Specific Reactivation of Latent HIV-1 by dCas9-SunTag-VP64-mediated Guide RNA Targeting the HIV-1 Promoter.

HIV-1 escapes antiretroviral agents by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. Transcriptional activation is prerequisite for reactivation and the eradication of latent HIV-1 proviruses. dCas9-SunTag-VP64 transcriptional system has been reported that it can robustly activate the expression of an endogenous gene using a single guide RNA (sgRNA). Here, we systematically investigated the potential of dCas9-SunTag-VP64 with the designed sgRNAs for reactivating latent HIV-1. We found dCas9-SunTag-VP64 with sgRNA 4 or sgRNA 5 targeted from -164 to -146 or -124 to -106 bp upstream of the transcription start sites of HIV-1 could induce high expression of luciferase reporter gene after screening of sgRNAs targeting different regions of the HIV-1 promoter. Further, we confirmed that dCas9-SunTag-VP64 with sgRNA 4 or sgRNA 5 can effectively reactivate latent HIV-1 transcription in several latently infected human T-cell lines. Moreover, we confirmed that the reactivation of latent HIV-1 by dCas9-SunTag-VP64 with the designed sgRNA occurred through specific binding to the HIV-1 LTR promoter without genotoxicity and global T-cell activation. Taken together, our data demonstrated dCas9-SunTag-VP64 system can effectively and specifically reactivate latent HIV-1 transcription, suggesting that this strategy could offer a novel approach to anti-HIV-1 latency.

A stratified analysis of a deep learning algorithm in the diagnosis of diabetic retinopathy in a real-world study.

BACKGROUND: The aim of our research was to prospectively explore the clinical value of a deep learning algorithm (DLA) to detect referable diabetic retinopathy (DR) in different subgroups stratified by types of diabetes, blood pressure, sex, BMI, age, glycosylated hemoglobin (HbA1c), diabetes duration, urine albumin-to-creatinine ratio (UACR), and estimated glomerular filtration rate (eGFR) at a real-world diabetes center in China. METHODS: A total of 1147 diabetic patients from Shanghai General Hospital were recruited from October 2018 to August 2019. Retinal fundus images were graded by the DLA, and the detection of referable DR (moderate nonproliferative DR or worse) was compared with a reference standard generated by one certified retinal specialist with more than 12 years of experience. The performance of DLA across different subgroups stratified by types of diabetes, blood pressure, sex, BMI, age, HbA1c, diabetes duration, UACR, and eGFR was evaluated. RESULTS: For all 1674 gradable images, the area under the receiver operating curve, sensitivity, and specificity of the DLA for referable DR were 0.942 (95% CI, 0.920-0.964), 85.1% (95% CI, 83.4%-86.8%), and 95.6% (95% CI, 94.6%-96.6%), respectively. The DLA showed consistent performance across most subgroups, while it showed superior performance in the subgroups of patients with type 1 diabetes, UACR ≥ 30 mg/g, and eGFR < 90 mL/min/1.73m2 . CONCLUSIONS: This study showed that the DLA was a reliable alternative method for the detection of referable DR and performed superior in patients with type 1 diabetes and diabetic nephropathy who were prone to DR.

Quercetin synergistically reactivates human immunodeficiency virus type 1 latency by activating nuclear factor­κB.

Highly active antiretroviral therapy (HAART) is very effective in suppressing human immunodeficiency virus type 1 (HIV­1) replication. However, the treatment is required to be administered for the remainder of an individual's lifetime due to latent HIV­1 reservoirs. The 'shock­and­kill' strategy, which involves using agents to reactivate latent HIV­1 and subsequently killing latently infected cells in the presence of HAART, was recently proposed. Unfortunately, no agents have currently demonstrated an ability to reactivate latent HIV­1 in vivo in the absence of toxicity. Therefore, the identification of novel latency activators is required. In order to identify a potential novel agent, the present study investigated the effect of quercetin on latent HIV­1 reactivation using an established model of HIV­1 latency. As a marker for reactivation of HIV­1 in C11 Jurkat cells, the expression of green fluorescent protein, controlled by HIV­1 long terminal repeat, was observed by fluorescence microscopy. The results of the present study demonstrated that quercetin effectively reactivated latent HIV­1 gene expression alone, and led to synergistic reactivation when combined with prostratin or valproic acid. In addition, the present study provides evidence that quercetin may reactivate HIV­1 expression by inducing nuclear factor­κB nuclear translocation, and that the toxicity of quercetin is lower when compared with various additional activators of HIV­1. Combined, the results of the present study indicate that quercetin may be an effective agent to disrupt HIV­1 latency and may be useful in future eradication strategies.

Specific and Stable Suppression of HIV Provirus Expression In Vitro by Chimeric Zinc Finger DNA Methyltransferase 1.

HIV-1 inserts its proviral DNA into the infected host cells, by which HIV proviral DNA can then be duplicated along with each cell division. Thus, provirus cannot be eradicated completely by current antiretroviral therapy. We have developed an innovative strategy to silence the HIV provirus by targeted DNA methylation on the HIV promoter region. We genetically engineered a chimeric DNA methyltransferase 1 composed of designed zinc-finger proteins to become ZF2 DNMT1. After transient transfection of the molecular clone encoding this chimeric protein into HIV-1 infected or latently infected cells, efficient suppression of HIV-1 expression by the methylation of CpG islands in 5'-LTR was observed and quantified. The effective suppression of HIV in latently infected cells by ZF2-DNMT1 is stable and can last through about 40 cell passages. Cytotoxic caused by ZF2-DNMT1 was only observed during cellular proliferation. Taken together, our results demonstrate the potential of this novel approach for anti-HIV-1 therapy.

The BET inhibitor OTX015 reactivates latent HIV-1 through P-TEFb.

None of the currently used anti-HIV-1 agents can effectively eliminate latent HIV-1 reservoirs, which is a major hurdle to a complete cure for AIDS. We report here that a novel oral BET inhibitor OTX015, a thienotriazolodiazepine compound that has entered phase Ib clinical development for advanced hematologic malignancies, can effectively reactivate HIV-1 in different latency models with an EC50 value 1.95-4.34 times lower than JQ1, a known BET inhibitor that can reactivate HIV-1 latency. We also found that OTX015 was more potent when used in combination with prostratin. More importantly, OTX015 treatment induced HIV-1 full-length transcripts and viral outgrowth in resting CD4(+) T cells from infected individuals receiving suppressive antiretroviral therapy (ART), while exerting minimal toxicity and effects on T cell activation. Finally, biochemical analysis showed that OTX015-mediated activation of HIV-1 involved an increase in CDK9 occupancy and RNAP II C-terminal domain (CTD) phosphorylation. Our results suggest that the BET inhibitor OTX015 may be a candidate for anti-HIV-1-latency therapies.

TRIM5α H43Y Polymorphism and Susceptibility to HIV-1 Infection: A Meta-Analysis.

TRIM5α is an antiviral factor that can greatly limit HIV-1 infection. Although several researchers have investigated whether TRIM5α H43Y polymorphism influences the risk of HIV-1 infection, no definite conclusion has ever been drawn. In this research, we performed a meta-analysis to generate a more robust estimate of the association between TRIM5α H43Y and susceptibility to HIV-1 infection. In total, six studies including 1,713 HIV-1 patients and 1,814 controls were included. TRIM5α H43Y polymorphisms of all individuals were genotyped. Odds ratios (ORs) with 95% confidence intervals were presented as the result of analysis. ORs for the main analysis were 0.82 (95% CI: 0.63-1.08) in the allelic comparison, 0.57 (95% CI: 0.34-0.95) in the homozygote comparison, 0.82 (95% CI: 0.57-1.16) in the dominant model, and 0.56 (95% CI: 0.33-0.93) in the recessive model. In the subgroup analysis by ethnicity, significantly decreased risks of infection were detected in the Asian population (homozygote comparison: 0.50, 95% CI: 0.28-0.89; recessive model: 0.49, 95% CI: 0.28-0.87), whereas such effects were not observed in the non-Asian population. Our meta-analysis indicates that TRIM5α H43Y polymorphism is associated with a decreased risk of HIV-1 infection in the homozygote comparison and recessive model. This polymorphism may act as a protective factor against HIV-1 infection, especially in Asians.

Designed transcription activator-like effector proteins efficiently induced the expression of latent HIV-1 in latently infected cells.

HIV latency is the foremost barrier to clearing HIV infection from patients. Reactivation of latent HIV-1 represents a promising strategy to deplete these viral reservoirs. Here, we report a novel approach to reactivate latent HIV-1 provirus using artificially designed transcription activator-like effector (TALE) fusion proteins containing a DNA-binding domain specifically targeting the HIV-1 promoter and the herpes simplex virus-based transcriptional activator VP64 domain. We engineered four TALE genes (TALE1-4) encoding TALE proteins, each specifically targeting different 20-bp DNA sequences within the HIV-1 promoter, and we constructed four TALE-VP64 expression vectors corresponding to TALE1-4. We found that TALE1-VP64 effectively reactivated HIV-1 gene expression in latently infected C11 and A10.6 cells. We further confirmed that TALE1-VP64 reactivated latent HIV-1 via specific binding to the HIV-LTR promoter. Moreover, we also found that TALE1-VP64 did not affect cell proliferation or cell cycle distribution. Taken together, our data demonstrated that TALE1-VP64 can specifically and effectively reactivate latent HIV-1 transcription, suggesting that this strategy may provide a novel approach for anti-HIV-1 latency therapy in the future.

Two cellular microRNAs, miR-196b and miR-1290, contribute to HIV-1 latency.

Understanding the mechanism of HIV-1 latency is crucial to the viral reservoir eradication. Human cellular miRNAs can modulate HIV-1 expression by targeting of viral RNAs or host gene transcripts. To identify miRNAs modulating HIV-1 latency, we determined the miRNA expression profiles of HIV-1 latently infected and productively infected cells by microarray and qRT-PCR. Among the differentially expressed miRNAs, miR-196b and miR-1290 targeted the 3' untranslated region of HIV-1 and affected its expression. Ectopic expression of these two miRNAs efficiently suppressed HIV-1 production and infectivity. Specific inhibitors of these miRNAs substantially counteracted their effects on HIV-1, as measured either as viral production and infectivity in HEK-293T cells or as HIV-1 RNA expression or viral production in cells isolated from HIV-1-infected individuals. Our study emphasizes the role of cellular miRNAs in HIV-1 latency regulation, and it suggests that inhibitors of miR-196b and miR-1290 could be used to activate latent HIV-1.