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A large body of work in the last four decades has revealed the key pillars of HIV-1 transcription control at the initiation and elongation steps. Here, I provide a recount of this collective knowledge starting with the genomic elements (DNA and nascent TAR RNA stem-loop) and transcription factors (cellular and the viral transactivator Tat), and later transitioning to the assembly and regulation of transcription initiation and elongation complexes, and the role of chromatin structure. Compelling evidence support a core HIV-1 transcriptional program regulated by the sequential and concerted action of cellular transcription factors and Tat to promote initiation and sustain elongation, highlighting the efficiency of a small virus to take over its host to produce the high levels of transcription required for viral replication. I summarize new advances including the use of CRISPR-Cas9, genetic tools for acute factor depletion, and imaging to study transcriptional dynamics, bursting and the progression through the multiple phases of the transcriptional cycle. Finally, I describe current challenges to future major advances and discuss areas that deserve more attention to both bolster our basic knowledge of the core HIV-1 transcriptional program and open up new therapeutic opportunities.

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A 12-keto-type oleanolic acid derivative (4) has been identified as a potent anti-human immunodeficiency virus type-1 (HIV-1) compound that demonstrates synergistic effects with several types of HIV-1 neutralizing antibodies. In the present study, we used a common key synthetic intermediate to carry out the late-stage derivatization of an anti-HIV compound based on the chemical structure of a 12-keto-type oleanolic acid derivative. To execute this strategy, we designed a diketo-type oleanolic acid derivative (5) for chemoselective transformation, targeting the carboxy group and the hydroxyl group on the statine unit, as well as the 3-carbonyl group on the oleanolic acid unit, as orthogonal synthetic handles. We carried out four types of chemoselective transformations, leading to identification of the indole-type derivative (16) as a novel potent anti-HIV compound. In addition, further optimization of the ß-hydroxyl group on the statine unit provided the R-4-isobutyl γ-amino acid-type derivative (6), which exhibited potent anti-HIV activity comparable to that of 4 but with reduced cytotoxicity.

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BACKGROUND: Human immunodeficiency virus-1 infection still remains a global health threat. While antiretroviral therapy is the primary treatment option, concerns about the emergence of drug-resistance mutations and treatment failure in HIV-infected patients persist. OBJECTIVE: In this study, we investigated the development of drug resistance in HIV-1-infected individuals receiving antiretroviral therapy for 6-10 years. METHODS: In this cross-sectional study, we evaluated 144 people living with HIV-1 who had received antiretroviral therapy for at least 6 years. Plasma specimens were collected, and the HIV-1 viral load and drug-resistance mutations were assessed using molecular techniques. RESULTS: The demographic and epidemiological characteristics of the participants were also analyzed: Twelve [8.3%) of the studied patients showed a viral load over 1000 copies per/mL, which indicates the suboptimal response to antiretroviral therapy. Significant correlations were found between viral load and CD4 count, as well as epidemiological factors, such as vertical transmission, history of imprisonment, and needle stick injuries. Drug resistance mutations were detected in 10 (83.3%) of patients who failed on antiretroviral therapy, with the most common mutations observed against nucleoside reverse transcriptase inhibitors (5 (41.7%)) and non-nucleoside reverse transcriptase inhibitors (9 (75%)). Phylogenetic analysis revealed that 12 patients who failed treatment were infected with CRF35_AD. CONCLUSION: Our study provides important insights into the characteristics and development of drug resistance in HIV-1-infected individuals receiving long-term antiretroviral therapy in Iran. The findings underline the need for regular viral load monitoring, individualized treatment selection, and targeted interventions to optimize treatment outcomes and prevent the further spread of drug-resistant strains.

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@#Acquired immune deficiency syndrome,or AIDS,has been a major infectious disease that troubles the public health in a global scale. Human immunodeficiency virus type 1(HIV-1)is the causative reagent responsible for AIDS development. Even though the highly active anti-retroviral therapy(HAART,or the cocktail therapy)that has been widely applied could effectively suppress the infection and replication of HIV-1,the infected people suffer from other related diseases,such as the HIV-associated neurocognitive disorder(HAND). This paper mainly focused on the function of an important regulatory protein of HIV-1,trans-activator of transcription(Tat),and its correlation with HIV-1 replication and HAND development,so as to clarify the importance of developing anti-AIDS drugs targeting Tat protein

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As a natural mutation of the human ccr5 gene has been shown to confer resistance to human immunodeficiency virus type 1 (HIV-1) infection, a new avenue has opened in the development of alternative treatment approaches through genome editing. One of the two chemokine co-receptors of the plasma membrane is utilized by HIV-1 to infect CD4+ cells. HIV-1 strains that utilize CCR5 circulate in early infection, and strains that utilize CXCR4 circulate at advanced stages. A complex relationship may exist in the expression regulation of the receptors and may affect virus replication in cells that normally do not express CCR5 on the membrane, such as the MT-4 cell line. MT-4 cells were used to study the effect of ccr5 modification HIV-1 replication in vitro. Genetic modification of ccr5 in MT-4 cells was shown to increase the activities of HIV-1 strains, especially in homozygote. The results indicate that genome editing should be performed with caution in human cells and that the issue needs comprehensive investigation.

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Vesicular stomatitis virus (VSV) remains an attractive platform for a potential HIV-1 vaccine but hurdles remain, such as selection of a highly immunogenic HIV-1 Envelope (Env) with a maximal surface expression on recombinant rVSV particles. An HIV-1 Env chimera with the transmembrane domain (TM) and cytoplasmic tail (CT) of SIVMac239 results in high expression on the approved Ebola vaccine, rVSV-ZEBOV, also harboring the Ebola Virus (EBOV) glycoprotein (GP). Codon-optimized (CO) Env chimeras derived from a subtype A primary isolate (A74) are capable of entering a CD4+/CCR5+ cell line, inhibited by HIV-1 neutralizing antibodies PGT121, VRC01, and the drug, Maraviroc. The immunization of mice with the rVSV-ZEBOV carrying the CO A74 Env chimeras results in anti-Env antibody levels as well as neutralizing antibodies 200-fold higher than with the NL4-3 Env-based construct. The novel, functional, and immunogenic chimeras of CO A74 Env with the SIV_Env-TMCT within the rVSV-ZEBOV vaccine are now being tested in non-human primates.

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A promising direction in the treatment of HIV infection is a gene therapy approach based on the insertion of antiviral genes aimed at inhibiting HIV replication into the genome of host cells. We obtained six constructs of lentiviral vectors with different arrangements of three antiviral genes: microRNAs against the CCR5 gene, the gene encoding the C-peptide, and the gene encoding the modified human TRIM5a protein. We found that despite containing the same genes, these vectors were produced at different titers and had different effects on cell viability, transduction efficiency, and expression stability. Comparative evaluation of the antiviral activity of three of the six developed vectors that showed stable expression was carried out using the continuous SupT1 lymphocytic cell line. All of the vectors protected cells from HIV infection: the viral load was several orders of magnitude lower than in control cells, and with one vector, complete cessation of virus growth in modified cells was achieved.

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(1) Background: The HIV-1 latent reservoir harboring replication-competent proviruses is the major barrier in the quest for an HIV-1 infection cure. HIV-1 infection at all stages of disease progression is associated with immune activation and dysfunctional production of proinflammatory soluble factors (cytokines and chemokines), and it is expected that during HIV-1 infection, different immune components and immune cells, in turn, participate in immune responses, subsequently activating downstream biological pathways. However, the functional interaction between HIV-1 integration and the activation of host biological pathways is presently not fully understood. (2) Methods: In this work, I used genes targeted by proviruses from published datasets to seek enriched immunologic signatures and host biological pathways alongside HIV-1 infections based on MSigDb and KEGG over-representation analysis. (3) Results: I observed that different combinations of immunologic signatures of immune cell types and proinflammatory soluble factors appeared alongside HIV-1 infections associated with antiretroviral therapy. Moreover, enriched KEGG pathways were often related to "cancer specific types", "immune system", "infectious disease viral", and "signal transduction". (4) Conclusions: The observations in this work suggest that the gene sets harboring provirus integration sites may define specific immune cells and proinflammatory soluble factors during HIV-1 infections associated with antiretroviral therapy.

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We earlier reported substantial progress in designing gp120 antagonists. Notably, we discovered that NBD-14189 is not only the most active gp120 antagonist but also shows antiviral activity against HIV-1 Reverse Transcriptase (RT). We also confirmed its binding to HIV-1 RT by X-ray crystallography. The dual inhibition is highly significant because, intriguingly, this compound bridges the dNTP and NNRTI-binding sites and inhibits the polymerase activity of isolated RT in the enzymatic assay. This novel finding is expected to lead to new avenues in designing a novel class of HIV-1 dual inhibitors. Therefore, we needed to advance this inhibitor to preclinical assessment. To this end, we report the pharmacokinetics (PK) study of NBD-14189 in rats and dogs. Subsequently, we assessed the toxicity and therapeutic efficacy in vivo in the SCID-hu Thy/Liv mouse model. The PK data indicated a favorable half-life (t1/2) and excellent oral bioavailability (%F = 61%). NBD-14189 did not show any measurable toxicity in the mice, and treatment reduced HIV replication at 300 mg/kg per day in the absence of clear evidence of protection from HIV-mediated human thymocyte depletion. The data indicated the potential of this inhibitor as an anti-HIV-1 agent and needs to be assessed in a non-human primate (NHP) model.

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Viruses are now recognized as bona fide etiologic factors of human cancer. Carcinogenic viruses include Epstein- Barr virus (EBV), high-risk human papillomaviruses (HPVs), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus type 1 (HTLV-1), human immunodeficiency virus type 1 (HIV-1, indirectly), and several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. Tumor viruses establish long-term persistent infections in humans, and cancer is an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, supporting the concept that cancer results from the accumulation of multiple cooperating events, in which human cancer viruses display different, often opposing roles. The laboratory mouse Mus musculus is one of the best in vivo experimental systems for modeling human pathology, including viral infections and cancer. However, mice are unsusceptible to infection with the known carcinogenic viruses. Many murine models were developed to overcome this limitation and to address various aspects of virus-associated carcinogenesis, from tumors resulting from xenografts of human tissues and cells, including cancerous and virus infected, to genetically engineered mice susceptible to viral infections and associated cancer. The review considers the main existing models, analyzes their advantages and drawbacks, describes their applications, outlines the prospects of their further development.

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Viruses are now recognized as bona fide etiologic factors of human cancer. Carcinogenic viruses include Epstein-Barr virus (EBV), high-risk human papillomaviruses (HPVs), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus type 1 (HTLV-1), human immunodeficiency virus type 1 (HIV-1, indirectly), and several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. Tumor viruses establish long-term persistent infections in humans, and cancer is an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, supporting the concept that cancer results from the accumulation of multiple cooperating events, in which human cancer viruses display different, often opposing roles. The laboratory mouse Mus musculus is one of the best in vivo experimental systems for modeling human pathology, including viral infections and cancer. However, mice are unsusceptible to infection with the known carcinogenic viruses. Many murine models were developed to overcome this limitation and to address various aspects of virus-associated carcinogenesis, from tumors resulting from xenografts of human tissues and cells, including cancerous and virus infected, to genetically engineered mice susceptible to viral infections and associated cancer. The review considers the main existing models, analyzes their advantages and drawbacks, describes their applications, outlines the prospects of their further development.

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The heterocyclic indole structure has been shown to be one of the most promising scaffolds, offering various medicinal advantages from its wide range of biological activity. Nonetheless, the significance of 3-oxindole has been less known. In this study, a series of novel 3-oxindole-2-carboxylates were synthesized and their antiviral activity against human immunodeficiency virus-1 (HIV-1) infection was evaluated. Among these, methyl (E)-2-(3-chloroallyl)-4,6-dimethyl-one (6f) exhibited the most potent inhibitory effect on HIV-1 infection, with a half-maximal inhibitory concentration (IC50) of 0.4578 µM but without severe cytotoxicity (selectivity index (SI) = 111.37). The inhibitory effect of these compounds on HIV-1 infection was concordant with their inhibitory effect on the viral replication cycle. Mode-of-action studies have shown that these prominent derivatives specifically inhibited the Tat-mediated viral transcription on the HIV-1 LTR promoter instead of reverse transcription or integration. Overall, our findings indicate that 3-oxindole derivatives could be useful as a potent scaffold for the development of a new class of anti-HIV-1 agents.

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Fission yeast can be used as a cell-based system for high-throughput drug screening. However, higher drug concentrations are often needed to achieve the same effect as in mammalian cells. Our goal here was to improve drug sensitivity so reduced drugs could be used. Three different methods affecting drug uptakes were tested using an FDA-approved HIV-1 protease inhibitor (PI) drug Darunavir (DRV). First, we tested whether spheroplasts without cell walls increase the drug sensitivity. Second, we examined whether electroporation could be used. Although small improvements were observed, neither of these two methods showed significant increase in the EC50 values of DRV compared with the traditional method. In contrast, when DRV was tested in a mutant strain PR836 that lacks key proteins regulating cellular efflux, a significant increase in the EC50 was observed. A comparison of nine FDA-approved HIV-1 PI drugs between the wild-type RE294 strain and the mutant PR836 strain showed marked enhancement of the drug sensitivities ranging from an increase of 0.56 log to 2.48 logs. Therefore, restricting cellular efflux through the adaption of the described fission yeast mutant strain enhances the drug sensitivity, reduces the amount of drug used, and increases the chance of success in future drug discovery.

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BACKGROUND: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) has been studied extensively for its role in mediating leukocyte rolling through interactions with its cognate receptor, P-selectin. Recently, PSGL-1 was identified as a novel HIV-1 host restriction factor, particularly when expressed at high levels in the HIV envelope. Importantly, while the potent antiviral activity of PSGL-1 has been clearly demonstrated in various complementary model systems, the breadth of PSGL-1 incorporation across genetically diverse viral isolates and clinical isolates has yet to be described. Additionally, the biological activity of virion-incorporated PSGL-1 has also yet to be shown. RESULTS: Herein we assessed the levels of PSGL-1 on viruses produced through transfection with various amounts of PSGL-1 plasmid DNA (0-250 ng), compared to levels of PSGL-1 on viruses produced through infection of T cell lines and primary PBMC. We found that very low levels of PSGL-1 plasmid DNA (< 2.5 ng/well) were necessary to generate virus models that could closely mirror the phenotype of viruses produced via infection of T cells and PBMC. Unique to this study, we show that PSGL-1 is incorporated in a broad range of HIV-1 and SIV isolates and that virions with incorporated PSGL-1 are detectable in plasma from viremic HIV-1-infected individuals, corroborating the relevance of PSGL-1 in natural infection. Additionally, we show that PSGL-1 on viruses can bind its cognate selectin receptors, P-, E-, and L-selectins. Finally, we show viruses with endogenous levels of PSGL-1 can be captured by P-selectin and transferred to HIV-permissive bystander cells, highlighting a novel role for PSGL-1 in HIV-1 infection. Notably, viruses which contained high levels of PSGL-1 were noninfectious in our hands, in line with previous findings reporting the potent antiviral activity of PSGL-1. CONCLUSIONS: Our results indicate that levels of PSGL-1 incorporation into virions can vary widely among model systems tested, and that careful tailoring of plasmid levels is required to recapitulate physiological systems when using pseudovirus models. Taken together, our data suggest that PSGL-1 may play diverse roles in the physiology of HIV-1 infection, particularly due to the functionally active state of PSGL-1 on virion surfaces and the breadth of PSGL-1 incorporation among a wide range of viral isolates.

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Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag-PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag-PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]; however, previous studies suggested that HTLV-1 Gag-PM interactions and therefore virus assembly are less dependent on PI(4,5)P2. We have recently shown that PI(4,5)P2 binds directly to HTLV-1 unmyristoylated MA [myr(-)MA] and that myr(-)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P2. Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA-membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P2-binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P2. Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly.

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Exogenous retroviruses such as human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia virus type 1 (HTLV-1) and bovine leukemia virus (BLV) can cause various diseases including immunodeficiency, inflammatory diseases and hematologic malignancies. These retroviruses persistently infect their hosts. Therefore, they need to evade host immune surveillance. One way in which these viruses might avoid immune detection is to utilize functional RNAs, rather than proteins, for certain activities, because RNAs are not recognized by the host immune system. HTLV-1 encodes the HTLV-1 bZIP factor (HBZ) gene in the antisense strand of the provirus. The HBZ protein is constantly expressed in HTLV-1 carriers and patients with adult T-cell leukemia-lymphoma, and it plays critical roles in pathogenesis. However, HBZ not only encodes this protein, but also functions as mRNA. Thus, HBZ gene mRNA is bifunctional. HIV-1 and BLV also encode long non-coding RNAs as antisense transcripts. In this review, we reshape our current understanding of how these antisense transcripts function and how they influence disease pathogenesis.

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Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.

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Retroviral Gag targeting to the plasma membrane (PM) for assembly is mediated by the N-terminal matrix (MA) domain. For many retroviruses, Gag-PM interaction is dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, it has been shown that for human T-cell leukemia virus type 1 (HTLV-1), Gag binding to membranes is less dependent on PI(4,5)P2 than HIV-1, suggesting that other factors may modulate Gag assembly. To elucidate the mechanism by which HTLV-1 Gag binds to the PM, we employed NMR techniques to determine the structure of unmyristoylated MA (myr(-)MA) and to characterize its interactions with lipids and liposomes. The MA structure consists of four α-helices and unstructured N- and C-termini. We show that myr(-)MA binds to PI(4,5)P2 via the polar head and that binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups on the inositol ring, indicating that the MA-IP binding is governed by charge-charge interactions. The IP binding site was mapped to a well-defined basic patch formed by lysine and arginine residues. Using an NMR-based liposome binding assay, we show that PI(4,5)P2and phosphatidylserine enhance myr(-)MA binding in a synergistic fashion. Confocal microscopy data revealed formation of puncta on the PM of Gag expressing cells. However, G2A-Gag mutant, lacking myristoylation, is diffuse and cytoplasmic. These results suggest that although myr(-)MA binds to membranes, myristoylation appears to be key for formation of HTLV-1 Gag puncta on the PM. Altogether, these findings advance our understanding of a key mechanism in retroviral assembly.

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Successful combination antiretroviral therapies (cART) eliminate active replicating HIV-1, slow down disease progression, and prolong lives. However, cART effectiveness could be compromised by the emergence of viral multidrug resistance, suggesting the need for new drug discoveries. The objective of this study was to further demonstrate the utility of the fission yeast cell-based systems that we developed previously for the discovery and testing of HIV protease (PR) inhibitors (PIs) against wild-type or multi-PI drug resistant M11PR that we isolated from an infected individual. All thirteen FDA-approved single-agent and fixed-dose combination HIV PI drugs were tested. The effect of these drugs on HIV PR activities was tested in pure compounds or formulation drugs. All FDA-approved PI drugs, except for a prodrug FPV, were able to suppress the wild-type PR-induced cellular and enzymatic activities. Relative drug potencies measured by EC50 in fission yeast were discussed in comparison with those measured in human cells. In contrast, none of the FDA-approved drugs suppressed the multi-PI drug resistant M11PR activities. Results of this study show that fission yeast is a reliable cell-based system for the discovery and testing of HIV PIs and further demonstrate the need for new PI drugs against viral multi-PI resistance.