HIV-1 transcriptional modulation: novel host factors and prospective therapeutic strategies.

PURPOSE OF REVIEW: This review highlights advances in HIV transcription and epigenetic latency mechanisms and outlines current therapeutic approaches to eliminate or block the HIV-1 latent reservoir. RECENT FINDINGS: Novel host factors have been reported to modulate HIV-1 transcription and latency. Chromatin affinity purification strategies followed by mass spectrometry (ChAP-MS) identified the chaperone protein p32 to play an important role in HIV-1 transcriptional regulation via interactions with the viral transcriptional activator Tat. Similarly, an shRNA screen identified the methyltransferase SMYD5 contributing to HIV-1 transcriptional activation also by modulating Tat activity. These new factors, among others, represent potential druggable targets that could be explored in the 'block-and-lock' or 'shock-and-kill' approaches. SUMMARY: The HIV-1 latent reservoir is established early after infection, persists during antiretroviral therapy, and is the source of viral rebound after treatment interruption. An HIV cure requires either eliminating this reservoir or blocking latent proviral reactivation in the absence of antiretroviral therapy (ART). Understanding the mechanisms and key-players modulating HIV transcriptional and reactivation may facilitate therapeutic advancements. Here we summarize, the latest findings on host factors' roles in HIV transcriptional regulation.

The chaperone protein p32 stabilizes HIV-1 Tat and strengthens the p-TEFb/RNAPII/TAR complex promoting HIV transcription elongation.

HIV gene expression is modulated by the combinatorial activity of the HIV transcriptional activator, Tat, host transcription factors, and chromatin remodeling complexes. To identify host factors regulating HIV transcription, we used specific single-guide RNAs and endonuclease-deficient Cas9 to perform chromatin affinity purification of the integrated HIV promoter followed by mass spectrometry. The scaffold protein, p32, also called ASF/SF2 splicing factor-associated protein, was identified among the top enriched factors present in actively transcribing HIV promoters but absent in silenced ones. Chromatin immunoprecipitation analysis confirmed the presence of p32 on active HIV promoters and its enhanced recruitment by Tat. HIV uses Tat to efficiently recruit positive transcription elongation factor b (p-TEFb) (CDK9/CCNT1) to TAR, an RNA secondary structure that forms from the first 59 bp of HIV transcripts, to enhance RNAPII transcriptional elongation. The RNA interference of p32 significantly reduced HIV transcription in primary CD4+T cells and in HIV chronically infected cells, independently of either HIV splicing or p32 anti-splicing activity. Conversely, overexpression of p32 specifically increased Tat-dependent HIV transcription. p32 was found to directly interact with Tat's basic domain enhancing Tat stability and half-life. Conversely, p32 associates with Tat via N- and C-terminal domains. Likely due its scaffold properties, p32 also promoted Tat association with TAR, p-TEFb, and RNAPII enhancing Tat-dependent HIV transcription. In sum, we identified p32 as a host factor that interacts with and stabilizes Tat protein, promotes Tat-dependent transcriptional regulation, and may be explored for HIV-targeted transcriptional inhibition.

Cure and Long-Term Remission Strategies.

The majority of virally suppressed individuals will experience rapid viral rebound upon antiretroviral therapy (ART) interruption, providing a strong rationale for the development of cure strategies. Moreover, despite ART virological control, HIV infection is still associated with chronic immune activation, inflammation, comorbidities, and accelerated aging. These effects are believed to be due, in part, to low-grade persistent transcription and trickling production of viral proteins from the pool of latent proviruses constituting the viral reservoir. In recent years there has been an increasing interest in developing what has been termed a functional cure for HIV. This approach entails the long-term, durable control of viral expression in the absence of therapy, preventing disease progression and transmission, despite the presence of detectable integrated proviruses. One such strategy, the block-and-lock approach for a functional cure, proposes the epigenetic silencing of proviral expression, locking the virus in a profound latent state, from which reactivation is very unlikely. The proof-of-concept for this approach was demonstrated with the use of a specific small molecule targeting HIV transcription. Here we review the principles behind the block-and-lock approach and some of the additional strategies proposed to silence HIV expression.

The Block-and-Lock Strategy for Human Immunodeficiency Virus Cure: Lessons Learned from Didehydro-Cortistatin A.

Antiretroviral therapy effectively controls human immunodeficiency virus (HIV) infection. However, a reservoir of latently infected cells persists under suppressive therapy, constituting a major barrier to an HIV cure. The block-and-lock approach to a functional cure aims at the transcriptional and epigenetic silencing of proviruses, blocking viral reactivation in the absence of therapy, preventing disease progression and transmission, despite the presence of detectable integrated proviruses. This approach has been put forward for exploration based on the activity of didehydro-cortistatin A, an inhibitor of the HIV transcriptional activator Tat. Here we review the mechanisms by which didehydro-cortistatin A inhibition of Tat's feedback loop transcriptional amplification results in epigenetic silencing of the HIV promoter, and we discuss the benefits and limitations of the block-and-lock approach for an HIV cure.

The XPB Subunit of the TFIIH Complex Plays a Critical Role in HIV-1 Transcription and XPB Inhibition by Spironolactone Prevents HIV-1 Reactivation from Latency.

HIV transcription requires assembly of cellular transcription factors at the HIV-1promoter. The TFIIH general transcription factor facilitates transcription initiation by opening the DNA strands around the transcription start site and phosphorylating the C-terminal domain for RNA polymerase II (RNAPII) for activation. Spironolactone (SP), an FDA approved aldosterone antagonist, triggers the proteasomal degradation of the XPB subunit of TFIIH, and concurrently suppresses acute HIV infection in vitro Here we investigated SP as a possible block-and-lock agent for a functional cure aimed at the transcriptional silencing of the viral reservoir. The long-term activity of SP was investigated in primary and cell line models of HIV-1 latency and reactivation. We show that SP rapidly inhibits HIV-1 transcription by reducing RNAPII recruitment to the HIV-1 genome. shRNA knockdown of XPB confirmed XPB degradation as the mechanism of action. Unfortunately, long-term pre-treatment with SP does not result in epigenetic suppression of HIV upon SP treatment interruption, since virus rapidly rebounds when XPB reemerges; however, SP alone without ART maintains the transcriptional suppression. Importantly, SP inhibits HIV reactivation from latency in both cell line models and resting CD4+T cells isolated from aviremic infected individuals upon cell stimulation with latency reversing agents. Furthermore, long-term treatment with concentrations of SP that potently degrade XPB does not lead to global dysregulation of cellular mRNA expression. Overall, these results suggest that XPB plays a key role in HIV transcriptional regulation and XPB degradation by SP strengthens the potential of HIV transcriptional inhibitors in block-and-lock HIV cure approaches.IMPORTANCE Antiretroviral therapy (ART) effectively reduces an individual's HIV loads to below the detection limit, nevertheless rapid viral rebound immediately ensues upon treatment interruption. Furthermore, virally suppressed individuals experience chronic immune activation from ongoing low-level virus expression. Thus, the importance of identifying novel therapeutics to explore in block-and-lock HIV functional cure approaches, aimed at the transcriptional and epigenetic silencing of the viral reservoir to block reactivation from latency. We investigated the potential of repurposing the FDA-approved spironolactone (SP), as one such drug. SP treatment rapidly degrades a host transcription factor subunit, XPB, inhibiting HIV transcription and blocking reactivation from latency. Long-term SP treatment does not affect cellular viability, cell cycle progression or global cellular transcription. SP alone blocks HIV transcription in the absence of ART but does not delay rebound upon drug removal as XPB rapidly reemerges. This study highlights XPB as a novel drug target in block-and-lock therapeutic approaches.

Key Players in HIV-1 Transcriptional Regulation: Targets for a Functional Cure.

HIV-1 establishes a life-long infection when proviral DNA integrates into the host genome. The provirus can then either actively transcribe RNA or enter a latent state, without viral production. The switch between these two states is governed in great part by the viral protein, Tat, which promotes RNA transcript elongation. Latency is also influenced by the availability of host transcription factors, integration site, and the surrounding chromatin environment. The latent reservoir is established in the first few days of infection and serves as the source of viral rebound upon treatment interruption. Despite effective suppression of HIV-1 replication by antiretroviral therapy (ART), to below the detection limit, ART is ineffective at reducing the latent reservoir size. Elimination of this reservoir has become a major goal of the HIV-1 cure field. However, aside from the ideal total HIV-1 eradication from the host genome, an HIV-1 remission or functional cure is probably more realistic. The "block-and-lock" approach aims at the transcriptional silencing of the viral reservoir, to render suppressed HIV-1 promoters extremely difficult to reactivate from latency. There are unfortunately no clinically available HIV-1 specific transcriptional inhibitors. Understanding the mechanisms that regulate latency is expected to provide novel targets to be explored in cure approaches.

Author Correction: Sex-specific innate immune selection of HIV-1 in utero is associated with increased female susceptibility to infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sex-specific innate immune selection of HIV-1 in utero is associated with increased female susceptibility to infection.

Female children and adults typically generate more efficacious immune responses to vaccines and infections than age-matched males, but also suffer greater immunopathology and autoimmune disease. We here describe, in a cohort of > 170 in utero HIV-infected infants from KwaZulu-Natal, South Africa, fetal immune sex differences resulting in a 1.5-2-fold increased female susceptibility to intrauterine HIV infection. Viruses transmitted to females have lower replicative capacity (p = 0.0005) and are more type I interferon-resistant (p = 0.007) than those transmitted to males. Cord blood cells from females of HIV-uninfected sex-discordant twins are more activated (p = 0.01) and more susceptible to HIV infection in vitro (p = 0.03). Sex differences in outcome include superior maintenance of aviraemia among males (p = 0.007) that is not explained by differential antiretroviral therapy adherence. These data demonstrate sex-specific innate immune selection of HIV associated with increased female susceptibility to in utero infection and enhanced functional cure potential among infected males.

Modeling HIV-1 Latency Using Primary CD4+ T Cells from Virally Suppressed HIV-1-Infected Individuals on Antiretroviral Therapy.

The low frequency of latently HIV-infected cells in vivo limits the testing of potential HIV cure strategies using cells from successfully suppressed individuals. To date, primary cell models of latency use cells infected in vitro Primary CD4+ T cell models carrying an individual's endogenous HIV reservoir that recapitulate in vivo conditions of HIV latency are still outstanding. We developed a primary CD4+ T cell model of HIV latency derived from memory CD4+ T cells isolated from virally suppressed HIV-infected individuals that recapitulates HIV-1 latency and viral reactivation events. This model is based on the expansion of primary CD4+ T cells up to 300-fold in cell number. These cells reestablish a resting state without active virus production after extended culture and maintain a stable number of total HIV proviruses. The ability of these cells to respond to various classes of latency-reversing agents is similar to that of ex vivo CD4+ T cells directly isolated from blood. Importantly, viral outgrowth assays confirmed the ability of these expanded cells to produce replication-competent endogenous virus. In sum, this model recapitulates ex vivo viral reactivation conditions, captures the variability between individuals with different HIV reservoirs, and provides large numbers of cells for testing multiple agents from a single donor. The use of this novel model will allow accurate exploration of novel cure approaches aimed either at promoting viral reactivation or maintaining sustained latency.IMPORTANCE Primary cell models of HIV latency have been very useful to identify mechanisms contributing to HIV latency and to evaluate potential HIV cure strategies. However, the current models utilize in vitro infection with exogenous virus that does not fully recapitulate virus reactivation profiles of endogenous HIV in in vivo-infected CD4+ T cells. In contrast, obtaining sufficient amounts of CD4+ T cells from HIV-infected individuals to interrogate the HIV reservoir in vitro requires leukapheresis. In the model we propose here, in vitro expansion and extended culture of primary CD4+ T cells isolated from virally suppressed HIV-infected individuals enable obtaining large numbers of cells harboring endogenous latent HIV reservoirs without performing leukapheresis. This model captures the variability of HIV reservoirs seeded in different individuals and should be useful to evaluate future HIV cure strategies.

Recovery of effective HIV-specific CD4+ T-cell activity following antiretroviral therapy in paediatric infection requires sustained suppression of viraemia.

BACKGROUND: The success of increasing access to antiretroviral therapy (ART) in paediatric HIV infection prompts the question of the potential for eradication of HIV infection in this age group. 'Shock-and-kill' HIV cure approaches, currently in development, may depend upon an effective antiviral T-cell response to eradicate virus-infected cells. METHOD: We here investigate the ability of HIV-infected children receiving ART from early childhood (median 24 months' age) to generate effective HIV-specific CD4 and CD8 T-cell immune responses that would facilitate future immune-based cure therapies. RESULTS: Initial analysis of ART-naive HIV-infected children demonstrated that maintenance of normal-for-age absolute CD4 T-cell counts was strongly linked to high IL-2 production and polyfunctional HIV-specific CD4 T-cell responses (P < 0.0001 in each case). Low viral load was, similarly, strongly associated with markedly low IFN-γ and high IL-2 HIV-specific CD4 T-cell responses (P < 0.0001). In children receiving ART, establishment of this immune profile (high IL-2 and low IFN-γ HIV-specific T-cell production) was strongly related to the duration of viraemic suppression. Failure to suppress viraemia on ART, and even the successful suppression of viraemia interrupted by the occurrence of transient viraemia of more than 1000 HIV copies/ml, was associated with an immune profile of high IFN-γ and low IL-2 HIV-specific T-cell responses and low polyfunctionality. CONCLUSION: These data are consistent with recovery of functional CD4 T-cell responses in ART-treated children, in contrast to relative lack of CD4 T-cell function recovery described in ART-treated adults. However, the challenges of achieving long-term suppression of viraemia in ART-treated children through adolescence remain daunting.

Aplicación de la digitopuntura en pacientes con cefalea migrañosa. Policlínico Contreras, Matanzas, 2014 / Digitopuncture application in patients with migrainous cephalalgia. Contreras Policlinic, Matanzas, 2014

Introducción: la cefalea es toda sensación dolorosa que tiene lugar en la parte superior de la cabeza, desde el reborde orbitario hasta la nuca. Constituye la primera causa de acudir al neurólogo y uno de los motivos más frecuentes en consultas de Medicina Interna. La cefalea tipo tensión es frecuente en la práctica diaria y la más invalidante desde los puntos de vistas físico, social, económico y psicológico. Objetivo: mostrar la actuación de Enfermería en el alivio de la migraña y describir la efectividad de la digitopuntura en pacientes con cefalea migrañosa, del Policlínico Contreras, primer trimestre de 2014. Materiales y métodos: estudio observacional, descriptivo y transversal. El grupo de estudio, 120 pacientes que acudieron a la consulta de Medicina Natural y Tradicional. Resultados: la edad de mayor incidencia 25-34 años, con 47.4 %. Prevaleció el sexo femenino, en un 70 %; la duración del dolor mostró un 43.3 % de afectación. Transcurrió entre las 4 y 8 horas, y siempre tuvo relación con el estrés, en 44.2 %; en un 69.2 % existió relación del dolor con la ingestión de alimentos. Se alivió con el sueño el 61.7 %, y el 52,5 % alcanzaron estado evolutivo excelente. Conclusiones: la aplicación de la digitopuntura a pacientes con cefalea migrañosa contribuyó a que los casos estudiados evolucionaran satisfactoriamente, expresando alivio del dolor. El tratamiento demostró efectividad y contribuyó a la disminución de drogas utilizadas en esta entidad, disminución de gastos económicos que implica el consumo de las mismas; lograr reincorporar a la vida laboral y social en un menor tiempo al paciente (AU).

Introduction: cephalalgia is any painful sensation occurring in the superior part of the head, from the orbital ridge to the nape. It is the first cause of visiting a neurologist and one of the most frequent causes of Internal Medicine consultations. The tension-kind cephalalgia is frequent in daily practice and the most invaliding one from the physical, social, economic and psychological point of view. Aim: to show Nursery behavior in migraine alleviation and to describe the digitopuncture effectiveness in patients with migrainous cephalalgia from Contreras Policlinic in the first trimester of 2014. Materials and methods: observational, descriptive, cross-sectional study. The studied group was 120 patients assisting the consultation of Natural and Traditional Medicine. Outcomes: the 25-34 age group was the one that showed higher incidence, with 47.4 %. The female sex prevailed with 70 %; pain lasting had 44.3 % of affectation. It lasted between 4 and 8 hours, always related to stress in 44.2 %. There it was a relation between pain and food intake in 69.2 %. 61.7 % of the cases were lessened with sleep, and 52.5 % achieved an excellent evolving status. Conclusions: Acupuncture application to patients with migrainous cephalalgia contributed to the satisfactory evolution of the studied cases: The patients expressed pain relieve. The treatment showed effectiveness and facilitated the decrease of drug usage in this entity, also diminishing the economic expenses its use implies; the patients reincorporated to social and working life in a shorter time period (AU).

Aplicación de la digitopuntura en pacientes con cefalea migrañosa. Policlínico Contreras, Matanzas, 2014 / Digitopuncture application in patients with migrainous cephalalgia. Contreras Policlinic, Matanzas, 2014

Introducción: la cefalea es toda sensación dolorosa que tiene lugar en la parte superior de la cabeza, desde el reborde orbitario hasta la nuca. Constituye la primera causa de acudir al neurólogo y uno de los motivos más frecuentes en consultas de Medicina Interna. La cefalea tipo tensión es frecuente en la práctica diaria y la más invalidante desde los puntos de vistas físico, social, económico y psicológico. Objetivo: mostrar la actuación de Enfermería en el alivio de la migraña y describir la efectividad de la digitopuntura en pacientes con cefalea migrañosa, del Policlínico Contreras, primer trimestre de 2014. Materiales y métodos: estudio observacional, descriptivo y transversal. El grupo de estudio, 120 pacientes que acudieron a la consulta de Medicina Natural y Tradicional. Resultados: la edad de mayor incidencia 25-34 años, con 47.4 %. Prevaleció el sexo femenino, en un 70 %; la duración del dolor mostró un 43.3 % de afectación. Transcurrió entre las 4 y 8 horas, y siempre tuvo relación con el estrés, en 44.2 %; en un 69.2 % existió relación del dolor con la ingestión de alimentos. Se alivió con el sueño el 61.7 %, y el 52,5 % alcanzaron estado evolutivo excelente. Conclusiones: la aplicación de la digitopuntura a pacientes con cefalea migrañosa contribuyó a que los casos estudiados evolucionaran satisfactoriamente, expresando alivio del dolor. El tratamiento demostró efectividad y contribuyó a la disminución de drogas utilizadas en esta entidad, disminución de gastos económicos que implica el consumo de las mismas; lograr reincorporar a la vida laboral y social en un menor tiempo al paciente (AU).

Introduction: cephalalgia is any painful sensation occurring in the superior part of the head, from the orbital ridge to the nape. It is the first cause of visiting a neurologist and one of the most frequent causes of Internal Medicine consultations. The tension-kind cephalalgia is frequent in daily practice and the most invaliding one from the physical, social, economic and psychological point of view. Aim: to show Nursery behavior in migraine alleviation and to describe the digitopuncture effectiveness in patients with migrainous cephalalgia from Contreras Policlinic in the first trimester of 2014. Materials and methods: observational, descriptive, cross-sectional study. The studied group was 120 patients assisting the consultation of Natural and Traditional Medicine. Outcomes: the 25-34 age group was the one that showed higher incidence, with 47.4 %. The female sex prevailed with 70 %; pain lasting had 44.3 % of affectation. It lasted between 4 and 8 hours, always related to stress in 44.2 %. There it was a relation between pain and food intake in 69.2 %. 61.7 % of the cases were lessened with sleep, and 52.5 % achieved an excellent evolving status. Conclusions: Acupuncture application to patients with migrainous cephalalgia contributed to the satisfactory evolution of the studied cases: The patients expressed pain relieve. The treatment showed effectiveness and facilitated the decrease of drug usage in this entity, also diminishing the economic expenses its use implies; the patients reincorporated to social and working life in a shorter time period (AU).

Sex Differences in Antiretroviral Therapy Initiation in Pediatric HIV Infection.

The incidence and severity of infections in childhood is typically greater in males. The basis for these observed sex differences is not well understood, and potentially may facilitate novel approaches to reducing disease from a range of conditions. We here investigated sex differences in HIV-infected children in relation to antiretroviral therapy (ART) initiation and post-treatment outcome. In a South African cohort of 2,101 HIV-infected children, we observed that absolute CD4+ count and CD4% were significantly higher in ART-naïve female, compared to age-matched male, HIV-infected children. Absolute CD4 count and CD4% were also significantly higher in HIV-uninfected female versus male neonates. We next showed that significantly more male than female children were initiated on ART (47% female); and children not meeting criteria to start ART by >5 yrs were more frequently female (59%; p<0.001). Among ART-treated children, immune reconstitution of CD4 T-cells was more rapid and more complete in female children, even after adjustment for pre-ART absolute CD4 count or CD4% (p=0.011, p=0.030, respectively). However, while ART was initiated as a result of meeting CD4 criteria less often in females (45%), ART initiation as a result of clinical disease in children whose CD4 counts were above treatment thresholds occurred more often in females (57%, p<0.001). The main sex difference in morbidity observed in children initiating ART above CD4 thresholds, above that of TB disease, was as a result of wasting and stunting observed in females with above-threshold CD4 counts (p=0.002). These findings suggest the possibility that optimal treatment of HIV-infected children might incorporate differential CD4 treatment thresholds for ART initiation according to sex.

Discordant Impact of HLA on Viral Replicative Capacity and Disease Progression in Pediatric and Adult HIV Infection.

HLA class I polymorphism has a major influence on adult HIV disease progression. An important mechanism mediating this effect is the impact on viral replicative capacity (VRC) of the escape mutations selected in response to HLA-restricted CD8+ T-cell responses. Factors that contribute to slow progression in pediatric HIV infection are less well understood. We here investigate the relationship between VRC and disease progression in pediatric infection, and the effect of HLA on VRC and on disease outcome in adult and pediatric infection. Studying a South African cohort of >350 ART-naïve, HIV-infected children and their mothers, we first observed that pediatric disease progression is significantly correlated with VRC. As expected, VRCs in mother-child pairs were strongly correlated (p = 0.004). The impact of the protective HLA alleles, HLA-B*57, HLA-B*58:01 and HLA-B*81:01, resulted in significantly lower VRCs in adults (p<0.0001), but not in children. Similarly, in adults, but not in children, VRCs were significantly higher in subjects expressing the disease-susceptible alleles HLA-B*18:01/45:01/58:02 (p = 0.007). Irrespective of the subject, VRCs were strongly correlated with the number of Gag CD8+ T-cell escape mutants driven by HLA-B*57/58:01/81:01 present in each virus (p = 0.0002). In contrast to the impact of VRC common to progression in adults and children, the HLA effects on disease outcome, that are substantial in adults, are small and statistically insignificant in infected children. These data further highlight the important role that VRC plays both in adult and pediatric progression, and demonstrate that HLA-independent factors, yet to be fully defined, are predominantly responsible for pediatric non-progression.

Long-range transcriptional control of an operon necessary for virulence-critical ESX-1 secretion in Mycobacterium tuberculosis.

The ESX-1 secretion system of Mycobacterium tuberculosis has to be precisely regulated since the secreted proteins, although required for a successful virulent infection, are highly antigenic and their continued secretion would alert the immune system to the infection. The transcription of a five-gene operon containing espACD-Rv3613c-Rv3612c, which is required for ESX-1 secretion and is essential for virulence, was shown to be positively regulated by the EspR transcription factor. Thus, transcription from the start site, found to be located 67 bp upstream of espA, was dependent upon EspR enhancer-like sequences far upstream (between 884 and 1,004 bp), which we term the espA activating region (EAR). The EAR contains one of the known binding sites for EspR, providing the first in vivo evidence that transcriptional activation at the espA promoter occurs by EspR binding to the EAR and looping out DNA between this site and the promoter. Regulation of transcription of this operon thus takes place over long regions of the chromosome. This regulation may differ in some members of the M. tuberculosis complex, including Mycobacterium bovis, since deletions of the intergenic region have removed the upstream sequence containing the EAR, resulting in lowered espA expression. Consequent differences in expression of ESX-1 in these bacteria may contribute to their various pathologies and host ranges. The virulence-critical nature of this operon means that transcription factors controlling its expression are possible drug targets.