A cascade toehold-mediated strand displacement strategy for label-free and sensitive non-enzymatic recycling amplification detection of the HIV-1 gene.

In this work, a label-free fluorescence biosensor for simple detection of the HIV-1 gene was proposed by using toehold-mediated strand displacement reactions (TMSDRs) combined with a non-enzymatic target recycling amplification strategy. In this system, two TMSDRs were used. In the presence of the HIV-1 gene, an autocatalytic DNA machine can be activated. This leads to the generation of numerous free G-rich sequences, which can associate with a fluorescent dye N-methylmesoporphyrin IX (NMM) to yield an amplified fluorescence signal for the target detection. This sensing platform showed a high sensitivity towards the HIV-1 gene with a detection limit as low as 1.9 pM without any labelling, immobilization, or washing steps. The designed sensing system also exhibits an excellent selectivity for the HIV-1 gene compared with other interference DNA sequences. Furthermore, the presented biosensor is robust and has been successfully applied for the detection of the HIV-1 gene in a real biological sample with satisfactory results, suggesting that this method is promising for simple and early clinical diagnosis of HIV infection. Thanks to its simplicity, cost-effectiveness and ultrasensitivity, our proposed sensing strategy provides a universal platform for the detection of other genes by substituting the target-recognition element.

HIV-1 causes CD4 cell death through DNA-dependent protein kinase during viral integration.

Human immunodeficiency virus-1 (HIV-1) has infected more than 60 million people and caused nearly 30 million deaths worldwide, ultimately the consequence of cytolytic infection of CD4(+) T cells. In humans and in macaque models, most of these cells contain viral DNA and are rapidly eliminated at the peak of viraemia, yet the mechanism by which HIV-1 induces helper T-cell death has not been defined. Here we show that virus-induced cell killing is triggered by viral integration. Infection by wild-type HIV-1, but not an integrase-deficient mutant, induced the death of activated primary CD4 lymphocytes. Similarly, raltegravir, a pharmacologic integrase inhibitor, abolished HIV-1-induced cell killing both in cell culture and in CD4(+) T cells from acutely infected subjects. The mechanism of killing during viral integration involved the activation of DNA-dependent protein kinase (DNA-PK), a central integrator of the DNA damage response, which caused phosphorylation of p53 and histone H2AX. Pharmacological inhibition of DNA-PK abolished cell death during HIV-1 infection in vitro, suggesting that processes which reduce DNA-PK activation in CD4 cells could facilitate the formation of latently infected cells that give rise to reservoirs in vivo. We propose that activation of DNA-PK during viral integration has a central role in CD4(+) T-cell depletion, raising the possibility that integrase inhibitors and interventions directed towards DNA-PK may improve T-cell survival and immune function in infected individuals.

Beyond the replication-competent HIV reservoir: transcription and translation-competent reservoirs.

Recent years have seen a substantial increase in the number of tools available to monitor and study HIV reservoirs. Here, we discuss recent technological advances that enable an understanding of reservoir dynamics beyond classical assays to measure the frequency of cells containing provirus able to propagate a spreading infection (replication-competent reservoir). Specifically, we focus on the characterization of cellular reservoirs containing proviruses able to transcribe viral mRNAs (so called transcription-competent) and translate viral proteins (translation-competent). We suggest that the study of these alternative reservoirs provides complementary information to classical approaches, crucially at a single-cell level. This enables an in-depth characterization of the cellular reservoir, both following reactivation from latency and, importantly, directly ex vivo at baseline. Furthermore, we propose that the study of cellular reservoirs that may not contain fully replication-competent virus, but are able to produce HIV mRNAs and proteins, is of biological importance. Lastly, we detail some of the key contributions that the study of these transcription and translation-competent reservoirs has made thus far to investigations into HIV persistence, and outline where these approaches may take the field next.

Tools for Visualizing HIV in Cure Research.

PURPOSE OF REVIEW: The long-lived HIV reservoir remains a major obstacle for an HIV cure. Current techniques to analyze this reservoir are generally population-based. We highlight recent developments in methods visualizing HIV, which offer a different, complementary view, and provide indispensable information for cure strategy development. RECENT FINDINGS: Recent advances in fluorescence in situ hybridization techniques enabled key developments in reservoir visualization. Flow cytometric detection of HIV mRNAs, concurrently with proteins, provides a high-throughput approach to study the reservoir on a single-cell level. On a tissue level, key spatial information can be obtained detecting viral RNA and DNA in situ by fluorescence microscopy. At total-body level, advancements in non-invasive immuno-positron emission tomography (PET) detection of HIV proteins may allow an encompassing view of HIV reservoir sites. HIV imaging approaches provide important, complementary information regarding the size, phenotype, and localization of the HIV reservoir. Visualizing the reservoir may contribute to the design, assessment, and monitoring of HIV cure strategies in vitro and in vivo.

Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique.

UNLABELLED: HIV-1 establishes a pool of latently infected cells early following infection. New therapeutic approaches aiming at diminishing this persisting reservoir by reactivation of latently infected cells are currently being developed and tested. However, the reactivation kinetics of viral mRNA and viral protein production, and their respective consequences for phenotypical changes in infected cells that might enable immune recognition, remain poorly understood. We adapted a novel approach to assess the dynamics of HIV-1 mRNA and protein expression in latently and newly infected cells on the single-cell level by flow cytometry. This technique allowed the simultaneous detection of gagpol mRNA, intracellular p24 Gag protein, and cell surface markers. Following stimulation of latently HIV-1-infected J89 cells with human tumor necrosis factor alpha (hTNF-α)/romidepsin (RMD) or HIV-1 infection of primary CD4(+) T cells, four cell populations were detected according to their expression levels of viral mRNA and protein. gagpol mRNA in J89 cells was quantifiable for the first time 3 h after stimulation with hTNF-α and 12 h after stimulation with RMD, while p24 Gag protein was detected for the first time after 18 h poststimulation. HIV-1-infected primary CD4(+) T cells downregulated CD4, BST-2, and HLA class I expression at early stages of infection, proceeding Gag protein detection. In conclusion, here we describe a novel approach allowing quantification of the kinetics of HIV-1 mRNA and protein synthesis on the single-cell level and phenotypic characterization of HIV-1-infected cells at different stages of the viral life cycle. IMPORTANCE: Early after infection, HIV-1 establishes a pool of latently infected cells, which hide from the immune system. Latency reversal and immune-mediated elimination of these latently infected cells are some of the goals of current HIV-1 cure approaches; however, little is known about the HIV-1 reactivation kinetics following stimulation with latency-reversing agents. Here we describe a novel approach allowing for the first time quantification of the kinetics of HIV-1 mRNA and protein synthesis after latency reactivation or de novo infection on the single-cell level using flow cytometry. This new technique furthermore enabled the phenotypic characterization of latently infected and de novo-infected cells dependent on the presence of viral RNA or protein.

Global landscape of HIV-human protein complexes.

Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV-human protein-protein interactions involving 435 individual human proteins, with ∼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.

Thermus thermophilus-derived protein tags that aid in preparation of insoluble viral proteins.

The expression and solubilization of insoluble proteins have been facilitated by the introduction of protein tags. In our analyses of viral protein R (Vpr) of human immunodeficiency virus 1 (HIV-1), however, several conventional tag proteins enhanced its expression but failed to solubilize it. Therefore, we decided to explore whether proteins derived from Thermus thermophilus HB8 (T. th.), a highly heat-stable bacterium, could be used as tag proteins to enhance the solubilization of Vpr. Based on the data accumulated during the recent structural genomics project of T. th., we selected 15 T. th. proteins with high expression levels and solubilities. From this group, we identified a T. th. tag protein that expressed Vpr in a soluble form. Furthermore, two T. th. tag proteins, including the identified one, were found to solubilize the extremely insoluble membrane-spanning domain of the envelope protein of HIV-1. When green fluorescent protein (GFP) was used as a passenger protein of T. th. tags, the brightness and stability of GFP were similar to those of untagged GFP, suggesting that the T. th. tags do not negatively affect the function of the passenger protein. Thus, data of structural genomics can be applied to generate a customized versatile protein tag for protein analyses.

HIV-1 Vpu inhibits accumulation of the envelope glycoprotein within clathrin-coated, Gag-containing endosomes.

Several viruses encode ion channels that both modulate the trafficking of envelope glycoprotein(s) and stimulate the release of virions from cells. HIV-1 Vpu enhances virion release and inhibits the endosomal accumulation of the viral structural protein Gag. We investigated whether Vpu affects the subcellular distribution of Env as well as Gag. Env and Vpu colocalized with each other, in part within the trans-Golgi network. In the absence of Vpu, Env accumulated more extensively within clathrin-coated endosomal structures. These structures had several features consistent with an endosomal viral assembly domain: they contained Gag, including proteolytically processed viral matrix protein; the tetraspanins CD63 and CD81; the adaptor protein complex AP-3; and AIP1/ALIX, a cellular cofactor for viral budding. These endosomes labelled incompletely with Env derived from the cell surface, suggesting that some Env reaches this compartment without transiting the plasma membrane. Consistent with this, endosomal accumulation of Env was not blocked by dominant-negative Eps15, an inhibitor of AP-2-mediated endocytosis. Although these data are potentially explained by greater endocytosis of mature virions in the absence of Vpu, they also raise the possibility that Vpu inhibits the transport of Env and Gag to late endosomes, leading to viral assembly at the plasma membrane.

Identification, Localization, and Quantification of HIV Reservoirs Using Microscopy.

The major barrier to eradicating human immunodeficiency virus-1 (HIV) infection is the generation and extended survival of HIV reservoirs. In order to eradicate HIV infection, it is essential to detect, quantify, and characterize circulating and tissue-associated viral reservoirs in infected individuals. Currently, PCR-based technologies and Quantitative Viral Outgrowth Assays (Q-VOA) are the gold standards to detect viral reservoirs. However, these methods are limited to detecting circulating viral reservoirs, and it has been shown that they misrepresent the size of the reservoirs, largely because they detect only one component of the HIV life cycle and are unable to detect viral reservoirs in tissues. Here, we described the use of multiple detection systems to identify integrated HIV DNA or viral mRNA and several HIV proteins in circulating and tissue reservoirs using improved staining and microscopy techniques. We believe that this imaging-based approach for detecting HIV reservoirs will lead to breakthroughs necessary to eradicate these reservoirs. © 2018 by John Wiley & Sons, Inc.

Longitudinal quasispecies analysis of viral variants in HIV type 1 dually infected individuals highlights the importance of sequence identity in viral recombination.

Little is known regarding the likelihood of recombination between any given pair of nonidentical HIV-1 viruses in vivo. The present study analyzes the HIV-1 quasispecies in the C1C2 region of env, the vif-vpr-vpu accessory gene region, and the reverse transcriptase region of pol. These sequences were amplified from samples obtained sequentially over a 12- to 33-month period from five dually HIV-1-infected subjects. Analysis of an average of 248 clones amplified from each subject revealed no recombinants within the three loci studied of the subtype-discordant infecting strains, whose genetic diversity was >11% in env. In contrast, two subjects who were initially coinfected by two subtype-concordant variants with genetic diversity of 7.4% in env were found to harbor 10 unique recombinants of these strains, as exhibited by analysis of the env gene. The frequent recombination observed among the subtype-concordant strains studied herein correlates with prior sequence analyses that have commonly found higher rates of recombination at loci bearing the most conserved sequences, demonstrating an important role for sequence identity in HIV-1 recombination. Viral load analysis revealed that the samples studied contained an average of 8125 virus copies/ml (range, 882-31,626 copies/ml), signifying that the amount of viral RNA in the samples was not limiting for studying virus diversity. These data reveal that recombination between genetically distant strains may not be an immediate or common outcome to dual infection in vivo and suggest critical roles for viral and host factors such as viral fitness, virus diversity, and host immune responses that may contribute to limiting the frequency of intersubtype recombination during in vivo dual infection.

Estimating diagnostic accuracy of multiple binary tests with an imperfect reference standard.

The goal in diagnostic medicine is often to estimate the diagnostic accuracy of multiple experimental tests relative to a gold standard reference. When a gold standard reference is not available, investigators commonly use an imperfect reference standard. This paper proposes methodology for estimating the diagnostic accuracy of multiple binary tests with an imperfect reference standard when information about the diagnostic accuracy of the imperfect test is available from external data sources. We propose alternative joint models for characterizing the dependence between the experimental tests and discuss the use of these models for estimating individual-test sensitivity and specificity as well as prevalence and multivariate post-test probabilities (predictive values). We show using analytical and simulation techniques that, as long as the sensitivity and specificity of the imperfect test are high, inferences on diagnostic accuracy are robust to misspecification of the joint model. The methodology is demonstrated with a study examining the diagnostic accuracy of various HIV-antibody tests for HIV.

Macronutrientes: relación inversa entre consumo de proteínas vegetales y presión arterial de adolescentes que acuden al Centro de Atención a Pacientes con Enfermedades Infectocontagiosas "Dra. Elsa La Corte" Facultad de Odontología (CAPEI/UCV) / Macronutrients: inverse relationship between vegetable protein intake and blood preassure of adolescents that attend CAPEI/UCV

Existen evidencias que apuntan a la existencia de una asociación inversa entre la ingesta de proteínas vegetales y la presión arterial. En este estudio, los datos de adolescentes VIH +, que acuden a la consulta del Centro de Atención a Pacientes con Enfermedades Infectocontagiosas "Dra. Elsa La Corte" (CAPEI) de la Universidad Central de Venezuela (UCV), fueron analizados para estudiar la relación entre el consumo de proteínas vegetales y la presión arterial tanto sistólica como diastólica, ajustando por índice de masa corporal (IMC) y consumo de energía. Estudio transversal en 43 adolescentes VIH+ con edades entre 15 y 18 años en ambos sexos, que acudieron al CAPEI en el año 2009. Se analizó la media de dos lecturas obtenidas con 5 minutos de intervalo en una visita. Se determinaron peso y altura y se calculó el IMC. Para la determinación de la ingesta de proteínas vegetales se aplicó la técnica de recordatorio de 24 horas. El análisis estadístico se basó en el modelo de regresión lineal. Los resultados muestran una asociación negativa y significativa entre el consumo de proteínas vegetales y la presión arterial sistólica y diastólica, después de ajustar por consumo de energía e IMC, las diferencias de presión arterial sistólica y diastólica asociada con una ingesta de proteínas vegetales de 57,6 kilocalorías por ciento (variación intercuartil) fue de -2,8 mm Hg y -2,4 mm Hg, respectivamente (p <0,05 para ambos). La promoción y planificación de dietas con altos contenidos de proteínas vegetales puede ser de utilidad para prevenir y controlar valores elevados de la presión arterial

Data are available that indicate an independent inverse relationship of dietary vegetable protein to blood pressure (BP). In this investigation data from HIV adolescents attending CAPEI/UCV, were analyzed to study the relationship between dietary vegetable protein and systolic/diastolic pressures, with control for body mass index (BMI) and calorie intake. This was a cross-sectional study with 43 HIV adolescents 15 to 18 years of age. BP was measured 2 times at 1 visit; height and weight were measured, and BMI was calculated; dietary data were obtained from 24-hour dietary recalls. Multivariate regression was applied.The results showed that with control for BMI and calorie intake, estimated average BP differences associated with a vegetable protein intake that was higher by 57,6 %kcal (interquartile range) were -2,8 mm Hg systolic and -2,4 mm Hg diastolic (p <0,05 both). Broad improvement in diets high in vegetable protein can be important in preventing and controlling high blood pressure