Human Immunodeficiency Virus Genome Research Articles

Evaluation of Rapid Molecular Viral Load Monitoring with ABBOTT m-PIMATM HIV-1/2 VL

World Health Organization recommends rapid results of Human Immunodeficiency Virus (HIV) viral load by using Point of Care (POC) testing, delivering faster decision-making to prevent transmission and suppress drug resistance and greater likelihood of starting therapy if the clinic provides care and treatment services. The m-PIMATM HIV-1/2VL cartridge is an RT-PCR POC test that detects HIV-1 and HIV-2 RNA viral load, recognizing the conserved-part 5'-UTR of the HIV genome. The standard, Roche Cobas®4800 system HIV-1, targets gag and LTR regions. This study aimed to determine the correlation, sensitivity, and specificity of Abbott m-PIMATM HIV-1/2VL compared to Roche Cobas®4800 system HIV-1. A cross-sectional study at Dharmais Hospital Laboratory. EDTA plasma obtained from 200 HIV patients, consisted of 80 high-viral-load samples (>1000 copies/mL), 80 low-viral-load samples (<1000 copies/mL), and 40 undetectable-viral-load samples (<14.2 copies/mL). Kappa Cohen was used to measure the categorical correlation. Sensitivity and specificity were calculated with diagnostic test. Dharmais Ethic Committee released ethical clearance No.0132/KEPK/IX/2020. The categorical correlation of Abbott m-PIMATM HIV-1/2 VL in determining the group of high or low viral load samples was 0.948 (very strong correlation). The categorical correlation deciding whether the HIV viral load sample was high, low, or undetectable was 0.714 (strong correlation). The categorical sensitivity of Abbott m-PIMATM HIV-1/2 VL compared to Roche Cobas®4800 system HIV-1 in categorizing HIV viral load was 97.5%, specificity of 97.5%. Abbott m-PIMATM HIV-1/2 VL performance in determining HIV RNA viral load group samples has a strong correlation compared to Roche Cobas®4800 system HIV-1. A larger study is required.

Zinc selenide based dual-channel SPR optical biosensor for HIV genome DNA hybridization detection

Simultaneous measurement of human immunodeficiency virus (HIV) genome DNA hybridization and the DNA melting temperature in a prism-based surface plasmon resonance (SPR) biosensor is modeled theoretically using a simple dual-channel construction. The proposed sensor consists of a BK7 prism coated with silver as a plasmonic material. The metal surface is divided into two channels to detect medium refractive index (RI) and temperature. One half is covered with zinc selenide (ZnSe) semiconductor to enhance the hybridization detection sensitivity and to protect silver from oxidation. The other half is covered with polydimethylsiloxane (PDMS) polymer to detect the temperature variations. The proposed sensor is optimized numerically, and the optimum structure provides an excellent sensitivity of 208 deg/RIU, thanks to the use of the ZnSe layer, which is greater than double the reported dual-channel prism-based sensor in thickness. The polymer channel shows high sensitivity to the temperature variations of − 0.125 deg/°C, which is nearly 10 times the response of the RI channel to temperature variations. The data obtained from the polymer channel is used to compensate for the thermal perturbations of the sensing medium RI, and at the same time, to monitor the increments of the temperature in order to avoid reaching the DNA melting temperature. A mathematical expression is provided to consider the effect of the temperature variations on the RI of the sensing medium to get a better accurate detection process. The DNA hybridization detection of HIV is theoretically discussed in detail starting from the preparation of the sensing medium with the different ingredients until the hybridization between probe and complementary target DNA (ct-DNA) molecules.

KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir

Human immunodeficiency virus (HIV) infection induces immunological dysfunction, which limits the elimination of HIV-infected cells during treated infection. Identifying and targeting dysfunctional immune cells might help accelerate the purging of the persistent viral reservoir. Here, we show that chronic HIV infection increases natural killer (NK) cell populations expressing the negative immune regulator KLRG1, both in peripheral blood and lymph nodes. Antiretroviral treatment (ART) does not reestablish these functionally impaired NK populations, and the expression of KLRG1 correlates with active HIV transcription. Targeting KLRG1 with specific antibodies significantly restores the capacity of NK cells to kill HIV-infected cells, reactivates latent HIV present in CD4+ Tcells co-expressing KLRG1, and reduces the intact HIV genomes in samples from ART-treated individuals. Our data support the potential use of immunotherapy against the KLRG1 receptor to impact the viral reservoir during HIV persistence.

Genomic analysis of circulating HIV and hepatitis C virus infections and coinfections in Cameroon: 2005–2006 and 2015–2016

IntroductionIn humans, RNA viruses are responsible for a wide range of acute, chronic, emerging and re-emerging infections. Human Immunodeficiency virus (HIV) and hepatitis C virus (HCV) rank as some of the most important public health challenges affecting Africa.MethodsWe performed enzyme-linked immune-sorbent assays to confirm positive specimens, and the genomic characterization on two cohorts of people living with HIV in Douala and Yaoundé for the periods 2005-2006 and 2015-2016. These groups were tested for co-infection with HCV using the enzyme-linked immunosorbent assays. Viral RNA was extracted from positive patients’ plasma samples by QIAGEN method, and specific primers were used to amplify the genes of interest on HIV and HCV genomes. The amplification products were subsequently cloned and sequenced. The nucleotide sequences were aligned, genotyped and phylogenetically analyzed.ResultsThe HIV isolate identified in this study belongs to HIV-1 group M Subtype A1. The HCV subtypes characterized in this study are 1h and 4t corresponding to the dominant strains that circulate in Cameroon. Phylogenetic analysis of the HCV NS5B gene showed that the study viruses cluster with Gabonese, Canadian, and previously sequenced viruses from Cameroon.Conclusion and perspectivesThese results shed light on the genetic diversity of HIV and HCV in Cameroon. Virulent HCV infections are common in Cameroon, and therefore there is a great need for further analysis of the viral evolutionary and spatio-temporal patterns.

CRISPR-Cas12b enables a highly efficient attack on HIV proviral DNA in T cell cultures

BackgroundThe novel endonuclease Cas12b was engineered for targeted genome editing in mammalian cells and is a promising tool for certain applications because of its small size, high sequence specificity and ability to generate relatively large deletions. We previously reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections upon attack of the integrated viral DNA genome by spCas9 and Cas12a. MethodsWe now tested the ability of the Cas12b endonuclease to suppress a spreading HIV infection in cell culture with anti-HIV gRNAs. Virus inhibition was tested in long-term HIV replication studies, which allowed us to test for viral escape and the potential for reaching a CURE of the infected T cells. FindingsWe demonstrate that Cas12b can achieve complete HIV inactivation with only a single gRNA, a result for which Cas9 required two gRNAs. When the Cas12b system is programmed with two antiviral gRNAs, the overall anti-HIV potency is improved and more grossly mutated HIV proviruses are generated as a result of multiple cut-repair actions. Such “hypermutated” HIV proviruses are more likely to be defective due to mutation of multiple essential parts of the HIV genome. We report that the mutational profiles of the Cas9, Cas12a and Cas12b endonucleases differ significantly, which may have an impact on the level of virus inactivation. These combined results make Cas12b the preferred editing system for HIV-inactivation. InterpretationThese results provide in vitro “proof of concept’ for CRISPR-Cas12b mediated HIV-1 inactivation.

Transmitted Drug Resistance Against Integrase Strand Transfer Inhibitors in Iranian HIV-Infected Naïve Patients.

Human Immunodeficiency Virus (HIV) has claimed the lives of millions of people during the past decades. While several antiretroviral drugs like Integrase Strand Transfer Inhibitors (INSTIs) have been introduced to control HIV, Transmitted Drug Resistance (TDR) in HIV genome caused failure in treatment. This study aimed to investigate TDR and natural occurring mutations (NOPs) in HIV integrase gene in Iranian HIV patients. In this cross-sectional study, blood samples of 30 HIV-positive patients who had never taken integrase inhibitors were considered for CD4 T cell count, RT real-time PCR, and, Nested PCR. The sequencing results were analyzed by CLC sequence viewer software and Stanford University HIV Drug Resistance Database. In all samples, nine NOPs with a high prevalence were found; however, we did not find any drug resistance mutations, except for a mutation in one sample, which showed a low resistance level. Subtype A1 was dominant in all samples. Based on the findings and compared to our previous study, all patients were sustainable to main integrase inhibitors, including bictegravir, raltegravir, bictegravir, elvitegravir and dolutegravir. It seems the resistant mutation pattern attributed to integrase inhibitors was not diffent among studied patients; hence, the prescription of such inhibitors helps physicians to control HIV infection in Iranian HIV-infected patients.

Monocyte-derived macrophages contain persistent latent HIV reservoirs

The development of persistent cellular reservoirs of latent human immunodeficiency virus (HIV) is a critical obstacle to viral eradication since viral rebound takes place once anti-retroviral therapy (ART) is interrupted. Previous studies show that HIV persists in myeloid cells (monocytes and macrophages) in blood and tissues in virologically suppressed people with HIV (vsPWH). However, how myeloid cells contribute to the size of the HIV reservoir and what impact they have on rebound after treatment interruption remain unclear. Here we report the development of a human monocyte-derived macrophage quantitative viral outgrowth assay (MDM-QVOA) and highly sensitive T cell detection assays to confirm purity. We assess the frequency of latent HIV in monocytes using this assay in a longitudinal cohort of vsPWH (n = 10, 100% male, ART duration 5–14 yr) and find half of the participants showed latent HIV in monocytes. In some participants, these reservoirs could be detected over several years. Additionally, we assessed HIV genomes in monocytes from 30 vsPWH (27% male, ART duration 5–22 yr) utilizing a myeloid-adapted intact proviral DNA assay (IPDA) and demonstrate that intact genomes were present in 40% of the participants and higher total HIV DNA correlated with reactivatable latent reservoirs. The virus produced in the MDM-QVOA was capable of infecting bystander cells resulting in viral spread. These findings provide further evidence that myeloid cells meet the definition of a clinically relevant HIV reservoir and emphasize that myeloid reservoirs should be included in efforts towards an HIV cure.

Eradication of human immunodeficiency virus-1 reservoir in the brain microglia.

Eradication of human immunodeficiency virus-1 reservoir in the brain microglia.

In-silico Structural Modeling of Human Immunodeficiency Virus Proteins.

Human immunodeficiency virus (HIV) is an infectious virus that depletes the CD4+ T lymphocytes of the immune system and causes a chronic life-treating disease-acquired immunodeficiency syndrome (AIDS). The HIV genome encodes different structural and accessory proteins involved in viral entry and life cycle. Determining the 3D structure of HIV proteins is essential for new target position finding, structure-based drug designing, and future planning for computational and laboratory experimentations. Hence, the study aims to predict the 3D structures of all the HIV structural and accessory proteins using computational homology modeling to understand better the structural basis of HIV proteins interacting with host cells and viral replication. The sequences of HIV capsid, matrix, nucleocapsid, p6, reverse transcriptase, invertase, protease, gp120, gp41, virus protein r, viral infectivity factor, virus protein unique, RNA splicing regulator, transactivator protein, negative regulating factor, and virus protein x proteins were retrieved from UniProt. The primary and secondary structures of HIV proteins were predicted by Expasy ProtParam and SOPMA web servers. For the homology modeling, the MODELLER predicted the 3D structures of HIV proteins using templates. Then, the modeled structures were validated by the Ramachandran plot, local and global quality estimation scores, QMEAN scores, and Z-scores. Most of the amino acid residues of HIV proteins were present in the most favored and generously allowed regions in the Ramachandran plots. The local and global quality scores and Z-scores of the HIV proteins confirmed the good quality of modeled structures. The 3D modeled structures of HIV proteins might help further investigate the possible treatment.

Internal RNA 2'O-methylation in the HIV-1 genome counteracts ISG20 nuclease-mediated antiviral effect.

RNA 2'O-methylation is a 'self' epitranscriptomic modification allowing discrimination between host and pathogen. Indeed, human immunodeficiency virus 1 (HIV-1) induces 2'O-methylation of its genome by recruiting the cellular FTSJ3 methyltransferase, thereby impairing detection by RIG-like receptors. Here, we show that RNA 2'O-methylations interfere with the antiviral activity of interferon-stimulated gene 20-kDa protein (ISG20). Biochemical experiments showed that ISG20-mediated degradation of 2'O-methylated RNA pauses two nucleotides upstream of and at the methylated residue. Structure-function analysis indicated that this inhibition is due to steric clash between ISG20 R53 and D90 residues andthe 2'O-methylated nucleotide. We confirmed that hypomethylated HIV-1 genomes produced in FTSJ3-KO cells were more prone to in vitro degradation by ISG20 than those produced in cells expressing FTSJ3. Finally, we found that reverse-transcription of hypomethylated HIV-1 was impaired in T cells by interferon-induced ISG20, demonstrating the direct antagonist effect of 2'O-methylation on ISG20-mediated antiviral activity.

Probe Capture Enrichment Methods for HIV and HCV Genome Sequencing and Drug Resistance Genotyping.

Human immunodeficiency virus (HIV) infections remain a significant public health concern worldwide. Over the years, sophisticated sequencing technologies such as next-generation sequencing (NGS) have emerged and been utilized to monitor the spread of HIV drug resistance (HIVDR), identify HIV drug resistance mutations, and characterize transmission dynamics. Similar applications also apply to the Hepatitis C virus (HCV), another bloodborne viral pathogen with significant intra-host genetic diversity. Several advantages to using NGS over conventional Sanger sequencing include increased data throughput, scalability, cost-effectiveness when batched sample testing is performed, and sensitivity for quantitative detection of minority resistant variants. However, NGS alone may fail to detect genomes from pathogens present in low copy numbers. As with all sequencing platforms, the primary determinant in achieving quality sequencing data is the quality and quantity of the initial template input. Samples containing degraded RNA/DNA and/or low copy number have been a consistent sequencing challenge. To overcome this limitation probe capture enrichment is a method that has recently been employed to target, enrich, and sequence the genome of a pathogen present in low copies, and for compromised specimens that contain poor quality nucleic acids. It involves the hybridization of sequence-specific DNA or RNA probes to a target sequence, which is followed by an enrichment step via PCR to increase the number of copies of the targeted sequences after which the samples are subjected to NGS procedures. This method has been performed on pathogens such as bacteria, fungus, and viruses and allows for the sequencing of complete genomes, with high coverage. Post NGS, data analysis can be performed through various bioinformatics pipelines which can provide information on genetic diversity, genotype, virulence, and drug resistance. This article reviews how probe capture enrichment helps to increase the likelihood of sequencing HIV and HCV samples that contain low viral loads and/or are compromised.

HIV Infection Predisposes to Increased Chances of HBV Infection: Current Understanding of the Mechanisms Favoring HBV Infection at Each Clinical Stage of HIV Infection.

Human immunodeficiency virus (HIV) selectively targets and destroys the infection-fighting CD4+ T-lymphocytes of the human immune system, and has a life cycle that encompasses binding to certain cells, fusion to that cell, reverse transcription of its genome, integration of its genome into the host cell DNA, replication of the HIV genome, assembly of the HIV virion, and budding and subsequent release of free HIV virions. Once a host is infected with HIV, the host’s ability to competently orchestrate effective and efficient immune responses against various microorganisms, such as viral infections, is significantly disrupted. Without modern antiretroviral therapy (ART), HIV is likely to gradually destroy the cellular immune system, and thus the initial HIV infection will inexorably evolve into acquired immunodeficiency syndrome (AIDS). Generally, HIV infection in a patient has an acute phase, a chronic phase, and an AIDS phase. During these three clinical stages, patients are found with relatively specific levels of viral RNA, develop rather distinctive immune conditions, and display unique clinical manifestations. Convergent research evidence has shown that hepatitis B virus (HBV) co-infection, a common cause of chronic liver disease, is fairly common in HIV-infected individuals. HBV invasion of the liver can be facilitated by HIV infection at each clinical stage of the infection due to a number of contributing factors, including having identical transmission routes, immunological suppression, gut microbiota dysbiosis, poor vaccination immune response to hepatitis B immunization, and drug hepatotoxicity. However, there remains a paucity of research investigation which critically describes the influence of the different HIV clinical stages and their consequences which tend to favor HBV entrenchment in the liver. Herein, we review advances in the understanding of the mechanisms favoring HBV infection at each clinical stage of HIV infection, thus paving the way toward development of potential strategies to reduce the prevalence of HBV co-infection in the HIV-infected population.

A quantitative analysis of the similarities and differences of the HIV/FIV gag genome

Among viruses, human immunodeficiency virus (HIV) presents the greatest challenge to humans. Here, we retrieved genome sequences from NCBI and were then run through LALIGN bioinformatics software to compute the E value, bit score, Waterman eggert score, and percent identity, which are four important indicators of how similar the sequences are. These 4 values are 3.1 x 10^-9,51.9, 241 and 55.4%.Bases 1600 to 1990 in HIV and bases 800 to 910 in FIV have a higher than normal similarity. This reflects that while the DNA sequences of the gag region of both the HIV and FIV genomes are rather similar, it is unlikely that this similarity is due to random chance; therefore, there are a noticeable number of differences. A better understanding of the level of similarity and differences in the gag region of the genome sequence would facilitate our understanding of structural and cellular behavioral differences between FIV and HIV, and in the long term, it will provide new insights into the differences observed in previous studies or even facilitate the development of an effective HIV treatment.

The TAR binding dynamics and its implication in Tat degradation mechanism

Human immunodeficiency virus (HIV) is a retrovirus that progressively attacks the human immune system. It is known that the HIV viral protein Tat recruits the host elongation factor, positive transcription elongation factor b (P-TEFb), onto the nascent HIV viral transactivation response element (TAR) RNA to overcome the elongation pause for active transcription of the entire viral genome. Interestingly, there exists an amplifying feedback loop between Tat and TAR—a reduction in Tat increases the elongation pause, resulting in more TAR RNA fragments instead of the entire viral genome transcript, and the TAR fragments as a scaffold for PRC2 complex in turn promote Tat ubiquitination and degradation. In this study, the structural ensembles and binding dynamics of various interfaces in the Tat/TAR/P-TEFb complex are probed by all-atom accelerated sampling molecular dynamics simulations. The results show that a protein-binding inhibitor F07#13 targeting the Tat/P-TEFb interface initiates the above feedback loop and shuts down the active transcription. Another RNA binding inhibitor, JB181, targeting the Tat/TAR interface, can prevent TAR from pulling down the Tat from P-TEFb protein and further reducing Tat degradation. The detailed mechanism of the complex dynamics helps elucidate how Tat and TAR coordinate the regulation between HIV genome transcription versus possible HIV latency.

Анализ частоты формирования мутаций резистентности ВИЧ, ассоциированных с устойчивостью к антиретровирусным препаратам

Актуальность. В Украине проводится широкомасштабная антиретровирусная терапия (АРТ) больных ВИЧ-инфекцией. Необходимо постоянное наблюдение за развитием устойчивости ВИЧ к антиретровирусным препаратам (АРВП). Цель: проанализировать влияние продолжительности АРТ, возраста и пола пациентов на частоту формирования мутаций резистентности (МР) ВИЧ к АРВП разных классов. Материал и методы. Исследовали образцы плазмы крови ВИЧ-инфицированных пациентов с вирусологической неэффективностью АРТ. Секвенирование генома ВИЧ проводили на тест-системах ViroSeqTM Genotyping System v.2.1 (Celera Diagnostics, США) согласно инструкции производителя. Результаты. Достоверно чаще (р ≤ 0,05) МР ВИЧ к АРВП выявлялись у ВИЧ-инфицированных мужчин (59,70 %), чем у женщин (40,3 %). Установлено, что в 59,9 % случаев вирусологическая неэффективность АРТ была обусловлена формированием МР ВИЧ хотя бы к одному из АРВП. Среди МР ВИЧ к ненуклеозидным ингибиторам обратной транскриптазы наиболее распространенными были G190S (75,8 %); K101E (72,7%); Y181C (68,1 %); K103N (38,8 %); среди МР к нуклеозидным ингибиторам обратной транскриптазы преобладали: M184V (69 %), K65R (61 %), D67N (20 %). МР ВИЧ к ингибитору протеазы встречались редко. Выводы. Показано, что продолжительность АРТ и возраст пациентов существенного влияния на устойчивость ВИЧ к АРВП не оказывали. Решающее значение имел генетический барьер АРВП: на фоне приема препаратов с низким генетическим барьером частота выявления МР ВИЧ составляла 5,3 %; прием препаратов с высоким генетическим барьером сопровождался формированием МР ВИЧ значительно реже — в 1,1 % случаев. Доказано, что биоповеденческие особенности пациентов-мужчин являются фактором, ассоциирующимся с повышенным риском формирования МР ВИЧ к АРВП.

Variations in the Abortive HIV-1 RNA Hairpin Do Not Impede Viral Sensing and Innate Immune Responses.

The highly conserved trans-acting response element (TAR) present in the RNA genome of human immunodeficiency virus 1 (HIV-1) is a stably folded hairpin structure involved in viral replication. However, TAR is also sensed by viral sensors, leading to antiviral immunity. While high variation in the TAR RNA structure renders the virus replication-incompetent, effects on viral sensing remain unclear. Here, we investigated the role of TAR RNA structure and stability on viral sensing. TAR mutants with deletions in the TAR hairpin that enhanced thermodynamic stability increased antiviral responses. Strikingly, TAR mutants with lower stability due to destabilization of the TAR hairpin also increased antiviral responses without affecting pro-inflammatory responses. Moreover, mutations that affected the TAR RNA sequence also enhanced specific antiviral responses. Our data suggest that mutations in TAR of replication-incompetent viruses can still induce immune responses via viral sensors, hereby underscoring the robustness of HIV-1 RNA sensing mechanisms.

Human Immunodeficiency Virus (HIV) Genetic Diversity Informs Stage of HIV-1 Infection Among Patients Receiving Antiretroviral Therapy in Botswana.

BackgroundHuman immunodeficiency virus (HIV)-1 genetic diversity increases during infection and can help infer the time elapsed since infection. However, the effect of antiretroviral treatment (ART) on the inference remains unknown.MethodsParticipants with estimated duration of HIV-1 infection based on repeated testing were sourced from cohorts in Botswana (n = 1944). Full-length HIV genome sequencing was performed from proviral deoxyribonucleic acid. We optimized a machine learning model to classify infections as < or >1 year based on viral genetic diversity, demographic, and clinical data.ResultsThe best predictive model included variables for genetic diversity of HIV-1 gag, pol, and env, viral load, age, sex, and ART status. Most participants were on ART. Balanced accuracy was 90.6% (95% confidence interval, 86.7%–94.1%). We tested the algorithm among newly diagnosed participants with or without documented negative HIV tests. Among those without records, those who self-reported a negative HIV test within <1 year were more frequently classified as recent than those who reported a test >1 year previously. There was no difference in classification between those self-reporting a negative HIV test <1 year, whether or not they had a record.ConclusionsThese results indicate that recency of HIV-1 infection can be inferred from viral sequence diversity even among patients on suppressive ART.

Investigation of drug resistance against protease, reverse transcriptase, and integrase inhibitors by next-generation sequencing in HIV-positive patients.

Our aim was to investigate the mutations in protease (PR), reverse transcriptase (RT), and integrase (IN) gene regions in human immunodeficiency virus (HIV) using a single amplicon via next-generation sequencing (NGS). The study included plasma samples from 49 HIV-1-positive patients, which were referred for HIV-1 drug resistance testing during 2017. A nested polymerase chain reaction (PCR) was performed after the RNA extraction and one-step reverse transcription stages. The sequencing of the HIV genome in the PR, RT, and IN gene regions was carried out using MiSeq NGS technology. Sanger sequencing (SS) was used to analyze resistance mutations in the PR and RT gene regions using aViroSeq HIV-1 Genotyping System. PCR products were analyzed with anABI3500 GeneticAnalyzer (Applied Biosystems). Resistance mutations detected with NGS at frequencies above 20% were identical to the SS results. Resistance to at least one antiretroviral (ARV) drug was 22.4% (11 of 49) with NGS and 10.2% (5 of 49) with SS. At least one low-frequency resistance mutation was detected in 18.3% (9 of 49) of the samples. Low-frequency resistance mutations resulted in virological failure in only one patient. The cost of the analyses was reduced by sample pooling and multiplex analysis using the MiSeq system.This is the first study in Turkey to use NGS technologies for the detection of resistance mutations in all three gene(PR, RT, IN) regions using a single amplicon. Our findings suggest that NGS is more sensitive and cost-effective than the SS method.

Region-specific effects of HIV-1 Tat on intrinsic electrophysiological properties of pyramidal neurons in mouse prefrontal cortex and hippocampus.

Human immunodeficiency virus (HIV)-1 transactivator of transcription protein (Tat) is a viral protein that promotes transcription of the HIV genome and possesses cell-signaling properties. Long-term exposure of central nervous system (CNS) tissue to HIV-1 Tat is theorized to contribute to HIV-associated neurodegenerative disorder (HAND). In the current study, we sought to directly evaluate the effect of HIV-1 Tat expression on the intrinsic electrophysiological properties of pyramidal neurons located in layer 2/3 of the medial prefrontal cortex and in area CA1 of the hippocampus. Toward that end, we drove Tat expression with doxycycline (100 mg·kg-1·day-1 ip) in inducible Tat (iTat) transgenic mice for 7 days and then performed single-cell electrophysiological studies in acute tissue slices made through the prefrontal cortex and hippocampus. Control experiments were performed in doxycycline-treated G-tg mice, which retain the tetracycline-sensitive promoter but do not express Tat. Our results indicated that the predominant effects of HIV-1 Tat expression are excitatory in medial prefrontal cortical pyramidal neurons yet inhibitory in hippocampal pyramidal neurons. Notably, in these two populations, HIV-1 Tat expression produced differential effects on neuronal gain, membrane time constant, resting membrane potential, and rheobase. Similarly, we also observed distinct effects on action potential kinetics and afterhyperpolarization, as well as on the current-voltage relationship in subthreshold voltage ranges. Collectively, these data provide mechanistic evidence of complex and region-specific changes in neuronal physiology by which HIV-1 Tat protein may promote cognitive deficits associated with HAND.NEW & NOTEWORTHY We drove expression of human immunodeficiency virus (HIV)-1 transactivator of transcription protein (Tat) protein in inducible Tat (iTat) transgenic mice for 7 days and then examined the effects on the intrinsic electrophysiological properties of pyramidal neurons located in the medial prefrontal cortex (mPFC) and in the hippocampus. Our results reveal a variety of specific changes that promote increased intrinsic excitability of layer II/III mPFC pyramidal neurons and decreased intrinsic excitability of hippocampal CA1 pyramidal neurons, highlighting both cell type and region-specific effects.

Evaluation of Phylogenetic Methods for Inferring the Direction of Human Immunodeficiency Virus (HIV) Transmission: HIV Prevention Trials Network (HPTN) 052.

Phylogenetic analysis can be used to assess human immunodeficiency virus (HIV) transmission in populations. We inferred the direction of HIV transmission using whole-genome HIV sequences from couples with known linked infection and known transmission direction. Complete next-generation sequencing (NGS) data were obtained for 105 unique index-partner sample pairs from 32 couples enrolled in the HIV Prevention Trials Network (HPTN) 052 study (up to 2 samples/person). Index samples were obtained up to 5.5 years before partner infection; partner samples were obtained near the time of seroconversion. The bioinformatics method, phyloscanner, was used to infer transmission direction. Analyses were performed using samples from individual sample pairs, samples from all couples (1 sample/person; group analysis), and all available samples (multisample group analysis). Analysis was also performed using NGS data from defined regions of the HIV genome (gag, pol, env). Using whole-genome NGS data, transmission direction was inferred correctly (index to partner) for 98 of 105 (93.3%) of the individual sample pairs, 99 of 105 (94.3%) sample pairs using group analysis, and 31 of the 32 couples (96.9%) using multisample group analysis. There were no cases where the incorrect transmission direction (partner to index) was inferred. The accuracy of the method was higher with greater time between index and partner sample collection. Pol region sequences performed better than env or gag sequences for inferring transmission direction. We demonstrate the potential of a phylogenetic method to infer the direction of HIV transmission between 2 individuals using whole-genome and pol NGS data.