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1.
J Acquir Immune Defic Syndr ; 94(2S): S5-S12, 2023 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-37707842

RESUMEN

BACKGROUND: There is an urgent need to increase diversity among scientific investigators in the HIV research field to be more reflective of communities highly affected by the HIV epidemic. Thus, it is critical to promote the inclusion and advancement of early-stage scholars from racial and ethnic groups underrepresented in HIV science and medicine. METHODS: To widen the HIV research career pathway for early-stage scholars from underrepresented minority groups, the National Institutes of Health supported the development of the Centers for AIDS Research (CFAR) Diversity, Equity, and Inclusion Pathway Initiative (CDEIPI). This program was created through partnerships between CFARs and Historically Black Colleges and Universities and other Minority Serving Institutions throughout the United States. RESULTS: Seventeen CFARs and more than 20 Historically Black Colleges and Universities and Minority Serving Institutions have participated in this initiative to date. Programs were designed for the high school (8), undergraduate (13), post baccalaureate (2), graduate (12), and postdoctoral (4) levels. Various pedagogical approaches were used including didactic seminar series, intensive multiday workshops, summer residential programs, and mentored research internship opportunities. During the first 18 months of the initiative, 257 student scholars participated in CDEIPI programs including 150 high school, 73 undergraduate, 3 post baccalaureate, 27 graduate, and 4 postdoctoral students. CONCLUSION: Numerous student scholars from a wide range of educational levels, geographic backgrounds, and racial and ethnic minority groups have engaged in CDEIPI programs. Timely and comprehensive program evaluation data will be critical to support a long-term commitment to this unique training initiative.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida , Infecciones por VIH , Estados Unidos , Humanos , Etnicidad , Diversidad, Equidad e Inclusión , Grupos Minoritarios
2.
J Acquir Immune Defic Syndr ; 94(2S): S80-S85, 2023 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-37707853

RESUMEN

BACKGROUND: The Texas Developmental Center for AIDS Research (D-CFAR) diversity program, termed the CFAR Diversity, Equity, and Inclusion Pathway Initiative (CDEIPI), was created in 2021 to engage high school students and graduate students from Underrepresented Minorities/Black, Indigenous, and People of Color populations. SETTING: The Texas D-CFAR CDEIPI has partnered with 2 Texas high schools with predominantly economically disadvantaged and minority student populations-Michael E. DeBakey High School for Health Professions in Houston, TX, and the South Texas Independent School District Medical Professions High School in Olmito, TX in the Rio Grande Valley. METHODS: A total of 370 high school student learners at both partner schools participated in presentations of research and career paths related to HIV-1 and SARS-CoV-2 during the 2021-2022 academic year. Afterward, learners completed anonymous surveys to share their self-reported interest in research degrees and careers. RESULTS: Learners reported increased knowledge of related science content and interest in research careers, including HIV-1 research, after each of the sessions. CONCLUSIONS: The programming has been of interest to student learners, and future additions intend to build upon the Texas D-CFAR CDEIPI.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida , COVID-19 , Infecciones por VIH , Seropositividad para VIH , VIH-1 , Humanos , Síndrome de Inmunodeficiencia Adquirida/epidemiología , Síndrome de Inmunodeficiencia Adquirida/prevención & control , Diversidad, Equidad e Inclusión , Texas/epidemiología , SARS-CoV-2 , Infecciones por VIH/epidemiología , Infecciones por VIH/prevención & control
3.
J Leukoc Biol ; 112(5): 1245-1259, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35362118

RESUMEN

The reservoirs of the HIV display cellular properties resembling long-lived immune memory cells that could be exploited for viral clearance. Our interest in developing a cure for HIV stems from the studies of immunologic memory against infections. We and others have found that long-lived immune memory cells employ prosurvival autophagy and antiapoptotic mechanisms to protect their longevity. Here, we describe the rationale for the development of an approach to clear HIV-1 by selective elimination of host cells harboring replication-competent HIV (SECH). While reactivation of HIV-1 in the host cells with latency reversing agents (LRAs) induces viral gene expression leading to cell death, LRAs also simultaneously up-regulate prosurvival antiapoptotic molecules and autophagy. Mechanistically, transcription factors that promote HIV-1 LTR-directed gene expression, such as NF-κB, AP-1, and Hif-1α, can also enhance the expression of cellular genes essential for cell survival and metabolic regulation, including Bcl-xL, Mcl-1, and autophagy genes. In the SECH approach, we inhibit the prosurvival antiapoptotic molecules and autophagy induced by LRAs, thereby allowing maximum killing of host cells by the induced HIV-1 proteins. SECH treatments cleared HIV-1 infections in humanized mice in vivo and in HIV-1 patient PBMCs ex vivo. SECH also cleared infections by the SIV in rhesus macaque PBMCs ex vivo. Research efforts are underway to improve the efficacy and safety of SECH and to facilitate the development of SECH as a therapeutic approach for treating people with HIV.


Asunto(s)
Infecciones por VIH , VIH-1 , Ratones , Animales , Latencia del Virus , FN-kappa B , Macaca mulatta , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/uso terapéutico , Factor de Transcripción AP-1 , Autofagia , Apoptosis , Linfocitos T CD4-Positivos , Activación Viral/genética
4.
Front Immunol ; 12: 693462, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34691016

RESUMEN

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019 in Wuhan, China, and then rapidly spread causing an unprecedented pandemic. A robust serological assay is needed to evaluate vaccine candidates and better understand the epidemiology of coronavirus disease (COVID-19). Methods: We used the full-length spike (S) protein of SARS-CoV-2 for the development of qualitative and quantitative IgG and IgA anti-S enzyme linked immunosorbent assays (ELISA). A total of 320 sera used for assay development were comprised of pandemic sera from SARS-CoV-2 infected adults (n=51) and pre-pandemic sera (n=269) including sera from endemic human coronavirus infected adults. Reverse cumulative curves and diagnostic test statistics were evaluated to define the optimal serum dilution and OD cutoff value for IgG anti-S and IgA anti-S ELISAs. The IgG and IgA anti-S, and three functional antibodies (ACE-2 receptor blocking antibody, lentipseudovirus-S neutralizing antibody, and SARS-CoV-2 neutralizing antibody) were measured using additional SARS-CoV-2 PCR positive sera (n=76) and surveillance sera (n=25). Lastly, the IgG and IgA anti-S levels were compared in different demographic groups. Results: The optimal serum dilution for the qualitative IgG anti-S ELISA was at 1:1024 yielding a 99.6% specificity, 92.2% sensitivity, 92.9% positive predictive value (PPV), and 99.6% negative predictive value (NPV) at a SARS-CoV-2 seroprevalence of 5%. The optimal serum dilution for the qualitative IgA anti-S ELISA was at 1:128 yielding a 98.9% specificity, 76.5% sensitivity, 78.3% PPV, and 98.8% NPV at the same seroprevalence. Significant correlations were demonstrated between the IgG and IgA (r=0.833 for concentrations, r=0.840 for titers) as well as between IgG and three functional antibodies (r=0.811-0.924 for concentrations, r=0.795-0.917 for titers). The IgG and IgA anti-S levels were significantly higher in males than females (p<0.05), and in adults with moderate/severe symptoms than in adults with mild/moderate symptoms (p<0.001). Conclusion: We developed a highly specific and sensitive IgG anti-S ELISA assay to SARS-CoV-2 using full length S protein. The IgG anti-S antibody level was strongly associated with IgA and functional antibody levels in adults with SARS-CoV-2 infection. Gender and disease severity, rather than age, play an important role in antibody levels.


Asunto(s)
Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , SARS-CoV-2/inmunología , Adulto , COVID-19/diagnóstico , Prueba Serológica para COVID-19 , Femenino , Células HEK293 , Humanos
6.
PLoS One ; 16(4): e0244771, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33914760

RESUMEN

Although current antiretroviral therapies (ART) are successful in controlling HIV-1 infection, a stable viral reservoir reactivates when ART is discontinued. Consequently, there is a major research effort to develop approaches to disrupt the latent viral reservoir and enhance the immune system's ability to clear HIV-1. A number of small molecules, termed latency reversal agents (LRAs), have been identified which can reactivate latent HIV-1 in cell lines and patients' cells ex vivo. However, clinical trials have suggested that combinations of LRAs will be required to efficiently reactivate HIV-1 in vivo, especially LRAs that act synergistically by functioning through distinct pathways. To identify novel LRAs, we used an image-based assay to screen a natural compound library for the ability to induce a low level of aggregation of resting primary CD4+ T cells from healthy donors. We identified celastrol as a novel LRA. Celastrol functions synergistically with other classes of LRA to reactivate latent HIV-1 in a Jurkat cell line, suggesting a novel mechanism in its LRA activity. Additionally, celastrol does not appear to activate resting CD4+ T cells at levels at which it can reactivate latent HIV-1. Celastrol appears to represent a novel class of LRAs and it therefore can serve as a lead compound for LRA development.


Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Triterpenos Pentacíclicos/farmacología , Latencia del Virus/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Descubrimiento de Drogas , VIH-1/fisiología , Humanos , Células Jurkat
7.
Virology ; 540: 88-96, 2020 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-31759187

RESUMEN

HIV-1 is dependent upon cellular proteins to mediate the many processes required for viral replication. One such protein, PACS1, functions to localize Furin to the trans-Golgi network where Furin cleaves HIV-1 gp160 Envelope into gp41 and gp120. We show here that PACS1 also shuttles between the nucleus and cytoplasm, associates with the viral Rev protein and its cofactor CRM1, and contributes to nuclear export of viral transcripts. PACS1 appears specific to the Rev-CRM1 pathway and not other retroviral RNA export pathways. Over-expression of PACS1 increases nuclear export of unspliced viral RNA and significantly increases p24 expression in HIV-1-infected Jurkat CD4+ T cells. SiRNA depletion and over-expression experiments suggest that PACS1 may promote trafficking of HIV-1 GagPol RNA to a pathway distinct from that of translation on polyribosomes.


Asunto(s)
Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Interacciones Huésped-Patógeno , ARN Viral/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Carioferinas/metabolismo , Unión Proteica , Transporte de Proteínas , Transporte de ARN , Receptores Citoplasmáticos y Nucleares/metabolismo , Replicación Viral , Proteína Exportina 1
8.
J Virol ; 93(21)2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31375595

RESUMEN

Influenza A virus (IAV) nonstructural protein 1 (NS1), a potent antagonist of the host immune response, is capable of interacting with RNA and a wide range of cellular proteins. NS1 consists of an RNA-binding domain (RBD) and an effector domain (ED) separated by a flexible linker region (LR). H5N1-NS1 has a characteristic 5-residue deletion in the LR, with either G (minor group) or E (major group) at the 71st position, and non-H5N1-NS1 contains E71 with an intact linker. Based on the orientation of the ED with respect to the RBD, previous crystallographic studies have shown that minor group H5N1-NS1(G71), a non-H5N1-NS1 [H6N6-NS1(E71)], and the LR deletion mutant H6N6-NS1(Δ80-84/E71) mimicking the major group H5N1-NS1 exhibit "open," "semiopen," and "closed" conformations, respectively, suggesting that NS1 exhibits a strain-dependent conformational preference. Here we report the first crystal structure of a naturally occurring H5N1-NS1(E71) and show that it adopts an open conformation similar to that of the minor group of H5N1-NS1 [H5N1-NS1(G71)]. We also show that H6N6-NS1(Δ80-84/E71) under a different crystallization condition and H6N6-NS1(Δ80-84/G71) also exhibit open conformations, suggesting that NS1 can adopt an open conformation irrespective of E or G at the 71st position. Our single-molecule fluorescence resonance energy transfer (FRET) analysis to investigate the conformational preference of NS1 in solution showed that all NS1 constructs predominantly exist in an open conformation. Further, our coimmunoprecipitation and binding studies showed that they all bind to cellular factors with similar affinities. Taken together, our studies suggest that NS1 exhibits strain-independent structural plasticity that allows it to interact with a wide variety of cellular ligands during viral infection.IMPORTANCE IAV is responsible for several pandemics over the last century and continues to infect millions annually. The frequent rise in drug-resistant strains necessitates exploring novel targets for developing antiviral drugs that can reduce the global burden of influenza infection. Because of its critical role in the replication and pathogenesis of IAV, nonstructural protein 1 (NS1) is a potential target for developing antivirals. Previous studies suggested that NS1 adopts strain-dependent "open," "semiopen," and "closed" conformations. Here we show, based on three crystal structures, that NS1 irrespective of strain differences can adopt an open conformation. We further show that NS1 from different strains primarily exists in an open conformation in solution and binds to cellular proteins with a similar affinity. Together, our findings suggest that conformational polymorphism facilitated by a flexible linker is intrinsic to NS1, and this may be the underlying factor allowing NS1 to bind several cellular factors during IAV replication.


Asunto(s)
Virus de la Influenza A/química , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Ligandos , Mutación , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo
9.
mBio ; 10(4)2019 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-31289189

RESUMEN

Didehydro-cortistatin A (dCA) is a human immunodeficiency virus type 1 (HIV-1) Tat inhibitor that functions by selectively binding to the RNA binding domain of Tat. In addition to inhibiting viral replication, dCA can drive HIV-1 into a state of "deep latency" in which latent viruses are refractory to reactivation. Mousseau et al. (G. Mousseau, R. Aneja, M. A. Clementz, S. Mediouni, et al., mBio 10:e01750-18, 2019, https://doi.org/10.1128/mBio.01750-18) have now selected dCA-resistant (dCAr) viruses in vitro Remarkably, dCAr viruses do not contain mutations in Tat or the viral transactivation-responsive element (TAR) RNA element that is targeted by Tat. Rather, the viruses contain a combination of mutations in the viral long terminal repeat (LTR) and Nef and Vpr proteins that result in an increase in basal RNA polymerase II (Pol II) transcription of integrated HIV-1. Interestingly, dCAr viruses may be deficient in the establishment of latent infection because of their elevated basal Pol II transcription. dCA holds promise for strategies to achieve a functional cure of HIV-1 infection and justifies efforts to develop additional Tat inhibitors.


Asunto(s)
VIH-1/genética , Compuestos Heterocíclicos de 4 o más Anillos , Humanos , Isoquinolinas , Mutación , ARN Viral , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética
10.
Infect Dis Model ; 4: 215-226, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31236525

RESUMEN

Bordetella pertussis (B. pertussis) is the causative agent of pertussis, also referenced as whooping cough. Although pertussis has been appropriately controlled by routine immunization of infants, it has experienced a resurgence since the beginning of the 21st century. Given that elucidating the immune response to pertussis is a crucial factor to improve therapeutic and preventive treatments, we re-analyzed a time course microarray dataset of B. pertussis infection by applying a newly developed dynamic data analysis pipeline. Our results indicate that the immune response to B. pertussis is highly dynamic and heterologous across different organs during infection. Th1 and Th17 cells, which are two critical types of T helper cell populations in the immune response to B. pertussis, and follicular T helper cells (TFHs), which are also essential for generating antibodies, might be generated at different time points and distinct locations after infection. This phenomenon may indicate that different lymphoid organs may have their unique functions during infection. These findings provide a better understanding of the basic immunology of bacterial infection, which may provide valuable insights for the improvement of pertussis vaccine design in the future.

12.
Virol J ; 16(1): 22, 2019 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-30786885

RESUMEN

BACKGROUND: The regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. The importance of CycT1 and the Positive Transcription Elongation Factor b (P-TEFb) complex for HIV replication is well-established, but regulation of CycT1 expression and protein levels during HIV replication and latency establishment in CD4 T cells is less characterized. METHODS: To better define the regulation of CycT1 levels during HIV replication in CD4 T cells, multiparameter flow cytometry was utilized to study the interaction between HIV replication (intracellular p24) and CycT1 of human peripheral blood memory CD4 T cells infected with HIV in vitro. CycT1 was further examined in CD4 T cells of human lymph nodes. RESULTS: In activated (CD3+CD28 costimulation) uninfected blood memory CD4 T cells, CycT1 was most significantly upregulated in maximally activated (CD69+CD25+ and HLA.DR+CD38+) cells. In memory CD4 T cells infected with HIV in vitro, two distinct infected populations of p24+CycT1+ and p24+CycT1- cells were observed during 7 days infection, suggestive of different phases of productive HIV replication and subsequent latency establishment. Intriguingly, p24+CycT1- cells were the predominant infected population in activated CD4 T cells, raising the possibility that productively infected cells may transition into latency subsequent to CycT1 downregulation. Additionally, when comparing infected p24+ cells to bystander uninfected p24- cells (after bulk HIV infections), HIV replication significantly increased T cell activation (CD69, CD25, HLA.DR, CD38, and Ki67) without concomitantly increasing CycT1 protein levels, possibly due to hijacking of P-TEFb by the viral Tat protein. Lastly, CycT1 was constitutively expressed at higher levels in lymph node CD4 T cells compared to blood T cells, potentially enhancing latency generation in lymphoid tissues. CONCLUSIONS: CycT1 is most highly upregulated in maximally activated memory CD4 T cells as expected, but may become less associated with T cell activation during HIV replication. The progression into latency may further be predicated by substantial generation of p24+CycT1- cells during HIV replication.


Asunto(s)
Linfocitos T CD4-Positivos/virología , Ciclina T/genética , Infecciones por VIH/inmunología , Latencia del Virus/fisiología , Replicación Viral/fisiología , Linfocitos T CD4-Positivos/inmunología , Citometría de Flujo , Regulación de la Expresión Génica , VIH-1/fisiología , Interacciones Microbiota-Huesped , Humanos , Factor B de Elongación Transcripcional Positiva/genética , Activación Transcripcional
13.
Transcription ; 10(2): 111-117, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30375919

RESUMEN

Studies of RNA Polymerase II (Pol II) transcription of the HIV-1 genome are of clinical interest, as the insight gained may lead to strategies to selectively reactivate latent viruses in patients in whom viral replication is suppressed by antiviral drugs. Such a targeted reactivation may contribute to a functional cure of infection. This review discusses five Cyclin-dependent kinases - CDK7, CDK9, CDK11, CDK2, and CDK8 - involved in transcription and processing of HIV-1 RNA. CDK7 is required for Pol II promoter clearance of reactivated viruses; CDK7 also functions as an activating kinase for CDK9 when resting CD4+ T cells harboring latent HIV-1 are activated. CDK9 is targeted by the viral Tat protein and is essential for productive Pol II elongation of the HIV-1 genome. CDK11 is associated with the TREX/THOC complex and it functions in the 3' end processing and polyadenylation of HIV-1 transcripts. CDK2 phosphorylates Tat and CDK9 and this stimulates Tat activation of Pol II transcription. CDK8 may stimulate Pol II transcription of the HIV-1 genome through co-recruitment with NF-κB to the viral promoter. Some notable open questions are discussed concerning the roles of these CDKs in HIV-1 replication and viral latency.


Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , VIH-1/genética , ARN Polimerasa II/metabolismo , Transcripción Genética/genética , Humanos , Poliadenilación
14.
Virology ; 522: 193-198, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30036787

RESUMEN

The related NEAT1_1 and NEAT1_2 long noncoding RNAs (lnc RNAs) have been recently implicated in innate immunity against viral infection. We used CRISPR-Cas9 to generate Jurkat CD4+ T cell lines with a knockout (KO) of the NEAT1 gene. Viabilities of NEAT1 KO Jurkat lines were indistinguishable from parental Jurkat cells, as was the induction of CD69 after T cell activation. The KO lines were however more sensitive to the induction of apoptosis than parental Jurkat cells. HIV-1 replication was higher in the KO lines than parental Jurkat cells, demonstrating an anti-HIV function of NEAT1 lncRNAs. We observed a strong down-regulation of NEAT1 lncRNAs following activation of resting peripheral blood mononuclear cells and purified CD4+ T cells. These findings indicate that HIV-1 infection exploits the normal down-regulation of anti-viral NEAT1 lncRNAs in activated CD4+ T cells to enhance viral replication.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , Evasión Inmune , ARN Largo no Codificante/metabolismo , Replicación Viral , Regulación hacia Abajo , Técnicas de Inactivación de Genes , Humanos , Factores Inmunológicos/genética , Factores Inmunológicos/metabolismo , Células Jurkat , ARN Largo no Codificante/genética
15.
mBio ; 9(3)2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29764948

RESUMEN

Viral pneumonias cause profound worldwide morbidity, necessitating novel strategies to prevent and treat these potentially lethal infections. Stimulation of intrinsic lung defenses via inhalation of synergistically acting Toll-like receptor (TLR) agonists protects mice broadly against pneumonia, including otherwise-lethal viral infections, providing a potential opportunity to mitigate infectious threats. As intact lung epithelial TLR signaling is required for the inducible resistance and as these cells are the principal targets of many respiratory viruses, the capacity of lung epithelial cells to be therapeutically manipulated to function as autonomous antiviral effectors was investigated. Our work revealed that mouse and human lung epithelial cells could be stimulated to generate robust antiviral responses that both reduce viral burden and enhance survival of isolated cells and intact animals. The antiviral protection required concurrent induction of epithelial reactive oxygen species (ROS) from both mitochondrial and dual oxidase sources, although neither type I interferon enrichment nor type I interferon signaling was required for the inducible protection. Taken together, these findings establish the sufficiency of lung epithelial cells to generate therapeutically inducible antiviral responses, reveal novel antiviral roles for ROS, provide mechanistic insights into inducible resistance, and may provide an opportunity to protect patients from viral pneumonia during periods of peak vulnerability.IMPORTANCE Viruses are the most commonly identified causes of pneumonia and inflict unacceptable morbidity, despite currently available therapies. While lung epithelial cells are principal targets of respiratory viruses, they have also been recently shown to contribute importantly to therapeutically inducible antimicrobial responses. This work finds that lung cells can be stimulated to protect themselves against viral challenges, even in the absence of leukocytes, both reducing viral burden and improving survival. Further, it was found that the protection occurs via unexpected induction of reactive oxygen species (ROS) from spatially segregated sources without reliance on type I interferon signaling. Coordinated multisource ROS generation has not previously been described against viruses, nor has ROS generation been reported for epithelial cells against any pathogen. Thus, these findings extend the potential clinical applications for the strategy of inducible resistance to protect vulnerable people against viral infections and also provide new insights into the capacity of lung cells to protect against infections via novel ROS-dependent mechanisms.


Asunto(s)
Células Epiteliales/inmunología , Subtipo H3N2 del Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Especies Reactivas de Oxígeno/inmunología , Animales , Células Epiteliales/virología , Femenino , Humanos , Subtipo H3N2 del Virus de la Influenza A/genética , Gripe Humana/genética , Gripe Humana/virología , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Pulmón/citología , Pulmón/inmunología , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología
16.
AIDS Res Hum Retroviruses ; 34(1): 103-110, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29084447

RESUMEN

The latent HIV-1 reservoir of memory CD4+ T cells that persists during combination antiviral therapy prevents a cure of infection. Insight into mechanisms of latency and viral reactivation are essential for the rational design of strategies to reduce the latent reservoir. In this study, we quantified the levels of >2,600 proteins in the CCL19 primary CD4+ T cell model of HIV-1 latency. We profiled proteins under conditions that promote latent infection and after cells were treated with phorbol 12-myristate 13-acetate (PMA) + ionomycin, which is known to efficiently induce reactivation of latent HIV-1. In an analysis of cells from two healthy blood donors, we identified 61 proteins that were upregulated ≥2-fold, and 36 proteins that were downregulated ≥2-fold under conditions in which latent viruses were reactivated. These differentially expressed proteins are, therefore, candidates for cellular factors that regulate latency or viral reactivation. Two unexpected findings were obtained from the proteomic data: (1) the interactions among the majority of upregulated proteins are largely undetermined in published protein-protein interaction networks and (2) downregulated proteins are strongly associated with Gene Ontology terms related to mitochondrial protein synthesis. This proteomic data set provides a useful resource for future mechanistic studies of HIV-1 latency.


Asunto(s)
Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Activación Viral , Latencia del Virus , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Quimiocina CCL19/genética , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Infecciones por VIH/virología , VIH-1/genética , Humanos , Ionomicina/farmacología , Mapas de Interacción de Proteínas , Proteómica , Acetato de Tetradecanoilforbol/farmacología , Replicación Viral
17.
Curr Pharm Des ; 23(28): 4098-4102, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28677507

RESUMEN

The general mechanism involved in Tat activation of RNA Polymerase II (RNAP II) elongation of the integrated HIV-1 was elucidated over 20 years ago. This mechanism involves Tat binding to the TAR RNA element that forms at the 5' end of viral transcripts and recruiting a general RNAP II elongation factor termed as PTEFb. This elongation factor consists of CDK9 and Cyclin T1, and when recruited by Tat to TAR RNA, CDK9 was proposed to phosphorylate the carboxyl terminal domain of RNAP II and thereby activate elongation. Research in the past two decades has shown that the mechanism of Tat action is considerably more complicated than this simple model. In metabolically active cells, CDK9 and Cyclin T1 are now known to be largely sequestered in a RNA-protein complex termed the 7SK RNP. CDK9 and Cyclin T1 are released from the 7SK RNP by mechanisms not yet fully elucidated and along with Tat, bind to TAR RNA and orchestrate the assembly of a Super Elongation Complex (SEC) containing several additional proteins. CDK9 in the SEC then phosphorylates multiple substrates in the RNAP II complex to activate elongation. Importantly for therapeutic strategies, CDK9 and Cyclin T1 functions are down-regulated in resting CD4+ T cells that harbor latent HIV-1, and their up-regulation is required for reactivation of latent virus. Current strategies for a functional cure of HIV-1 infection therefore are likely to require development of latency reversal agents that up-regulate CDK9 and Cyclin T1 function in resting CD4+ T cells.


Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Linfocitos T CD4-Positivos/virología , Ciclina T/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , Regulación hacia Abajo/genética , Diseño de Fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , ARN Polimerasa II/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Latencia del Virus/efectos de los fármacos
18.
Nucleic Acids Res ; 45(16): 9348-9360, 2017 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-28637181

RESUMEN

In eukaryotic cells, the gene expression status is strictly controlled by epigenetic modifications on chromatin. The repressive status of chromatin largely contributes to HIV latency. Studies have shown that modification of histone H3K27 acts as a key molecular switch for activation or suppression of many cellular genes. In this study, we found that K27-acetylated histone H3 specifically recruited Super Elongation Complex (SEC), the transcriptional elongation complex essential for HIV-1 long terminal repeat (LTR)-mediated and general cellular transcription. Interestingly, H3K27 acetylation further stimulates H3R26 methylation, which subsequently abrogates the recruitment of SEC, forming a negative feedback regulatory loop. Importantly, by inhibiting methyltransferase activity of CARM1, the enzyme responsible for H3R26 methylation, HIV-1 transcription is reactivated in several HIV latency cell models, including a primary resting CD4+ T cell model. When combined with other latency disrupting compounds such as JQ1 or vorinostat/SAHA, the CARM1 inhibitor achieved synergistic effects on HIV-1 activation. This study suggests that coordinated and dynamic modifications at histone H3K27 and H3R26 orchestrate HIV-1 LTR-mediated transcription, and potentially opens a new avenue to disrupt latent HIV-1 infection by targeting specific epigenetic enzymes.


Asunto(s)
Regulación Viral de la Expresión Génica , VIH-1/genética , Código de Histonas , Histonas/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Latencia del Virus/genética , Acetilación , Compuestos de Bencilideno/química , Compuestos de Bencilideno/farmacología , Línea Celular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Duplicado del Terminal Largo de VIH , VIH-1/efectos de los fármacos , VIH-1/metabolismo , VIH-1/fisiología , Humanos , Metilación , Piperidonas/química , Piperidonas/farmacología , Elongación de la Transcripción Genética , Factores de Transcripción/metabolismo , Latencia del Virus/efectos de los fármacos
19.
BMC Bioinformatics ; 18(1): 74, 2017 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-28143596

RESUMEN

BACKGROUND: Gene regulatory interactions are of fundamental importance to various biological functions and processes. However, only a few previous computational studies have claimed success in revealing genome-wide regulatory landscapes from temporal gene expression data, especially for complex eukaryotes like human. Moreover, recent work suggests that these methods still suffer from the curse of dimensionality if a network size increases to 100 or higher. RESULTS: Here we present a novel scalable algorithm for identifying genome-wide gene regulatory network (GRN) structures, and we have verified the algorithm performances by extensive simulation studies based on the DREAM challenge benchmark data. The highlight of our method is that its superior performance does not degenerate even for a network size on the order of 104, and is thus readily applicable to large-scale complex networks. Such a breakthrough is achieved by considering both prior biological knowledge and multiple topological properties (i.e., sparsity and hub gene structure) of complex networks in the regularized formulation. We also validate and illustrate the application of our algorithm in practice using the time-course gene expression data from a study on human respiratory epithelial cells in response to influenza A virus (IAV) infection, as well as the CHIP-seq data from ENCODE on transcription factor (TF) and target gene interactions. An interesting finding, owing to the proposed algorithm, is that the biggest hub structures (e.g., top ten) in the GRN all center at some transcription factors in the context of epithelial cell infection by IAV. CONCLUSIONS: The proposed algorithm is the first scalable method for large complex network structure identification. The GRN structure identified by our algorithm could reveal possible biological links and help researchers to choose which gene functions to investigate in a biological event. The algorithm described in this article is implemented in MATLAB Ⓡ , and the source code is freely available from https://github.com/Hongyu-Miao/DMI.git .


Asunto(s)
Algoritmos , Redes Reguladoras de Genes , Células Epiteliales/metabolismo , Células Epiteliales/virología , Humanos , Virus de la Influenza A/fisiología , Factores de Transcripción/metabolismo
20.
J Virol Methods ; 242: 1-8, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28034670

RESUMEN

Despite intensive study, it is unclear which mechanisms are responsible for latent HIV infection in vivo. One potential mechanism is inhibition of HIV transcriptional elongation, which results in short abortive transcripts containing the trans-activation response (TAR) region. Because the relative levels of total (including short) and processive transcripts provide measures of HIV transcriptional initiation and elongation, there is a compelling need for techniques that accurately measure both. Nonetheless, prior assays for total transcripts have been semi-quantitative and have seen limited application to patient samples. This manuscript reports the validation of quantitative reverse transcription (RT) droplet digital PCR assays for measurement of total (TAR) and processive (R-U5/gag) HIV transcripts. Traditional RT priming strategies can efficiently detect the TAR region on long HIV transcripts but detect <4% of true short transcripts. The TAR assay presented here utilizes an initial polyadenylation step, which provides an accessible RT priming site and detects short and long transcripts with approximately equal efficiency (70%). By applying these assays to blood samples from 8 ART-treated HIV+ individuals, total HIV transcripts were detected at levels >10-fold higher than elongated transcripts, implying a substantial block to transcriptional elongation in vivo. This approach may be applied to other difficult-to-prime RNA targets.


Asunto(s)
Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH , VIH-1/genética , ARN Viral/sangre , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación Viral de la Expresión Génica , Infecciones por VIH/sangre , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , VIH-1/aislamiento & purificación , Humanos , Masculino , Poliadenilación , Transcripción Reversa , Transcripción Genética , Latencia del Virus
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