The HIV-1 Transcriptional Program: From Initiation to Elongation Control.

A large body of work in the last four decades has revealed the key pillars of HIV-1 transcription control at the initiation and elongation steps. Here, I provide a recount of this collective knowledge starting with the genomic elements (DNA and nascent TAR RNA stem-loop) and transcription factors (cellular and the viral transactivator Tat), and later transitioning to the assembly and regulation of transcription initiation and elongation complexes, and the role of chromatin structure. Compelling evidence support a core HIV-1 transcriptional program regulated by the sequential and concerted action of cellular transcription factors and Tat to promote initiation and sustain elongation, highlighting the efficiency of a small virus to take over its host to produce the high levels of transcription required for viral replication. I summarize new advances including the use of CRISPR-Cas9, genetic tools for acute factor depletion, and imaging to study transcriptional dynamics, bursting and the progression through the multiple phases of the transcriptional cycle. Finally, I describe current challenges to future major advances and discuss areas that deserve more attention to both bolster our basic knowledge of the core HIV-1 transcriptional program and open up new therapeutic opportunities.

Viral cis-regulatory elements as sensors of cellular states and environmental cues.

To withstand a hostile cellular environment and replicate, viruses must sense, interpret, and respond to many internal and external cues. Retroviruses and DNA viruses can intercept these cues impinging on host transcription factors via cis-regulatory elements (CREs) in viral genomes, allowing them to sense and coordinate context-specific responses to varied signals. Here, we explore the characteristics of viral CREs, the classes of signals and host transcription factors that regulate them, and how this informs outcomes of viral replication, immune evasion, and latency. We propose that viral CREs constitute central hubs for signal integration from multiple pathways and that sequence variation between viral isolates can rapidly rewire sensing mechanisms, contributing to the variability observed in patient outcomes.

Dynamic bidirectional regulation of NLRC3 and gammaherpesviruses during viral latency in B lymphocytes.

While most NOD-like receptors (NLRs) are predominately expressed by innate immune cells, NLRC3, an inhibitory NLR of immune signaling, exhibits the highest expression in lymphocytes. The role of NLRC3 or any NLRs in B lymphocytes is completely unknown. Gammaherpesviruses, including human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV-68), establish latent infection in B lymphocytes, which requires elevated NF-κB. This study shows that during latent EBV infection of human B cells, viral-encoded latent membrane protein 1 (LMP1) decreases NLRC3 transcript. LMP1-induced-NF-κB activation suppresses the promoter activity of NLRC3 via p65 binding to the promoter. Conversely, NLRC3 inhibits NF-κB activation by promoting the degradation of LMP1 in a proteasome-dependent manner. In vivo, MHV-68 infection reduces Nlrc3 transcripts in splenocytes, and Nlrc3-deficient mice show greater viral latency than controls. These results reveal a bidirectional regulatory circuit in B lymphocytes, where viral latent protein LMP1 reduces NLRC3 expression, while NLRC3 disrupts gammaherpesvirus latency, which is an important step for tumorigenesis.

A Canadian Survey of Research on HIV-1 Latency-Where Are We Now and Where Are We Heading?

Worldwide, almost 40 million people are currently living with HIV-1. The implementation of cART inhibits HIV-1 replication and reduces viremia but fails to eliminate HIV-1 from latently infected cells. These cells are considered viral reservoirs from which HIV-1 rebounds if cART is interrupted. Several efforts have been made to identify these cells and their niches. There has been little success in diminishing the pool of latently infected cells, underscoring the urgency to continue efforts to fully understand how HIV-1 establishes and maintains a latent state. Reactivating HIV-1 expression in these cells using latency-reversing agents (LRAs) has been successful, but only in vitro. This review aims to provide a broad view of HIV-1 latency, highlighting Canadian contributions toward these aims. We will summarize the research efforts conducted in Canadian labs to understand the establishment of latently infected cells and how this informs curative strategies, by reviewing how HIV latency is established, which cells are latently infected, what methodologies have been developed to characterize them, how new compounds are discovered and evaluated as potential LRAs, and what clinical trials aim to reverse latency in people living with HIV (PLWH).

Breaking the Silence: Regulation of HIV Transcription and Latency on the Road to a Cure.

Antiretroviral therapy (ART) has brought the HIV/AIDS epidemic under control, but a curative strategy for viral eradication is still needed. The cessation of ART results in rapid viral rebound from latently infected CD4+ T cells, showing that control of viral replication alone does not fully restore immune function, nor does it eradicate viral reservoirs. With a better understanding of factors and mechanisms that promote viral latency, current approaches are primarily focused on the permanent silencing of latently infected cells ("block and lock") or reactivating HIV-1 gene expression in latently infected cells, in combination with immune restoration strategies to eliminate HIV infected cells from the host ("shock and kill"). In this review, we provide a summary of the current, most promising approaches for HIV-1 cure strategies, including an analysis of both latency-promoting agents (LPA) and latency-reversing agents (LRA) that have shown promise in vitro, ex vivo, and in human clinical trials to reduce the HIV-1 reservoir.

HIV RNA/DNA Levels at Diagnosis Can Predict Immune Reconstitution: A Longitudinal Analysis.

BACKGROUND: HIV DNA mirrors the number of infected cells and the size of the HIV viral reservoir. The aim of this study was to evaluate the effect of pre-cART HIV DNA levels as a predictive marker of immune reconstitution and on the post-cART CD4 counts trends. METHODS: HIV DNA was isolated from PBMCs and quantified by real-time PCR. Immune reconstitution was assessed up to four years. Piecewise-linear mixed models were used to describe CD4 count changes. RESULTS: 148 people living with HIV (PLWH) were included. The highest rate of immune reconstitution was observed during the first trimester. There was a trend showing that high HIV RNA level resulted in greater increase in CD4 count, especially during the first trimester of cART (difference above vs. below median 15.1 cells/µL/month; 95% CI -1.4-31.5; p = 0.073). Likewise, higher HIV DNA level would predict greater CD4 increases, especially after the first trimester (difference above vs. below median 1.2 cells/µL/month; 95% CI -0.1-2.6; p = 0.071). Higher DNA and RNA levels combined were significantly associated with greater CD4 increase past the first trimester (difference high/high vs. low/low 2.1 cells/µL/month; 95% CI 0.3-4.0; p = 0.024). In multivariable analysis, lower baseline CD4 counts predicted a greater CD4 rise. CONCLUSIONS: In successfully treated PLWH, pre-cART HIV DNA and HIV RNA levels are predictors of immune reconstitution.

The Natural Stilbenoid (-)-Hopeaphenol Inhibits HIV Transcription by Targeting Both PKC and NF-κB Signaling and Cyclin-Dependent Kinase 9.

Despite effective combination antiretroviral therapy (cART), people living with HIV (PLWH) continue to harbor replication-competent and transcriptionally active virus in infected cells, which in turn can lead to ongoing viral antigen production, chronic inflammation, and increased risk of age-related comorbidities. To identify new agents that may inhibit postintegration HIV beyond cART, we screened a library of 512 pure compounds derived from natural products and identified (-)-hopeaphenol as an inhibitor of HIV postintegration transcription at low to submicromolar concentrations without cytotoxicity. Using a combination of global RNA sequencing, plasmid-based reporter assays, and enzyme activity studies, we document that hopeaphenol inhibits protein kinase C (PKC)- and downstream NF-κB-dependent HIV transcription as well as a subset of PKC-dependent T-cell activation markers, including interleukin-2 (IL-2) cytokine and CD25 and HLA-DRB1 RNA production. In contrast, it does not substantially inhibit the early PKC-mediated T-cell activation marker CD69 production of IL-6 or NF-κB signaling induced by tumor necrosis factor alpha (TNF-α). We further show that hopeaphenol can inhibit cyclin-dependent kinase 9 (CDK9) enzymatic activity required for HIV transcription. Finally, it inhibits HIV replication in peripheral blood mononuclear cells (PBMCs) infected in vitro and dampens viral reactivation in CD4+ cells from PLWH. Our study identifies hopeaphenol as a novel inhibitor that targets a subset of PKC-mediated T-cell activation pathways in addition to CDK9 to block HIV expression. Hopeaphenol-based therapies could complement current antiretroviral therapy otherwise not targeting cell-associated HIV RNA and residual antigen production in PLWH.

miRNAs in Herpesvirus Infection: Powerful Regulators in Small Packages.

microRNAs are a class of small, single-stranded, noncoding RNAs that regulate gene expression. They can be significantly dysregulated upon exposure to any infection, serving as important biomarkers and therapeutic targets. Numerous human DNA viruses, along with several herpesviruses, have been found to encode and express functional viral microRNAs known as vmiRNAs, which can play a vital role in host-pathogen interactions by controlling the viral life cycle and altering host biological pathways. Viruses have also adopted a variety of strategies to prevent being targeted by cellular miRNAs. Cellular miRNAs can act as anti- or proviral components, and their dysregulation occurs during a wide range of infections, including herpesvirus infection. This demonstrates the significance of miRNAs in host herpesvirus infection. The current state of knowledge regarding microRNAs and their role in the different stages of herpes virus infection are discussed in this review. It also delineates the therapeutic and biomarker potential of these microRNAs in future research directions.

Reviewing the Potential Links between Viral Infections and TDP-43 Proteinopathies.

Transactive response DNA binding protein 43 kDa (TDP-43) was discovered in 2001 as a cellular factor capable to inhibit HIV-1 gene expression. Successively, it was brought to new life as the most prevalent RNA-binding protein involved in several neurological disorders, such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite the fact that these two research areas could be considered very distant from each other, in recent years an increasing number of publications pointed out the existence of a potentially important connection. Indeed, the ability of TDP-43 to act as an important regulator of all aspects of RNA metabolism makes this protein also a critical factor during expression of viral RNAs. Here, we summarize all recent observations regarding the involvement of TDP-43 in viral entry, replication and latency in several viruses that include enteroviruses (EVs), Theiler's murine encephalomyelitis virus (TMEV), human immunodeficiency virus (HIV), human endogenous retroviruses (HERVs), hepatitis B virus (HBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), West Nile virus (WNV), and herpes simplex virus-2 (HSV). In particular, in this work, we aimed to highlight the presence of similarities with the most commonly studied TDP-43 related neuronal dysfunctions.

The CMV-encoded G protein-coupled receptors M33 and US28 play pleiotropic roles in immune evasion and alter host T cell responses.

Introduction: Human cytomegalovirus (HCMV) is a global health threat due to its ubiquity and lifelong persistence in infected people. During latency, host CD8+ T cell responses to HCMV continue to increase in a phenomenon known as memory inflation. We used murine CMV (MCMV) as a model for HCMV to characterize the memory inflation response to wild-type MCMV (KP) and a latency-defective mutant (ΔM33stop), which lacks M33, an MCMV chemokine receptor homolog. M33 is essential for normal reactivation from latency and this was leveraged to determine whether reactivation in vivo contributes to T cell memory inflation. Methods: Mice were infected with wild-type or mutant MCMV and T cell responses were analyzed by flow cytometry at acute and latent time points. Ex vivo reactivation and cytotoxicity assays were carried out to further investigate immunity and virus replication. Quantitative reverse-transcriptase polymerase chain reaction (q-RTPCR) was used to examine gene expression during reactivation. MHC expression on infected cells was analyzed by flow cytometry. Finally, T cells were depleted from latently-infected B cell-deficient mice to examine the in vivo difference in reactivation between wild-type and ΔM33stop. Results: We found that ΔM33stop triggers memory inflation specific for peptides derived from the immediate-early protein IE1 but not the early protein m164, in contrast to wild-type MCMV. During ex vivo reactivation, gene expression in DM33stop-infected lung tissues was delayed compared to wild-type virus. Normal gene expression was partially rescued by substitution of the HCMV US28 open reading frame in place of the M33 gene. In vivo depletion of T cells in immunoglobulin heavy chain-knockout mice resulted in reactivation of wild-type MCMV, but not ΔM33stop, confirming the role of M33 during reactivation from latency. Further, we found that M33 induces isotype-specific downregulation of MHC class I on the cell surface suggesting previously unappreciated roles in immune evasion. Discussion: Our results indicate that M33 is more polyfunctional than previously appreciated. In addition to its role in reactivation, which had been previously described, we found that M33 alters viral gene expression, host T cell memory inflation, and MHC class I expression. US28 was able to partially complement most functions of M33, suggesting that its role in HCMV infection may be similarly pleotropic.

First report of canine morbillivirus infection of adipose tissue-derived stem cells from dogs with distemper.

Background and Aim: Ribonucleic acid viruses remain latent in different cell types, including mesenchymal stem cells; however, the distemper virus remains undetected in these cells. This study aimed to determine whether adipose stem cells (ASCs) from dogs with distemper disease are infected with the canine morbillivirus (CM). Materials and Methods: Twelve dogs with the neurological phase of the disease and who were positive for CM by reverse transcription polymerase chain reaction (RT-PCR), were studied. ASCs from adipose tissue of the lesser omentum of these infected dogs were isolated and characterized. Direct fluorescence was used to detect the viral antigen in cell cultures. Flow cytometry and RT-PCR identified detectable quantities of the virus in two cultures, while electron microscopy confirmed the CM particles within ASCs. Results: This study revealed that ASCs of the omentum of dogs with distemper disease can be infected with CM, indicating their possible involvement in this virus latency and persistence. This suggests that its detection should be considered within the quality control process of stem cells intended for regenerative medicine. Conclusion: To the best of our knowledge, this is the first study that demonstrates that omentum ASCs from dogs with distemper disease can be infected with CM and may be involved in viral latency or persistence. Our study also suggests that the detection of CM should be considered within the quality control process of stem cells intended for regenerative medicine.

Sex differences in HIV-1 persistence and the implications for a cure.

Of the 38 million people currently living with Human Immunodeficiency Virus type-1 (HIV-1), women, especially adolescents and young women, are disproportionally affected by the HIV-1 pandemic. Acquired immunodeficiency syndrome (AIDS) - related illnesses are the leading cause of death in women of reproductive age worldwide. Although combination antiretroviral therapy (cART) can suppress viral replication, cART is not curative due to the presence of a long-lived viral reservoir that persists despite treatment. Biological sex influences the characteristics of the viral reservoir as well as the immune responses to infection, factors that can have a significant impact on the design and quantification of HIV-1 curative interventions in which women are grossly underrepresented. This mini-review will provide an update on the current understanding of the impact of biological sex on the viral reservoir and will discuss the implications of these differences in the context of the development of potential HIV-1 curative strategies, with a focus on the shock and kill approach to an HIV-1 cure. This mini-review will also highlight the current gaps in the knowledge of sex-based differences in HIV-1 persistence and will speculate on approaches to address them to promote the development of more scalable, effective curative approaches for people living with HIV-1.

Expression of the human herpesvirus 6A latency-associated transcript U94A impairs cytoskeletal functions in human neural cells.

Many neurodegenerative diseases have a multifactorial etiology and variable course of progression that cannot be explained by current models. Neurotropic viruses have long been suggested to play a role in these diseases, although their exact contributions remain unclear. Human herpesvirus 6A (HHV-6A) is one of the most common viruses detected in the adult brain, and has been clinically associated with multiple sclerosis (MS), and, more recently, Alzheimer's disease (AD). HHV-6A is a ubiquitous viral pathogen capable of infecting glia and neurons. Primary infection in childhood is followed by the induction of latency, characterized by expression of the U94A viral transcript in the absence of viral replication. Here we examine the effects of U94A on cells of the central nervous system. We found that U94A expression inhibits the migration and impairs cytoplasmic maturation of human oligodendrocyte precursor cells (OPCs) without affecting their viability, a phenotype that may contribute to the failure of remyelination seen in many patients with MS. A subsequent proteomics analysis of U94A expression OPCs revealed altered expression of genes involved in tubulin associated cytoskeletal regulation. As HHV-6A seems to significantly be associated with early AD pathology, we extended our initially analysis of the impact of U94A on human derived neurons. We found that U94A expression inhibits neurite outgrowth of primary human cortical neurons and impairs synapse maturation. Based on these data we suggest that U94A expression by latent HHV-6A in glial cells and neurons renders them susceptible to dysfunction and degeneration. Therefore, latent viral infections of the brain represent a unique pathological risk factor that may contribute to disease processes.

Methionine metabolism controls the B cell EBV epigenome and viral latency.

Epstein-Barr virus (EBV) subverts host epigenetic pathways to switch between viral latency programs, colonize the B cell compartment, and reactivate. Within memory B cells, the reservoir for lifelong infection, EBV genomic DNA and histone methylation marks restrict gene expression. But this epigenetic strategy also enables EBV-infected tumors, including Burkitt lymphomas, to evade immune detection. Little is known about host cell metabolic pathways that support EBV epigenome landscapes. We therefore used amino acid restriction, metabolomic, and CRISPR approaches to identify that an abundant methionine supply and interconnecting methionine and folate cycles maintain Burkitt EBV gene silencing. Methionine restriction, or methionine cycle perturbation, hypomethylated EBV genomes and de-repressed latent membrane protein and lytic gene expression. Methionine metabolism also shaped EBV latency gene regulation required for B cell immortalization. Dietary methionine restriction altered murine Burkitt xenograft metabolomes and de-repressed EBV immunogens in vivo. These results highlight epigenetic/immunometabolism crosstalk supporting the EBV B cell life cycle and suggest therapeutic approaches.

Lying low-chromatin insulation in persistent DNA virus infection.

Persistent virus infections are achieved when the intricate balance of virus replication, host-cell division and successful immune evasion is met. The genomes of persistent DNA viruses are either maintained as extrachromosomal episomes or can integrate into the host genome. Common to both these strategies of persistence is the chromatinisation of viral DNA by cellular histones which, like host DNA, are subject to epigenetic modification. Epigenetic repression of viral genes required for lytic replication occurs, while genes required for latent or persistent infection are maintained in an active chromatin state. Viruses utilise host-cell chromatin insulators, which function to maintain epigenetic boundaries and enforce this strict transcriptional programme. Here, we review insulator protein function in virus transcription control, focussing on CCCTC-binding factor (CTCF) and cofactors. We describe CTCF-dependent activities in virus transcription regulation through epigenetic and promoter-enhancer insulation, three-dimensional chromatin looping and manipulation of transcript splicing.

HibeRNAtion: HIV-1 RNA Metabolism and Viral Latency.

HIV-1 infection remains non-curative due to the latent reservoir, primarily a small pool of resting memory CD4+ T cells bearing replication-competent provirus. Pharmacological reversal of HIV-1 latency followed by intrinsic or extrinsic cell killing has been proposed as a promising strategy to target and eliminate HIV-1 viral reservoirs. Latency reversing agents have been extensively studied for their role in reactivating HIV-1 transcription in vivo, although no permanent reduction of the viral reservoir has been observed thus far. This is partly due to the complex nature of latency, which involves strict intrinsic regulation at multiple levels at transcription and RNA processing. Still, the molecular mechanisms that control HIV-1 latency establishment and maintenance have been almost exclusively studied in the context of chromatin remodeling, transcription initiation and elongation and most known LRAs target LTR-driven transcription by manipulating these. RNA metabolism is a largely understudies but critical mechanistic step in HIV-1 gene expression and latency. In this review we provide an update on current knowledge on the role of RNA processing mechanisms in viral gene expression and latency and speculate on the possible manipulation of these pathways as a therapeutic target for future cure studies.

Evidence of latent HPV infection in older Danish women with a previous history of cervical dysplasia.

INTRODUCTION: Understanding whether human papillomavirus (HPV) may establish latency in the uterine cervix is important. A better understanding of HPV natural history is useful for clinical counseling of women attending screening and to accurately inform health prevention strategies such as screening and HPV vaccination. We evaluated the extent of latent HPV infections in older women with a history of abnormal cytology. MATERIAL AND METHODS: We conducted a cross-sectional study in Aarhus, Denmark, from March 2013 through April 2015. Women were enrolled if they underwent cervical amputation or total hysterectomy because of benign disease. Prior to surgery, women completed a questionnaire and a cervical smear was collected for HPV testing and morphological assessment. For evaluation of latency (i.e., no evidence of active HPV infection, but HPV detected in the tissue), we selected women with a history of abnormal cervical cytology or histology, as these women were considered at increased risk of harboring a latent infection. Cervical tissue underwent extensive HPV testing using the SPF10-DEIA-LipA25 assay. RESULTS: Of 103 women enrolled, 26 were included in this analysis. Median age was 55 years (interquartile range [IQR] 52-65), and most women were postmenopausal and parous. The median number of sexual partners over the lifetime was six (IQR 3-10), and 85% reported no recent new sexual partner. Five women (19.2%) had evidence of active infection at the time of surgery, and 19 underwent latency evaluation. Of these, a latent infection was detected in 11 (57.9%), with HPV16 being the most prevalent type (50%). Nearly 80% (n = 14) of the 18 women with a history of previous low-grade or high-grade cytology with no treatment had an active or latent HPV infection, with latent infections predominating. HPV was detected in two of the six women with a history of high-grade cytology and subsequent excisional treatment, both as latent infections. CONCLUSIONS: HPV can be detected in cervical tissue specimens without any evidence of an active HPV infection, indicative of a latent, immunologically controlled infection. Modeling studies should consider including a latent state in their model when estimating the appropriate age to stop screening and when evaluating the impact of HPV vaccination.

New Aspects of the Virus Life Cycle and Clinical Utility of Next Generation Sequencing based HIV-1 Resistance Testing in the Genomic, the Proviral, and the Viral Reservoir of Peripheral Blood Mononuclear Cells.

BACKGROUND: Typically, genotypic resistance testing is recommended at the start of antiretroviral therapy and is even mandatory in cases of virologic failure. The material of choice is plasma viral RNA. However, in patients with low viremia (viral load < 500 copies/ml), resistance testing by population-based sequencing is very difficult. OBJECTIVE: Therefore, we aimed to investigate whether next generation sequencing (NGS) from proviral DNA and RNA could be an alternative. MATERIAL AND METHODS: EDTA blood samples (n = 36) from routine clinical viral load testing were used for the study. Viral loads ranged from 96 to 390,000 copies/mL, with 100% of samples having low viremia. Distribution of subtypes; A (n = 2), B (n = 16), C (n = 4), D (n = 2), G (1), CRF02 AG (n = 5), CRF01 AE (n = 5), undefined/mixed (n = 4). The extracted consensus sequences were uploaded to the Stanford HIV Drug Resistance Data Base and Geno2pheno for online analysis of drug resistance mutations and resistance factors. RESULTS: A total of 2476 variants or drug resistance mutations (DRMs) were detected with Sanger sequencing, compared with 2892 variants with NGS. An average of 822/1008 variants were identified in plasma viral RNA by Sanger or NGS sequencing, 834/956 in cellular viral RNA, and 820/928 in cellular viral DNA. CONCLUSION: Both methods are well suited for the detection of HIV substitutions or drug resistance mutations. Our results suggest that cellular RNA or cellular viral DNA is an informative alternative to plasma viral RNA for variant detection in patients with low viremia, as shown by the high correlation of variants in the different viral pools. We show that by using UDS, a plus of two DRMs per patient becomes visible, which can make a big difference in the assessment of the expected resistance behavior of the virus.

Pharmacological suppression of glycogen synthase kinase-3 reactivates HIV-1 from latency via activating Wnt/ß-catenin/TCF1 axis in CD4+ T cells.

ABSTRACTHIV-1 latency posts a major obstacle for HIV-1 eradication. Currently, no desirable latency reversing agents (LRAs) have been implicated in the "Shock and Kill" strategy to mobilize the latently infected cells to be susceptible for clearance by immune responses. Identification of key cellular pathways that modulate HIV-1 latency helps to develop efficient LRAs. In this study, we demonstrate that the Wnt downstream ß-catenin/TCF1 pathway is a crucial modulator for HIV-1 latency. The pharmacological activation of the ß-catenin/TCF1 pathway with glycogen synthase kinase-3 (GSK3) inhibitors promoted transcription of HIV-1 proviral DNA and reactivated latency in CD4+ T cells; the GSK3 kinase inhibitor 6-bromoindirubin-3'-oxime (6-BIO)-induced HIV-1 reactivation was subsequently confirmed in resting CD4+ T cells from cART-suppressed patients and SIV-infected rhesus macaques. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.

Latency Reversal and Clearance of Persistent HIV Infection.

Efforts to prevent and treat human immunodeficiency virus type 1 (HIV) infection have begun to blunt the spread of HIV infection. Potent, safe, and well-tolerated antiretroviral therapy (ART) allows those infected with HIV to attain a life expectancy similar to that of HIV-uninfected individuals. But the persistence of the quiescent retroviral genome, enforced by the natural proliferative responses of the immune system itself, and a delicate balance of regulators viral expression, mandates lifelong ART suppression to prevent rebound viremia and the return of disease.The approach to HIV eradication that has been studied the most extensively envisions adding therapies to induce the expression of quiescent HIV-1 genomes following the control of viremia by ART, paired with immunotherapies to clear persistent infection. Paired testing of latency reversal and clearance strategies has begun, but the field is still in its infancy and additional obstacles to HIV eradication may emerge. However, there is reason for optimism that together with advances in ART delivery and HIV prevention strategies, efforts in HIV cure research will markedly diminish the effect of the HIV pandemic on society.