Bioinformatics Insights on Viral Gene Expression Transactivation: From HIV-1 to SARS-CoV-2.

Viruses provide vital insights into gene expression control. Viral transactivators, with other viral and cellular proteins, regulate expression of self, other viruses, and host genes with profound effects on infected cells, underlying inflammation, control of immune responses, and pathogenesis. The multifunctional Tat proteins of lentiviruses (HIV-1, HIV-2, and SIV) transactivate gene expression by recruiting host proteins and binding to transacting responsive regions (TARs) in viral and host RNAs. SARS-CoV-2 nucleocapsid participates in early viral transcription, recruits similar cellular proteins, and shares intracellular, surface, and extracellular distribution with Tat. SARS-CoV-2 nucleocapsid interacting with the replication-transcription complex might, therefore, transactivate viral and cellular RNAs in the transcription and reactivation of self and other viruses, acute and chronic pathogenesis, immune evasion, and viral evolution. Here, we show, by using primary and secondary structural comparisons, that the leaders of SARS-CoV-2 and other coronaviruses contain TAR-like sequences in stem-loops 2 and 3. The coronaviral nucleocapsid C-terminal domains harbor a region of similarity to TAR-binding regions of lentiviral Tat proteins, and coronaviral nonstructural protein 12 has a cysteine-rich metal binding, dimerization domain, as do lentiviral Tat proteins. Although SARS-CoV-1 nucleocapsid transactivated gene expression in a replicon-based study, further experimental evidence for coronaviral transactivation and its possible implications is warranted.

HIV-1 Tat and morphine interactions dynamically shift striatal monoamine levels and exploratory behaviors over time.

Despite the advent of combination anti-retroviral therapy (cART), nearly half of people infected with HIV treated with cART still exhibit HIV-associated neurocognitive disorders (HAND). HAND can be worsened by co-morbid opioid use disorder. The basal ganglia are particularly vulnerable to HIV-1 and exhibit higher viral loads and more severe pathology, which can be exacerbated by co-exposure to opioids. Evidence suggests that dopaminergic neurotransmission is disrupted by HIV exposure, however, little is known about whether co-exposure to opioids may alter neurotransmitter levels in the striatum and if this in turn influences behavior. Therefore, we assayed motor, anxiety-like, novelty-seeking, exploratory, and social behaviors, and levels of monoamines and their metabolites following 2 weeks and 2 months of Tat and/or morphine exposure in transgenic mice. Morphine decreased dopamine levels, but significantly elevated norepinephrine, the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite 5-hydroxyindoleacetic acid, which typically correlated with increased locomotor behavior. The combination of Tat and morphine altered dopamine, DOPAC, and HVA concentrations differently depending on the neurotransmitter/metabolite and duration of exposure but did not affect the numbers of tyrosine hydroxylase-positive neurons in the mesencephalon. Tat exposure increased the latency to interact with novel conspecifics, but not other novel objects, suggesting the viral protein inhibits exploratory behavior initiation in a context-dependent manner. By contrast, and consistent with prior findings that opioid misuse can increase novelty-seeking behavior, morphine exposure increased the time spent exploring a novel environment. Finally, Tat and morphine interacted to affect locomotor activity in a time-dependent manner, while grip strength and rotarod performance were unaffected. Together, our results provide novel insight into the unique effects of HIV-1 Tat and morphine on monoamine neurochemistry that may underlie their divergent effects on motor and exploratory behavior.

Role of HIV-1 Tat Protein Interactions with Host Receptors in HIV Infection and Pathogenesis.

Each time the virus starts a new round of expression/replication, even under effective antiretroviral therapy (ART), the transactivator of viral transcription Tat is one of the first HIV-1 protein to be produced, as it is strictly required for HIV replication and spreading. At this stage, most of the Tat protein exits infected cells, accumulates in the extracellular matrix and exerts profound effects on both the virus and neighbor cells, mostly of the innate and adaptive immune systems. Through these effects, extracellular Tat contributes to the acquisition of infection, spreading and progression to AIDS in untreated patients, or to non-AIDS co-morbidities in ART-treated individuals, who experience inflammation and immune activation despite virus suppression. Here, we review the role of extracellular Tat in both the virus life cycle and on cells of the innate and adaptive immune system, and we provide epidemiological and experimental evidence of the importance of targeting Tat to block residual HIV expression and replication. Finally, we briefly review vaccine studies showing that a therapeutic Tat vaccine intensifies ART, while its inclusion in a preventative vaccine may blunt escape from neutralizing antibodies and block early events in HIV acquisition.

Fentanyl dysregulates neuroinflammation and disrupts blood-brain barrier integrity in HIV-1 Tat transgenic mice.

Opioid overdose deaths have dramatically increased by 781% from 1999 to 2021. In the setting of HIV, opioid drug abuse exacerbates neurotoxic effects of HIV in the brain, as opioids enhance viral replication, promote neuronal dysfunction and injury, and dysregulate an already compromised inflammatory response. Despite the rise in fentanyl abuse and the close association between opioid abuse and HIV infection, the interactive comorbidity between fentanyl abuse and HIV has yet to be examined in vivo. The HIV-1 Tat-transgenic mouse model was used to understand the interactive effects between fentanyl and HIV. Tat is an essential protein produced during HIV that drives the transcription of new virions and exerts neurotoxic effects within the brain. The Tat-transgenic mouse model uses a glial fibrillary acidic protein (GFAP)-driven tetracycline promoter which limits Tat production to the brain and this model is well used for examining mechanisms related to neuroHIV. After 7 days of fentanyl exposure, brains were harvested. Tight junction proteins, the vascular cell adhesion molecule, and platelet-derived growth factor receptor-ß were measured to examine the integrity of the blood brain barrier. The immune response was assessed using a mouse-specific multiplex chemokine assay. For the first time in vivo, we demonstrate that fentanyl by itself can severely disrupt the blood-brain barrier and dysregulate the immune response. In addition, we reveal associations between inflammatory markers and tight junction proteins at the blood-brain barrier.

HIV-1 Tat Induces Dysregulation of PGC1-Alpha and Sirtuin 3 Expression in Neurons: The Role of Mitochondrial Biogenesis in HIV-Associated Neurocognitive Disorder (HAND).

During the antiretroviral era, individuals living with HIV continue to experience milder forms of HIV-associated neurocognitive disorder (HAND). Viral proteins, including Tat, play a pivotal role in the observed alterations within the central nervous system (CNS), with mitochondrial dysfunction emerging as a prominent hallmark. As a result, our objective was to examine the expression of genes associated with mitophagy and mitochondrial biogenesis in the brain exposed to the HIV-1 Tat protein. We achieved this by performing bilateral stereotaxic injections of 100 ng of HIV-1 Tat into the hippocampus of Sprague-Dawley rats, followed by immunoneuromagnetic cell isolation. Subsequently, we assessed the gene expression of Ppargc1a, Pink1, and Sirt1-3 in neurons using RT-qPCR. Additionally, to understand the role of Tert in telomeric dysfunction, we quantified the activity and expression of Tert. Our results revealed that only Ppargc1a, Pink1, and mitochondrial Sirt3 were downregulated in response to the presence of HIV-1 Tat in hippocampal neurons. Interestingly, we observed a reduction in the activity of Tert in the experimental group, while mRNA levels remained relatively stable. These findings support the compelling evidence of dysregulation in both mitophagy and mitochondrial biogenesis in neurons exposed to HIV-1 Tat, which in turn induces telomeric dysfunction.

HIV-2 inhibits HIV-1 gene expression via two independent mechanisms during cellular co-infection.

IMPORTANCE: Twenty-five years after the first report that HIV-2 infection can reduce HIV-1-associated pathogenesis in dual-infected patients, the mechanisms are still not well understood. We explored these mechanisms in cell culture and showed first that these viruses can co-infect individual cells. Under specific conditions, HIV-2 inhibits HIV-1 through two distinct mechanisms, a broad-spectrum interferon response and an HIV-1-specific inhibition conferred by the HIV-2 TAR. The former could play a prominent role in dually infected individuals, whereas the latter targets HIV-1 promoter activity through competition for HIV-1 Tat binding when the same target cell is dually infected. That mechanism suppresses HIV-1 transcription by stalling RNA polymerase II complexes at the promoter through a minimal inhibitory region within the HIV-2 TAR. This work delineates the sequence of appearance and the modus operandi of each mechanism.

PRMT2 promotes HIV-1 latency by preventing nucleolar exit and phase separation of Tat into the Super Elongation Complex.

The HIV-1 Tat protein hijacks the Super Elongation Complex (SEC) to stimulate viral transcription and replication. However, the mechanisms underlying Tat activation and inactivation, which mediate HIV-1 productive and latent infection, respectively, remain incompletely understood. Here, through a targeted complementary DNA (cDNA) expression screening, we identify PRMT2 as a key suppressor of Tat activation, thus contributing to proviral latency in multiple cell line latency models and in HIV-1-infected patient CD4+ T cells. Our data reveal that the transcriptional activity of Tat is oppositely regulated by NPM1-mediated nucleolar retention and AFF4-induced phase separation in the nucleoplasm. PRMT2 preferentially methylates Tat arginine 52 (R52) to reinforce its nucleolar sequestration while simultaneously counteracting its incorporation into the SEC droplets, thereby leading to its functional inactivation to promote proviral latency. Thus, our studies unveil a central and unappreciated role for Tat methylation by PRMT2 in connecting its subnuclear distribution, liquid droplet formation, and transactivating function, which could be therapeutically targeted to eradicate latent viral reservoirs.

Disruption of blood-brain barrier: effects of HIV Tat on brain microvascular endothelial cells and tight junction proteins.

Although the widespread use of antiretroviral therapy (ART) has prolonged the life span of people living with HIV (PLWH), the incidence of HIV-associated neurocognitive disorders (HAND) in PLWH is also gradually increasing, seriously affecting the quality of life for PLWH. However, the pathogenesis of HAND has not been elucidated, which leaves HAND without effective treatment. HIV protein transactivator of transcription (Tat), as an important regulatory protein, is crucial in the pathogenesis of HAND, and its mechanism of HAND has received widespread attention. The blood-brain barrier (BBB) and its cellular component brain microvascular endothelial cells (BMVECs) play a necessary role in protecting the central nervous system (CNS), and their damage associated with Tat is a potential therapeutic target of HAND. In this review, we will study the Tat-mediated damage mechanism of the BBB and present multiple lines of evidence related to BMVEC damage caused by Tat.

HIV-1 Tat commandeers nuclear export of Rev-viral RNA complex by controlling hnRNPA2-mediated splicing.

IMPORTANCE: HIV-infected host cells impose varied degrees of regulation on viral replication, from very high to abortive. Proliferation of HIV in astrocytes is limited when compared to immune cells, such as CD4+ T lymphocytes. Understanding such differential regulation is one of the key questions in the field as these cells permit HIV persistence and rebound viremia, challenging HIV treatment and clinical cure. This study focuses on understanding the molecular mechanism behind such cell-specific disparities. We show that one of the key mechanisms is the regulation of heterogenous nuclear ribonucleoprotein A2, a host factor involved in alternative splicing and RNA processing, by HIV-1 Tat in CD4+ T lymphocytes, not observed in astrocytes. This regulation causes an increase in the levels of unspliced/partially spliced viral RNA and nuclear export of Rev-RNA complexes which results in high viral propagation in CD4+ T lymphocytes. The study reveals a new mechanism imposed by HIV on host cells that determines the fate of infection.

A truncated HIV Tat demonstrates potent and specific latency reversal activity.

A major barrier to HIV-1 cure is caused by the pool of latently infected CD4 T-cells that persist under combination antiretroviral therapy (cART). This latent reservoir is capable of producing replication-competent infectious viruses once prolonged suppressive cART is withdrawn. Inducing the reactivation of HIV-1 gene expression in T-cells harboring a latent provirus in people living with HIV-1 under cART may result in depletion of this latent reservoir due to cytopathic effects or immune clearance. Studies have investigated molecules that reactivate HIV-1 gene expression, but to date, no latency reversal agent has been identified to eliminate latently infected cells harboring replication-competent HIV in cART-treated individuals. Stochastic fluctuations in HIV-1 tat gene expression have been described and hypothesized to allow the progression into proviral latency. We hypothesized that exposing latently infected CD4+ T-cells to Tat would result in effective latency reversal. Our results indicate the capacity of a truncated Tat protein and mRNA to reactivate HIV-1 in latently infected T-cells ex vivo to a similar degree as the protein kinase C agonist: phorbol 12-myristate 13-acetate, without T-cell activation or any significant transcriptome perturbation.

TAT peptide at treatment-level concentrations crossed brain endothelial cell monolayer independent of receptor-mediated endocytosis or peptide-inflicted barrier disruption.

The peptide domain extending from residues 49 to 57 of the HIV-1 Tat protein (TAT) has been widely shown to facilitate cell entry of and blood-brain barrier (BBB) permeability to covalently bound macromolecules; therefore, TAT-linked therapeutic peptides trafficked through peripheral routes have been used to treat brain diseases in preclinical and clinical studies. Although the mechanisms underlying cell entry by similar peptides have been established to be temperature-dependent and cell-type specific and to involve receptor-mediated endocytosis, how these peptides cross the BBB remains unclear. Here, using an in vitro model, we studied the permeability of TAT, which was covalently bound to the fluorescent probe fluorescein isothiocyanate (FITC), and evaluated whether it crossed the "in vitro BBB", a monolayer of brain endothelial cells, and whether the mechanisms were similar to those involved in TAT entry into cells. Our results show that although TAT crossed the monolayer of brain endothelial cells in a temperature-dependent manner, in contrast to the reported mechanism of cell entry, it did not require receptor-mediated endocytosis. Furthermore, we revisited the hypothesis that TAT facilitates brain delivery of covalently bound macromolecules by causing BBB disruption. Our results demonstrated that the dose of TAT commonly used in preclinical and clinical studies did not exert an effect on BBB permeability in vitro or in vivo; however, an extremely high TAT concentration caused BBB disruption in vitro. In conclusion, the BBB permeability to TAT is temperature-dependent, but at treatment-level concentrations, it does not involve receptor-mediated endocytosis or BBB disruption.

HIV-1 subtype B Tat enhances NOTCH3 signaling in astrocytes to mediate oxidative stress, inflammatory response, and neuronal apoptosis.

NOTCH receptors are relevant to multiple neurodegenerative diseases. However, the roles and mechanisms of NOTCH receptors in HIV-associated neurocognitive disorder (HAND) remain largely unclear. Transactivator of transcription (Tat) induces oxidative stress and inflammatory response in astrocytes, thereby leading to neuronal apoptosis in the central nervous system. We determined that NOTCH3 expression was upregulated during subtype B or C Tat expression in HEB astroglial cells. Moreover, bioinformatics analysis of the Gene Expression Omnibus (GEO) dataset revealed that NOTCH3 mRNA expression in the frontal cortex tissues of HIV encephalitis patients was higher than that of HIV control patients. Of note, subtype B Tat, rather than subtype C Tat, interacted with the extracellular domain of the NOTCH3 receptor, thus activating NOTCH3 signaling. Downregulation of NOTCH3 attenuated subtype B Tat-induced oxidative stress and reactive oxygen species generation. In addition, we demonstrated that NOTCH3 signaling facilitated subtype B Tat-activated NF-κB signaling pathway, thereby mediating pro-inflammatory cytokines IL-6 and TNF-α production. Furthermore, downregulation of NOTCH3 in HEB astroglial cells protected SH-SY5Y neuronal cells from astrocyte-mediated subtype B Tat neurotoxicity. Taken together, our study clarifies the potential role of NOTCH3 in subtype B Tat-induced oxidative stress and inflammatory response in astrocytes, which could be a novel therapeutic target for the relief of HAND.

HIV-1 Transcriptional Activator Tat Inhibits IL2 Expression by Preventing the Presence of Pol II on the IL2 Promoter.

HIV-1 infection leads to a gradual loss of T helper cells, chronic immune activation, and eventual immune system breakdown. HIV-1 causes deregulation of the expression of IL-2, a cytokine important for T helper cell growth and survival, which is downregulated in HIV-1 patients. The present study addresses the regulation of IL2 expression via HIV-1 Tat transcriptional activator. We used J-LAT cells, a T cell line that serves as a latency model for studies of HIV-1 expression in T cells, and as controls a T cell line lacking HIV-1 elements and a T cell line with a stably integrated copy of the HIV-1-LTR promoter. We show that endogenously expressed Tat inhibits IL2 transcription in J-Lat cells via its presence in the ARRE-1/2 elements of the IL2 promoter and that the inhibition of IL2 expression is mediated by Tat inhibiting Pol II activity at the IL2 promoter, which is mediated by preventing the presence of Pol II at the ARRE-1/2 elements. Overall, Tat is present at the IL2 promoter, apart from its cognate HIV-1 LTR target. This supports our current knowledge of how HIV-1 affects the host transcriptional machinery and reflects the potential of Tat to disrupt transcriptional regulation of host genes to manipulate cell responses.

Protein Transduction System Based on Tryptophan-zipper against Intracellular Infections via Inhibiting Ferroptosis of Macrophages.

Cells penetrating molecules in living systems hold promise of capturing and eliminating threats and damage that can plan intracellular fate promptly. However, it remains challenging to construct cell penetration systems that are physiologically stable with predictable self-assembly behavior and well-defined mechanisms. In this study, we develop a core-shell nanoparticle using a hyaluronic acid (HA)-coated protein transduction domain (PTD) derived from the human immunodeficiency virus (HIV). This nanoparticle can encapsulate pathogens, transporting the PTD into macrophages via lipid rafts. PTD forms hydrogen bonds with the components of the membrane through TAT, which has a high density of positive charges and reduces the degree of membrane order through Tryptophan (Trp)-zipper binding to the acyl tails of phospholipid molecules. HA-encapsulated PTD increases the resistance to trypsin and proteinase K, thereby penetrating macrophages and eliminating intracellular infections. Interestingly, the nonagglutination mechanism of PTD against pathogens ensures the safe operation of the cellular system. Importantly, PTD can activate the critical pathway of antiferroptosis in macrophages against pathogen infection. The nanoparticles developed in this study demonstrate safety and efficacy against Gram-negative and Gram-positive pathogens in three animal models. Overall, this work highlights the effectiveness of the PTD nanoparticle in encapsulating pathogens and provides a paradigm for transduction systems-anti-intracellular infection therapy.

A Tripartite Complex HIV-1 Tat-Cyclophilin A-Capsid Protein Enables Tat Encapsidation That Is Required for HIV-1 Infectivity.

HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes. Nevertheless, it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolyl isomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated at the level of 200 to 250 copies of CypA/virion. Here, we found that a Tat-CypA-CA tripartite complex assembles in HIV-1-infected cells and allows Tat encapsidation into HIV virions (1 Tat/1 CypA). Biochemical and biophysical studies showed that high-affinity interactions drive the assembly of the Tat-CypA-CA complex that could be purified by size exclusion chromatography. We prepared different types of viruses devoid of transcriptionally active Tat. They showed a 5- to 10 fold decrease in HIV infectivity, and conversely, encapsidating Tat into ΔTat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreased the efficiency of reverse transcription by ~50% and transcription by more than 90%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust viral transcription and thus viral production at the beginning of cell infection, before neosynthesized Tat becomes available. IMPORTANCE The viral transactivating protein Tat has been shown to stimulate several steps of HIV gene expression. It was found to facilitate reverse transcription. Moreover, Tat is strictly required for the transcription of viral genes. Although the presence of Tat within HIV virions would undoubtedly favor these steps and therefore enable the incoming virus to boost initial viral production, whether and how Tat is present within virions has been a matter a debate. We here described and characterized a tripartite complex between Tat, HIV capsid protein, and the cellular chaperone cyclophilin A that enables efficient and specific Tat encapsidation within HIV virions. We further showed that Tat encapsidation is required for the virus to efficiently initiate infection and viral production. This effect is mainly due to the transcriptional activity of Tat.

Role of Dysregulated Autophagy in HIV Tat, Cocaine, and cART Mediated NLRP3 Activation in Microglia.

Despite the ability of combination antiretroviral therapy (cART) to suppress viremia, there is persistence low levels of HIV proteins such as Transactivator of transcription (Tat) in the central nervous system (CNS), contributing to glial activation and neuroinflammation. Accumulating evidence also implicates the role of drugs of abuse in exacerbating neurological complications associated with HIV-1. The combined effects of HIV Tat, drugs of abuse, and cART can thus create a toxic milieu in the CNS. The present study investigated the combinatorial effects of HIV-Tat, cocaine, and cART on autophagy and NLRP3 inflammasome activation. We selected a combination of three commonly used cART regimens: tenofovir, emtricitabine, and dolutegravir. Our results demonstrated that exposure of mouse primary microglia (MPMs) to these agents-HIV Tat (25 ng/ml), cocaine (1 µM), and cART (1 µM each) resulted in upregulation of autophagy markers: Beclin1, LC3B-II, and SQSTM1 with impaired lysosomal functioning involving increased lysosomal pH, decreased LAMP2 and cathepsin D, ultimately leading to dysregulated autophagy. Our findings also demonstrated activation of the NLRP3 signaling in microglia exposed to these agents. We further demonstrated that gene silencing of key autophagy protein BECN1 significantly blocked NLRP3-mediated activation of microglia. Silencing of NLRP3, however, failed to block HIV Tat, cocaine, and cART-mediated dysregulation of the autophagy-lysosomal axis; these in vitro phenomena were also validated in vivo using iTat mice administered cocaine and cART. This study thus underscores the cooperative effects of HIV Tat, cocaine, and cART in exacerbating microglial activation involving dysregulated autophagy and activation of the NLRP3 inflammasome signaling.

Disruption of the ADAM17/NF-κB feedback loop in astrocytes ameliorates HIV-1 Tat-induced inflammatory response and neuronal death.

A disintegrin and metalloproteinases (ADAMs) are involved in multiple neurodegenerative diseases. However, the roles and mechanisms of ADAMs in HIV-associated neurocognitive disorder (HAND) remain unclear. Transactivator of transcription (Tat) induces inflammatory response in astrocytes, thereby leading to neuronal apoptosis in the central nervous system. In this study, we determined that ADAM17 expression was upregulated during soluble Tat stimulus in HEB astroglial cells. Inhibition of ADAM17 suppressed Tat-induced pro-inflammatory cytokines production and rescued the astrocytes-derived conditioned media (ACM)-mediated SH-SY5Y neural cells apoptosis. Moreover, ADAM17 mediated Tat-triggered inflammatory response in a NF-κB-dependent manner. Conversely, Tat induced ADAM17 expression via NF-κB signaling pathway. In addition, pharmacological inhibition of NF-κB signaling inhibited Tat-induced inflammatory response, which could be rescued by overexpression of ADAM17. Taken together, our study clarifies the potential role of the ADAM17/NF-κB feedback loop in Tat-induced inflammatory response in astrocytes and the ACM-mediated neuronal death, which could be a novel therapeutic target for relief of HAND.

RNA conformational propensities determine cellular activity.

Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes1. These interactions are mediated by intermolecular contacts, which if disrupted, lead to alterations in cell physiology. Nevertheless, the formation of intermolecular contacts nearly universally requires changes in the conformations of the interacting biomolecules. As a result, binding affinity and cellular activity crucially depend both on the strength of the contacts and on the inherent propensities to form binding-competent conformational states2,3. Thus, conformational penalties are ubiquitous in biology and must be known in order to quantitatively model binding energetics for protein and nucleic acid interactions4,5. However, conceptual and technological limitations have hindered our ability to dissect and quantitatively measure how conformational propensities affect cellular activity. Here we systematically altered and determined the propensities for forming the protein-bound conformation of HIV-1 TAR RNA. These propensities quantitatively predicted the binding affinities of TAR to the RNA-binding region of the Tat protein and predicted the extent of HIV-1 Tat-dependent transactivation in cells. Our results establish the role of ensemble-based conformational propensities in cellular activity and reveal an example of a cellular process driven by an exceptionally rare and short-lived RNA conformational state.

Complex Formation of an RNA Aptamer with a Part of HIV-1 Tat through Induction of Base Triples in Living Human Cells Proven by In-Cell NMR.

An RNA aptamer that strongly binds to a target molecule has the potential to be a nucleic acid drug inside living human cells. To investigate and improve this potential, it is critical to elucidate the structure and interaction of RNA aptamers inside living cells. We examined an RNA aptamer for HIV-1 Tat (TA), which had been found to trap Tat and repress its function in living human cells. We first used in vitro NMR to examine the interaction between TA and a part of Tat containing the binding site for trans-activation response element (TAR). It was revealed that two U-A∗U base triples are formed in TA upon binding of Tat. This was assumed to be critical for strong binding. Then, TA in complex with a part of Tat was incorporated into living human cells. The presence of two U-A∗U base triples was also revealed for the complex in living human cells by in-cell NMR. Thus, the activity of TA in living human cells was rationally elucidated by in-cell NMR.

Loss of In Vivo Replication Fitness of HIV-1 Variants Resistant to the Tat Inhibitor, dCA.

HIV resistance to the Tat inhibitor didehydro-cortistatin A (dCA) in vitro correlates with higher levels of Tat-independent viral transcription and a seeming inability to enter latency, which rendered resistant isolates more susceptible to CTL-mediated immune clearance. Here, we investigated the ability of dCA-resistant viruses to replicate in vivo using a humanized mouse model of HIV infection. Animals were infected with WT or two dCA-resistant HIV-1 isolates in the absence of dCA and followed for 5 weeks. dCA-resistant viruses exhibited lower replication rates compared to WT. Viral replication was suppressed early after infection, with viral emergence at later time points. Multiplex analysis of cytokine and chemokines from plasma samples early after infection revealed no differences in expression levels between groups, suggesting that dCA-resistance viruses did not elicit potent innate immune responses capable of blocking the establishment of infection. Viral single genome sequencing results from plasma samples collected at euthanasia revealed that at least half of the total number of mutations in the LTR region of the HIV genome considered essential for dCA evasion reverted to WT. These results suggest that dCA-resistant viruses identified in vitro suffer a fitness cost in vivo, with mutations in LTR and Nef pressured to revert to wild type.