δnef-EGFP), there was no EGFP expression detected. These data are consistent with non-productive latent infection of CCL19-treated infected CD4+ T-cells. Treatment of cells with phytohemagluttinin (PHA)/IL-2 or CCL19, prior to infection with WT NL4.3, resulted in a mean fold change in unspliced (US) RNA at day 4 compared to day 0 of 21.2 and 1.1 respectively (p = 0.01; n = 5), and the mean expression of multiply spliced (MS) RNA was 56,000, and 5,000 copies/million cells respectively (p = 0.01; n = 5). In CCL19-treated infected CD4+ T-cells, MS-RNA was detected in the nucleus and not in the cytoplasm; in contrast to PHA/IL-2 activated infected cells where MS RNA was detected in both. Virus could be recovered from CCL19-treated infected CD4+ T-cells following mitogen stimulation (with PHA and phorbyl myristate acetate (PMA)) as well as TNFα, IL-7, prostratin and vorinostat.</p> <p>Conclusions</p> <p>In this model of CCL19-induced HIV latency, we demonstrate HIV integration without spontaneous production of infectious virus, detection of MS RNA in the nucleus only, and the induction of virus production with multiple activating stimuli. These data are consistent with <it>ex vivo </it>findings from latently infected CD4+ T-cells from patients on combination antiretroviral therapy, and therefore provide further support of this model as an excellent <it>in vitro </it>model of HIV latency.</p

Management of emergencies in general practice: Role of general practitioners

Introduction: Management of emergencies is an integral part of primary care. Being first contact care providers general practitioners may encounter any type of emergency. Acute attacks of asthma, myocardial infarction, anaphylactic shock, hypoglycemic coma, convulsions, head injuries and trauma are some of the common emergencies encountered by GPs. Updated knowledge, communication and procedural skills, trained paramedical staff, necessary equipment and medications and appropriate practice organization are vital to provide optimum care which may even save lives of patients. The wide range of problems and the rarity of the problems make it difficult for primary care doctors to be updated and competent in providing emergency care. Role of GP: Some of the emergencies can be managed completely at a general practice while others should be referred to hospital after initial management. The extent to which a patient should be managed may be determined by the degree of severity of the condition, expertise of the doctor and distance to the nearest hospital. Apart from pharmacological management, explanation about the condition and the need for admission and appropriate advice on care prior to admission are also vital components of management. Writing an appropriate referral, arranging transport facilities, informing the hospital about the referral are also important steps in the process as these measures could prevent crucial delays. Conclusion: Emergency care is a responsibility of primary care doctors and they should be knowledgeable and skilled and organize their practices to provide prompt and effective management whenever the need arises

Dynamics and consequences of the HTLV-1 proviral plus-strand burst.

Expression of the transcriptional transactivator protein Tax, encoded on the proviral plus-strand of human T-cell leukaemia virus type 1 (HTLV-1), is crucial for the replication of the virus, but Tax-expressing cells are rarely detected in fresh blood ex vivo. The dynamics and consequences of the proviral plus-strand transcriptional burst remain insufficiently characterised. We combined time-lapse live-cell imaging, single-cell tracking and mathematical modelling to study the dynamics of Tax expression at single-cell resolution in two naturally-infected, non-malignant T-cell clones transduced with a short-lived enhanced green fluorescent protein (d2EGFP) Tax reporter system. Five different patterns of Tax expression were observed during the 30-hour observation period; the distribution of these patterns differed between the two clones. The mean duration of Tax expression in the two clones was 94 and 417 hours respectively, estimated from mathematical modelling of the experimental data. Tax expression was associated with a transient slowing in cell-cycle progression and proliferation, increased apoptosis, and enhanced activation of the DNA damage response pathways. Longer-term follow-up (14 days) revealed an increase in the proportion of proliferating cells and a decrease in the fraction of apoptotic cells as the cells ceased Tax expression, resulting in a greater net expansion of the initially Tax-positive population. Time-lapse live-cell imaging showed enhanced cell-to-cell adhesion among Tax-expressing cells, and decreased cell motility of Tax-expressing cells at the single-cell level. The results demonstrate the within-clone and between-clone heterogeneity in the dynamics and patterns of HTLV-1 plus-strand transcriptional bursts and the balance of positive and negative consequences of the burst for the host cell

Treatment with flavopiridol for 1.5 hrs did not alter Tax protein expression.

Clone 11.63 cells were treated with 1nM flavopiridol for 1.5 hrs and then stained for Live/Dead and then Tax protein. (TIFF)</p

Flow sorting of provirus-expressing and non-expressing cells by an independent technique produced similar q4C profiles.

(A) q4C profiles of Tax−(upper panel) and Tax+ (lower panel) cells sorted from clone TBX4B after intracellular staining of Tax. (B) q4C profile of non-expressing (GFP-) (upper panel) and provirus-expressing (GFP+) (lower panel) cells isolated from d2EGFP-TBX4B clones, selected by GFP signal (without Tax staining). (TIFF)</p

Fusion transcripts between HTLV-1 provirus and host genome.

(A) Identification of splice sites of fusion transcripts in the plus-strand-expressing cells of clone d2EGFP-11.63 and (B) in timer protein reporter clone Timer-3.60. Plus-strand fusion transcripts between HTLV-1 exon1 (H1) and same sense, 3′ side host gene exon (blue) or novel host exons (green) are shown with fused sequences. (TIFF)</p

Quantitative 3C (3C-qPCR) analysis confirmed decreased frequencies of chromatin looping.

(A) q4C profile of Tax−cells of clone 11.63. The technical peak seen in the q4C viewpoint (VP) and two of the main peaks (Peak 1) and (Peak 2) identified in the output of the Tax−fraction of clone 11.63. (B) As control, the frequency of chromatin interactions was quantified by 3C-qPCR on sorted Tax−and Tax+ cells, using a primer pair and Taqman probe to detect the contacts between two regions: VP and another region in the provirus (S1 Table). There was no significant difference between Tax+ and Tax−cells (combined p value = 0.932, Fisher’s method of combining p values). (C and D) Primer pairs and probe were used to detect long-range chromatin contacts between the provirus and host genome region at Peak 1 (C) or Peak 2 (D). Results of 3C-qPCR of two biological replicates (rep) are shown. Peak1 contact frequency was significantly higher in Tax−cells than in Tax+ cells (combined p value 0.012, Fisher’s method). Peak2 contact frequency was significantly higher in Tax- cells than in Tax+ cells (combined p value 0.000607, Fisher’s method). (TIFF)</p

Expression of distant host genes correlates with expression of <i>tax</i>.

(A) Normalized mRNA read counts of two genes that lie >1.4 Mb from the provirus in clone Timer-TBX4B, in the four successive phases of the HTLV-1 plus-strand transcriptional burst: DN–double negative (HTLV-1 silent); blue–early burst; DP double-positive (mid-burst); red–late burst. Results of two independent experiments are shown. Expression of both SMC1B and RIBC2 closely followed the trajectory of the HTLV-1 burst in clone Timer-TBX4B, but not in the unrelated HTLV-1-infected clone Timer-3.60. Data from [11]. (B) Knockout of the CTCF binding site in the provirus in clone Timer-TBX4B (middle panel) abolished the transcription of both SMC1B and RIBC2 observed in the wild-type clone (lower panel). Results of two independent experiments are shown. Neither gene was expressed in an unrelated HTLV-1-infected clone ED. The results suggest that maintenance of a CTCF-dependent chromatin loop between the host genome and the provirus is required for the burst of transcription of these distant genes associated with the HTLV-1 plus-strand burst. (TIFF)</p

Proviral and host transcription and splicing in live-sorted T cell clones.

(A) RNA-seq analysis of HTLV-1 proviral expression in live-sorted d2EGFP clones (TBX4B, 11.50 and 11.63). (B) Tax expression measured by qPCR with primers specific for tax mRNA or 18S ribosomal RNA (18S rRNA). qPCR plots are expression values normalized to 18S rRNA. Data represent a mean of two biological replicates; error bars are SEM. AU—arbitrary units. (C) Host RNA expression 30kb on either side of the proviral integration site. On the horizontal axis, positive values denote positions extending from the 3′ LTR side of the provirus; negative values denote positions 5′ of the 5′LTR. Each row shows the transcription density (normalized RNA-seq read count) flanking that genomic position in the clone indicated at the right-hand side. In each case, transcription orientation and positions are shown relative to the integrated provirus. Read density shown in blue shows transcription in the same orientation as the proviral plus-strand (same sense); red shows transcription in the opposite sense to the proviral plus-strand (antisense). (D) Identification of splice sites of viral-host fusion transcripts in d2EGFP-TBX4B clone cells. Coverage tracks of same sense transcription (blue) and antisense transcription (red) in Integrative Genomics Viewer (IGV). Exons of PNPLA3 in the 3′ side of the integration site are highlighted in yellow. (E) Fusion transcripts between an HTLV-1 plus-strand major splice donor (red, proviral exon H1 or H2) and the canonical splice acceptor site in the host PNPLA3 gene (blue, PNPLA3 exon 3) were identified in GFP+ (HTLV-1 plus-strand-expressing) cells. To identify splice sites of fusion transcripts, reads were aligned to a reference genome (hg19) containing the HTLV-1 provirus (AB513134) genome in the TBX4B clone integration site at chr22:44323198. Fusion transcripts are shown with fused sequences.</p