The HIV-1 Transcriptional Program: From Initiation to Elongation Control.

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.

Tat-dependent conditionally replicating adenoviruses expressing diphtheria toxin A for specifically killing HIV-1-infected cells.

HIV-1 infection remains a public health problem with no cure. Although antiretroviral therapy (ART) is effective for suppressing HIV-1 replication, it requires lifelong drug administration due to a stable reservoir of latent proviruses and may cause serious side effects and drive the emergence of drug-resistant HIV-1 variants. Gene therapy represents an alternative approach to overcome the limitations of conventional treatments against HIV-1 infection. In this study, we constructed and investigated the antiviral effects of an HIV-1 Tat-dependent conditionally replicating adenovirus, which selectively replicates and expresses the diphtheria toxin A chain (Tat-CRAds-DTA) in HIV-1-infected cells both in vitro and in vivo. We found that Tat-CRAds-DTA could specifically induce cell death and inhibit virus replication in HIV-1-infected cells mediated by adenovirus proliferation and DTA expression. A low titer of progeny Tat-CRAds-DTA was also detected in HIV-1-infected cells. In addition, Tat-CRAds-DTA showed no apparent cytotoxicity to HIV-1-negative cells and demonstrated significant therapeutic efficacy against HIV-1 infection in a humanized mouse model. The findings in this study highlight the potential of Tat-CRAds-DTA as a new gene therapy for the treatment of HIV-1 infection.

From Glycolysis to Viral Defense: The Multifaceted Impact of Glycolytic Enzymes on Human Immunodeficiency Virus Type 1 Replication.

Viruses require host cells to replicate and proliferate, which indicates that viruses hijack the cellular machinery. Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4-positive T cells, and efficiently uses cellular proteins to replicate. Cells already have proteins that inhibit the replication of the foreign HIV-1, but their function is suppressed by viral proteins. Intriguingly, HIV-1 infection also changes the cellular metabolism to aerobic glycolysis. This phenomenon has been interpreted as a cellular response to maintain homeostasis during viral infection, yet HIV-1 efficiently replicates even in this environment. In this review, we discuss the regulatory role of glycolytic enzymes in viral replication and the impact of aerobic glycolysis on viral infection by introducing various host proteins involved in viral replication. Furthermore, we would like to propose a "glyceraldehyde-3-phosphate dehydrogenase-induced shock (G-shock) and kill strategy" that maximizes the antiviral effect of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to eliminate latently HIV-1-infected cells.

Role of I182, R187, and K188 Amino Acid Residues in the Catalytic Domain of HIV-1 Integrase in the Processes of Reverse Transcription and Integration.

Structural organization of HIV-1 integrase is based on a tetramer formed by two protein dimers. Within this tetramer, the catalytic domain of one subunit of the first dimer interacts with the N-terminal domain of the second dimer subunit. It is the tetrameric structure that allows both ends of the viral DNA to be correctly positioned relative to the cellular DNA and to realize catalytic functions of integrase, namely 3'-processing and strand transfer. However, during the HIV-1 replicative cycle, integrase is responsible not only for the integration stage, it is also involved in reverse transcription and is necessary at the stage of capsid formation of the newly formed virions. It has been suggested that HIV-1 integrase is a structurally dynamic protein and its biological functions depend on its structure. Accordingly, studying interactions between the domains of integrase that provide its tetrameric structure is important for understanding its multiple functions. In this work, we investigated the role of three amino acids of the catalytic domain, I182, R187, and K188, located in the contact region of two integrase dimers in the tetramer structure, in reverse transcription and integration. It has been shown that the R187 residue is extremely important for formation of the correct integrase structure, which is necessary at all stages of its functional activity. The I182 residue is necessary for successful integration and is not important for reverse transcription, while the K188 residue, on the contrary, is involved in formation of the integrase structure, which is important for the effective reverse transcription.

Glycan-Imprinted Nanoparticle as Artificial Neutralizing Antibody for Efficient HIV-1 Recognition and Inhibition.

The HIV-1 envelope is a heavily glycosylated class 1 trimeric fusion protein responsible for viral entry into CD4+ immune cells. Developing neutralizing antibodies against the specific envelope glycans is an alternative method for antiviral therapies. This work presents the first-ever development and characterization of artificial neutralizing antibodies using molecular imprinting technology to recognize and bind to the envelope protein of HIV-1. The prepared envelope glycan-imprinted nanoparticles (GINPs) can successfully prevent HIV-1 from infecting target cells by shielding the glycans on the envelope protein. In vitro experiments showed that GINPs have strong affinity toward HIV-1 (Kd = 36.7 ± 2.2 nM) and possess high anti-interference and specificity. GINPs demonstrate broad inhibition activity against both tier 1 and tier 2 HIV-1 strains with a pM-level IC50 and exhibit a significant inhibitory effect on long-term viral replication by more than 95%. The strategy provides a promising method for the inhibition and therapy of HIV-1 infection.

An Assessment of Trends in HIV-1 Prevalence and Incidence and Spatio-Temporal Analyses of HIV-1 Recent Infection Among MSM During the Surveillance Period Between 2018 and 2022 in Sichuan, China.

Background: Men who have sex with men (MSM) is one main type of high-risk activities facilitating HIV-1 transmission in Sichuan province. Previous works on HIV-1 incidence and prevalence among MSM only concentrated before 2018, the situation after that is unknown. In addition, the distribution of hot-spots related to current HIV-1 epidemic is also rarely known among MSM in Sichuan. Objective: To update trends of HIV-1 prevalence and incidence and to visualize hot-spots of ongoing transmission in Sichuan province during surveillance period among MSM between 2018 and 2022. Methods: Limiting Antigen Avidity assay was performed to detect recent infection within new HIV-1 diagnoses founded during surveillance period among MSM. The HIV-1 prevalence and incidence were calculated according to an extrapolation method proposed by publications and guidelines. Trend tests were performed using χ2 tests with linear-by-linear association. The spatial analysis was conducted with ArcGIS 10.7 to figure hot-spots of HIV-1 recent infections among MSM. Results: Between 2018 and 2022, 16,697 individuals participated in HIV-1 MSM sentinel surveillance program, of which 449 samples (98.25%) were tested with LAg-Avidity EIA, and 230 samples were classified as recent infection. Respectively, the overall prevalence and incidence were 2.74% and 3.69% (95% CI: 3.21, 4.16) and both had significant declining trends (p < 0.001). Luzhou city had a highest HIV-1 incidence (10.74%, 95% CI: 8.39, 13.10) over the study period and was recognized as a hot-spot for recent HIV-1 infection among MSM. Conclusion: During the surveillance period, both HIV-1 prevalence and incidence were declining. However, Luzhou city had an unusually high HIV-1 incidence and became an emerging hot-spot of recent HIV-1 infection among MSM. This finding suggested focused attention, cross-regional intervention strategies, and prevention programs are urgently required to curb the spread of ongoing transmission.

Late-Stage Derivatization of Oleanolic Acid-Based Anti-HIV-1 Compounds.

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.

Human Immunodeficiency Virus-1 Drug Resistance Mutations in Iranian Treatment-experienced Individuals.

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.

Machine learning-guided design of potent darunavir analogs targeting HIV-1 proteases: A computational approach for antiretroviral drug discovery.

In the pursuit of novel antiretroviral therapies for human immunodeficiency virus type-1 (HIV-1) proteases (PRs), recent improvements in drug discovery have embraced machine learning (ML) techniques to guide the design process. This study employs ensemble learning models to identify crucial substructures as significant features for drug development. Using molecular docking techniques, a collection of 160 darunavir (DRV) analogs was designed based on these key substructures and subsequently screened using molecular docking techniques. Chemical structures with high fitness scores were selected, combined, and one-dimensional (1D) screening based on beyond Lipinski's rule of five (bRo5) and ADME (absorption, distribution, metabolism, and excretion) prediction implemented in the Combined Analog generator Tool (CAT) program. A total of 473 screened analogs were subjected to docking analysis through convolutional neural networks scoring function against both the wild-type (WT) and 12 major mutated PRs. DRV analogs with negative changes in binding free energy ( ΔΔ G bind ) compared to DRV could be categorized into four attractive groups based on their interactions with the majority of vital PRs. The analysis of interaction profiles revealed that potent designed analogs, targeting both WT and mutant PRs, exhibited interactions with common key amino acid residues. This observation further confirms that the ML model-guided approach effectively identified the substructures that play a crucial role in potent analogs. It is expected to function as a powerful computational tool, offering valuable guidance in the identification of chemical substructures for synthesis and subsequent experimental testing.

Functions of HIV-1 Tat and its association with diseases / 中国生物制品学杂志

@#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

Higher Infectivity of the Human Immunodeficiency Virus in Sensitive Cells with a Modification of the CCR5 Gene.

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.

Exploring the HIV-1 Rev Recognition Element (RRE)-Rev Inhibitory Capacity and Antiretroviral Action of Benfluron Analogs.

Human immunodeficiency virus-type 1 (HIV-1) remains one of the leading contributors to the global burden of disease, and novel antiretroviral agents with alternative mechanisms are needed to cure this infection. Here, we describe an exploratory attempt to optimize the antiretroviral properties of benfluron, a cytostatic agent previously reported to exhibit strong anti-HIV activity likely based on inhibitory actions on virus transcription and Rev-mediated viral RNA export. After obtaining six analogs designed to modify the benzo[c]fluorenone system of the parent molecule, we examined their antiretroviral and toxicity properties together with their capacity to recognize the Rev Recognition Element (RRE) of the virus RNA and inhibit the RRE-Rev interaction. The results indicated that both the benzo[c] and cyclopentanone components of benfluron are required for strong RRE-Rev target engagement and antiretroviral activity and revealed the relative impact of these moieties on RRE affinity, RRE-Rev inhibition, antiviral action and cellular toxicity. These data provide insights into the biological properties of the benzo[c]fluorenone scaffold and contribute to facilitating the design of new anti-HIV agents based on the inhibition of Rev function.

The Use of CBD and Its Synthetic Analog HU308 in HIV-1-Infected Myeloid Cells.

Currently, there is no cure for human immunodeficiency virus type 1 (HIV-1) infection. However, combined antiretroviral therapy (cART) aids in viral latency and prevents the progression of HIV-1 infection into acquired immunodeficiency syndrome (AIDS). cART has extended many lives, but people living with HIV-1 (PLWH) face lifelong ailments such as HIV-associated neurocognitive disorders (HAND) that range from asymptomatic HAND to HIV-1-associated dementia. HAND has been attributed to chronic inflammation and low-level infection within the central nervous system (CNS) caused by proinflammatory cytokines and viral products. These molecules are shuttled into the CNS within extracellular vesicles (EVs), lipid bound nanoparticles, and are released from cells as a form of intercellular communication. This study investigates the impact of cannabidiol (CBD), as a promising and potential therapeutic for HAND patients, and a similar synthetic molecule, HU308, on the EVs released from HIV-1-infected myeloid cells as well as HIV-1-infected 3D neurospheres. The data shows that both CBD and HU308 decrease non-coding and coding viral RNA (TAR and env) as well as proinflammatory cytokines as IL-1ß and TNF-α mRNA. This decrease in viral RNA occurs in in vitro differentiated primary macrophages, in EVs released from HIV-1-infected cells monocytes, and infected neurospheres. Furthermore, a 3D neurosphere model shows an overall decrease in proinflammatory mRNA with HU308. Finally, using a humanized mouse model of HIV-1 infection, plasma viral RNA was shown to significantly decrease with HU308 alone and was most effective in combination with cART, even when compared to the typical cART treatment. Overall, CBD or HU308 may be a viable option to decrease EV release and associated cytokines which would dampen the virus spread and may be used in effective treatment of HAND in combination with cART.

Novel HIV-1 RNA biogenesis inhibitors identified by virtual pharmacophore-based screening.

The complex between the Rev protein of HIV-1 and the Rev Recognition Element (RRE) within the virus RNA promotes nuclear export of unspliced or incompletely spliced viral transcripts and is required for virus transmission. Here, we have screened a virtual collection of compounds using a pharmacophore based on the chemical similarity of previously characterized inhibitors to identify new chemical scaffolds blocking the RRE-Rev interaction. The best molecules discovered with this strategy inhibited the complex by binding to the RRE and exhibited substantial antiretroviral activity (between 0.582 and 11.3 µM EC50 values) likely associated to inhibitory actions on viral transcription and Rev function. These results have allowed us to identify structural features required for RRE-Rev inhibition as well as to add new compounds to the pool of possible candidates for developing antiretroviral agents based on blockage of HIV-1 RNA biogenesis.

Influence of HIV-1 Genomic RNA on the Formation of Gag Biomolecular Condensates.

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4+ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.

Optimal Expression, Function, and Immunogenicity of an HIV-1 Vaccine Derived from the Approved Ebola Vaccine, rVSV-ZEBOV.

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.

TRPV1 activation in human Langerhans cells and T cells inhibits mucosal HIV-1 infection via CGRP-dependent and independent mechanisms.

Upon its mucosal transmission, HIV type 1 (HIV-1) rapidly targets genital antigen-presenting Langerhans cells (LCs), which subsequently transfer infectious virus to CD4+ T cells. We previously described an inhibitory neuroimmune cross talk, whereby calcitonin gene-related peptide (CGRP), a neuropeptide secreted by peripheral pain-sensing nociceptor neurons innervating all mucosal epithelia and associating with LCs, strongly inhibits HIV-1 transfer. As nociceptors secret CGRP following the activation of their Ca2+ ion channel transient receptor potential vanilloid 1 (TRPV1), and as we reported that LCs secret low levels of CGRP, we investigated whether LCs express functional TRPV1. We found that human LCs expressed mRNA and protein of TRPV1, which was functional and induced Ca2+ influx following activation with TRPV1 agonists, including capsaicin (CP). The treatment of LCs with TRPV1 agonists also increased CGRP secretion, reaching its anti-HIV-1 inhibitory concentrations. Accordingly, CP pretreatment significantly inhibited LCs-mediated HIV-1 transfer to CD4+ T cells, which was abrogated by both TRPV1 and CGRP receptor antagonists. Like CGRP, CP-induced inhibition of HIV-1 transfer was mediated via increased CCL3 secretion and HIV-1 degradation. CP also inhibited direct CD4+ T cells HIV-1 infection, but in CGRP-independent manners. Finally, pretreatment of inner foreskin tissue explants with CP markedly increased CGRP and CCL3 secretion, and upon subsequent polarized exposure to HIV-1, inhibited an increase in LC-T cell conjugate formation and consequently T cell infection. Our results reveal that TRPV1 activation in human LCs and CD4+ T cells inhibits mucosal HIV-1 infection, via CGRP-dependent/independent mechanisms. Formulations containing TRPV1 agonists, already approved for pain relief, could hence be useful against HIV-1.

Comparative Evaluation of the Activity of Various Lentiviral Vectors Containing Three Anti-HIV Genes.

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.