P24 Production Research Articles

In vitro replicative potential of an HIV-1/MO intergroup recombinant virus compared to HIV-1/M and HIV-1/O parental viruses

Genetic recombination is one of the major evolution processes of HIV-1. Despite their great genetic divergence, HIV-1 groups M and O can generate HIV-1/MO intergroup recombinants. The current description of 20 HIV-1/MO unique recombinant forms suggests a possible benefit of the recombination. The aim of this work was to study in vitro the replicative potential of HIV-1/MO recombinant forms. This analysis was based on a simple recombination pattern, [Ogag/pol-Menv], harboring a breakpoint in Vpr. A chimeric infectious molecular clone, pOM-TB-2016 was synthesized from HIV-1/M subtype B and HIV-1/O subgroup T and recombinant viruses were obtained by transfection/co-culture. To compare the replicative potential of these viruses, two markers were monitored in culture supernatants: Reverse Transcriptase (RT) activity and P24 antigen concentration. The results showed a superiority of the group M parental virus compared to group O for both markers. In contrast, for the recombinant virus, RT activity data did not overlap with the concentration of P24 antigen, suggesting a hybrid behavior of the recombinant, in terms of enzyme activity and P24 production. These results highlighted many hypotheses about the impact of recombination on replicative potential and demonstrated again the significant plasticity of HIV genomes and their infinite possibility of evolution.

Nicotinamide mononucleotide impacts HIV-1 infection by modulating immune activation in T lymphocytes and humanized mice

HIV-1-associated immune activation drives CD4+ T cell depletion and the development of acquired immunodeficiency syndrome. We aimed to determine the role of nicotinamide mononucleotide (NMN), the direct precursor of nicotinamide adenine dinucleotide (NAD) co-enzyme, in CD4+ T cell modulation during HIV-1 infection. We examined HIV-1 integrated DNA or transcribed RNA, intracellular p24 protein, and T cell activation markers in CD4+ T cells including invitro HIV-1-infected cells, reactivated patient-derived cells, and in HIV-1-infected humanized mice, under NMN treatment. RNA-seq and CyTOF analyses were used for investigating the effect of NMN on CD4+ T cells. We found that NMN increased the intracellular NAD amount, resulting in suppressed HIV-1 p24 production and proliferation in infected CD4+ T cells, especially in activated CD25+CD4+ T cells. NMN also inhibited CD25 expression on reactivated resting CD4+ T cells derived from cART-treated people living with HIV-1 (PLWH). In HIV-1-infected humanized mice, the frequency of CD4+ T cells was reconstituted significantly by combined cART and NMN treatment as compared with cART or NMN alone, which correlated with suppressed hyperactivation of CD4+ T cells. Our results highlight the suppressive role of NMN in CD4+ T cell activation during HIV-1 infection. It warrants future clinical investigation of NMN as a potential treatment in combination with cART in PLWH. This work was supported by the Hong Kong Research Grants Council Theme-Based Research Scheme (T11-706/18-N), University Research Committee of The University of Hong Kong, the Collaborative Research with GeneHarbor (Hong Kong) Biotechnologies Limited and National Key R&D Program of China (Grant2021YFC2301900).

Long Noncoding RNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 Promotes HIV-1 Replication through Modulating microRNAs in Macrophages.

Macrophages can serve as a reservoir for human immunodeficiency-1 (HIV-1) virus in host cells, constituting a barrier to eradication, even in patients who are receiving antiretroviral therapy. Although many noncoding RNAs have been characterized as regulators in HIV-1/AIDS-induced immune response and pathogenesis, only a few long noncoding RNAs (lncRNAs) have demonstrated a close association with HIV-1 replication, and the molecular mechanisms remain unknown. In this study, we investigated how lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), related microRNAs, and key inflammatory genes alter HIV-1 replication in macrophages. Our data show that HIV-1 infection modulates the expression of miR-155 and miR-150-5p in a time-dependent manner, which is regulated by MALAT1. MALAT1 induced suppressor of cytokine signaling 1 (SOCS1) expression by sponging miR-150-5p in HIV-1-infected macrophages and stimulated inflammatory mediators triggering receptor expressed on myeloid cells/cold inducible RNA binding protein (TREM 1/CIRP) ligand/receptor. The RNA immunoprecipitation (RIP) assay validated the direct interaction within the MALAT1/miR-150-5p/SOCS1 axis. HIV-1 infection-mediated upregulation of MALAT1, SOCS1, and HIV-1 Gag was attenuated by SN50 (an NF-кB p50 inhibitor). MALAT1 antisense oligonucleotides (ASOs) suppressed HIV-1 p24 production and HIV-1 Gag gene expression and decreased expression of miR-155 and SOCS1, as well as the production of proinflammatory cytokines by HIV-1-infected macrophages. In conclusion, HIV-1 infection induces MALAT1, which attenuates miR-150-5p expression and increases SOCS1 expression, promoting HIV-1 replication and reactivation. These data provide new insights into how MALAT1 alters the macrophage microenvironment and subsequently promotes viral replication and suggest a potential role for targeting MALAT1 as a therapeutic approach to eliminate HIV-1 reservoirs. IMPORTANCE Viral reservoirs constitute an obstacle to curing HIV-1 diseases, despite antiretroviral therapy. Macrophages serve as viral reservoirs in HIV infection by promoting long-term replication and latency. Recent studies have shown that lncRNAs can modulate virus-host interactions, but the underlying mechanisms are not fully understood. In this study, we demonstrate how lncRNA MALAT1 contributes to HIV-1 replication through modulation of the miR-150/SOCS1 axis in human macrophages. Our findings have the potential to identify new therapies for eliminating HIV-1 reservoirs in immune cells.

Antiviral Response and HIV-1 Inhibition in Sickle Cell Disease

BACKGROUND: A number of host restriction factors controls HIV-1 infection. While many of these factors have been investigated in vitro, their role in controlling HIV-1 infection in vivo remains to be elucidated. Here, we investigate a naturally occurring HIV-1 restriction in Sickle cell disease (SCD), a hereditary disorder characterized by sickle cell hemoglobin polymerization and red blood cells hemolysis. We previously showed that SCD PBMCs express higher levels of ferroportin (FPN), and that reduced intracellular iron leads to the upregulation of antiviral restriction factors. Previously, HIV-1 infection was shown to inversely correlate with the expression levels of transferrin receptor (TFR), a marker of reduced intracellular iron. Iron chelation also potently inhibits HIV-1 replication as shown by us and others. Thus, iron metabolism is intrinsically connected to the HIV-1 infection and disease progression. Our study in vitro suggests that SCD environment leads to the antiviral state of T cells and macrophages, and suppression of HIV-1 infection. Here we analyzed HIV infection of SCD mice using mouse adapted EcoHIV and compared antiviral factors in SCD mouse spleenocytes and human SCD PBMCs. METHODS: The study was approved by Howard University IRB and IACUC committees. Townes SCD mice carrying human normal α (hα) and sickle hemoglobin beta (βS) genes and control Townes mice carrying human normal hα and βA genes were used in the study. EcoHIV virus obtained from David Volsky (Albert Einstein University) was constructed in pNL4-3 vector with the substitution of gp120 with the envelope-coding gp80 region from ectopic MLV. Whole blood samples were obtained from 9 SCD patients and 9 healthy individuals. RNA Seq analysis was conducted on Illumina® NextSeq 500 and the data were mapped and analyzed using Dragen software. Normalized gene counts were used for gene set enrichment analysis (GSEA) and Gene ontology (GO) analysis. RNA expression was measured by quantitative RT-PCR (Roche). Protein expression in mouse splenocytes was determined by ELISA. RESULTS: Infection of control mice with EcoHIV virus, which contained MLV envelope and was able to infect mouse cells, led to robust viral replication in spleen at 7 days post infection, as detected by the expression of HIV-1 Nef mRNA (Fig. 1) and Gag mRNA. We also detected HIV-1 p24 in splenic tissue. Comparing to the control, SCD mice showed significantly reduced EcoHIV replication both at the levels of RNA expression (Figs 1) and p24 production. GSEA analysis against the 72 genes that included all known HIV-1 restriction factors revealed strong enrichment of these genes in the human activated SCD PBMCs compared to the activated control PBMCs (Fig.2). These genes included APOBEC3A, HMOX1, CH25H, IFIT3, CDKN1A, APOBEC3B, IFITM3, SAMHD1 and TSPO. GSEA analysis also showed upregulation of several pathogen-associated molecular patterns sensing genes including NOD1, NLRC4, TRL7 and TLR9 in human PBMCS. In SCD mice splenocytes, we detected overexpression of FPN, and transferrin receptor (Tfr) genes indicating upregulation of iron metabolism similar to previously observed in human SCD PBMCs. In mouse splenocytes we detected upregulation of CDKN1A, HMOX1, IFITM3, CH25H and TSPO mRNAs. Protein expression analysis showed upregulation of FPN, TFR, HO-1, APOBEC, CH25H, IFIT3 and TSPO in SCD mouse splenocytes. CONCLUSION: Together, these data reveal, for the first time, that HIV-1 infection is suppressed in vivo in SCD mouse infected with the mouse-adapted EcoHIV. SCD mouse splenocytes expressed higher levels of FPN and TFR, and exhibited higher expression of antiviral factor, which were also observed in human SCD PBMCs. The observed upregulation of similar sets of antiviral factors in SCD PBCMs and SCD mice splenocytes suggests that these factors upregulation might be driven by the SCD-related processes such as hemolysis. Taken together, our findings point to a novel mechanism of upregulation of antiviral factors mediated by SCD that included induction of antiviral, heme- and iron- regulatory pathways. ACKNOWLEDGEMENTS: We thank Dr. David Volsky for the gift of EcoHIV and the George Washington University SPH Genomics Core for library preparation and sequencing. This work was supported by NIH grants 1R01HL125005, 5U54MD007597, 1P30AI117970-06 and 1SC1HL150685. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Microbial molecule ingress promotes neuroinflammation and brain CCR5 expression in persons with HIV-associated neurocognitive disorders

BackgroundSystemic inflammation accompanies HIV-1 infection, resulting in microbial translocation from different tissues. We investigated interactions between lentivirus infections, neuroinflammation and microbial molecule presence in the brain. MethodsBrain tissues from adult humans with (n = 22) and without HIV-1 (n = 11) infection as well as adult nonhuman primates (NHPs) with (n = 11) and without (n = 4) SIVmac251 infection were investigated by RT-PCR/ddPCR, immunofluorescence and western blotting. Studies of viral infectivity, host immune gene expression and viability were performed in primary human neural cells. FindingsAmong NHPs, SIV DNA quantitation in brain showed increased levels among animals with SIV encephalitis (n = 5) that was associated with bacterial genomic copy number as well as CCR5 and CASP1 expression in brain. Microbial DnaK and peptidoglycan were immunodetected in brains from uninfected and SIV-infected animals, chiefly in glial cells. Human microglia infected by HIV-1 showed increased p24 production after exposure to peptidoglycan that was associated CCR5 induction. HIV-1 Vpr application to human neurons followed by peptidoglycan exposure resulted in reduced mitochondrial function and diminished beta-III tubulin expression. In human brains, bacterial genome copies (250–550 copies/gm of tissue), were correlated with increased bacterial rRNA and GroEL transcript levels in patients with HIV-associated neurocognitive disorders (HAND). Glial cells displayed microbial GroEL and peptidoglycan immunoreactivity accompanied by CCR5 induction in brains from patients with HAND. InterpretationIncreased microbial genomes and proteins were evident in brain tissues from lentivirus-infected humans and animals and associated with neurological disease. Microbial molecule translocation into the brain might exacerbate neuroinflammatory disease severity and represent a driver of lentivirus-associated brain disease.

T-cell activation and B-cell interaction signatures in rectal tissues are associated with HIV replication in ex-vivo model of infection.

The rectal mucosa is a critical site of HIV vulnerability. We sought to identify transcriptomic features of rectal mucosal tissue prior to exposure associated with support or restriction of HIV replication. Rectal tissue from HIV-negative cis gender men ( n = 57) underwent concurrent RNAseq transcriptomic analyses (two biopsies/participant) and challenge with HIV in the ex-vivo explant model of infection (three biopsies challenged/participant) as part of a larger cohort study to understand the rectal mucosal immune environment among MSM. P24 was quantified in the explant supernatants over a culture period of 18 days via ELISA. Participant median p24 log area under the curve was correlated with bulk transcriptomic data (Illumina HiSeq3000) to identify associations between gene expression and p24 production. Significant differentially expressed genes (DEGs) were identified via DESeq2 analysis and analyzed with Reactome to identify pathways of interest. In total, 183 DEG (181 upregulated, two downregulated) were associated with higher p24 accumulation in the ex-vivo challenge model, including T-cell activation, B-cell function, and chemokine DEG. Reactome analysis of the upregulated genes identified 'Adaptive Immune System', 'Cytokine Signaling in Immune System', and 'Innate Immune System' as significantly upregulated pathways. For the first time, we identified rectal tissue transcriptomic signatures associated with increased p24 production utilizing an ex-vivo model. Our findings are highly relevant to HIV transmission and the early establishment of HIV reservoirs in humans, and future studies should examine the identified pathways as targets for new or improved biomedical prevention or treatment interventions.

Targeting Tat-TAR RNA Interaction for HIV-1 Inhibition.

The HIV-1 Tat protein interacts with TAR RNA and recruits CDK9/cyclin T1 and other host factors to induce HIV-1 transcription. Thus, Tat–TAR RNA interaction, which is unique for HIV-1, represents an attractive target for anti-HIV-1 therapeutics. To target Tat–TAR RNA interaction, we used a crystal structure of acetylpromazine bound to the bulge of TAR RNA, to dock compounds from the Enamine database containing over two million individual compounds. The docking procedure identified 173 compounds that were further analyzed for the inhibition of HIV-1 infection. The top ten inhibitory compounds with IC50 ≤ 6 µM were selected and the three least toxic compounds, T6780107 (IC50 = 2.97 μM), T0516-4834 (IC50 = 0.2 μM) and T5628834 (IC50 = 3.46 μM), were further tested for HIV-1 transcription inhibition. Only the T0516-4834 compound showed selective inhibition of Tat-induced HIV-1 transcription, whereas the T6780107 compound inhibited equally basal and Tat-induced transcription and the T5628834 compound only inhibited basal HIV-1 transcription. The compounds were tested for the inhibition of translation and showed minimal (<25%) effect. The T0516-4834 compound also showed the strongest inhibition of HIV-1 RNA expression and p24 production in CEM T cells and peripheral blood mononuclear cells infected with HIV-1 IIIB. Of the three compounds, only the T0516-4834 compound significantly disrupted Tat–TAR RNA interaction. Additionally, of the three tested compounds, T5628834 and, to a lesser extent, T0516-4834 disrupted Tat–CDK9/cyclin T1 interaction. None of the three compounds showed significant inhibition of the cellular CDK9 and cyclin T1 levels. In silico modelling showed that the T0516-4834 compound interacted with TAR RNA by binding to the bulge formed by U23, U25, C39, G26,C39 and U40 residues. Taken together, our study identified a novel benzoxazole compound that disrupted Tat–TAR RNA interaction and inhibited Tat-induced transcription and HIV-1 infection, suggesting that this compound might serve as a new lead for anti-HIV-1 therapeutics.

Plasmacytoid dendritic cells have divergent effects on HIV infection of initial target cells and induce a pro-retention phenotype.

Although HIV infection inhibits interferon responses in its target cells in vitro, interferon signatures can be detected in vivo soon after sexual transmission, mainly attributed to plasmacytoid dendritic cells (pDCs). In this study, we examined the physiological contributions of pDCs to early HIV acquisition using coculture models of pDCs with myeloid DCs, macrophages and the resting central, transitional and effector memory CD4 T cell subsets. pDCs impacted infection in a cell-specific manner. In myeloid cells, HIV infection was decreased via antiviral effects, cell maturation and downregulation of CCR5 expression. In contrast, in resting memory CD4 T cells, pDCs induced a subset-specific increase in intracellular HIV p24 protein expression without any activation or increase in CCR5 expression, as measured by flow cytometry. This increase was due to reactivation rather than enhanced viral spread, as blocking HIV entry via CCR5 did not alter the increased intracellular p24 expression. Furthermore, the load and proportion of cells expressing HIV DNA were restricted in the presence of pDCs while reverse transcriptase and p24 ELISA assays showed no increase in particle associated reverse transcriptase or extracellular p24 production. In addition, pDCs also markedly induced the expression of CD69 on infected CD4 T cells and other markers of CD4 T cell tissue retention. These phenotypic changes showed marked parallels with resident memory CD4 T cells isolated from anogenital tissue using enzymatic digestion. Production of IFNα by pDCs was the main driving factor for all these results. Thus, pDCs may reduce HIV spread during initial mucosal acquisition by inhibiting replication in myeloid cells while reactivating latent virus in resting memory CD4 T cells and retaining them for immune clearance.

Integrating high cell density cultures with adapted laboratory evolution for improved Gag‐HA virus‐like particles production in stable insect cell lines

Stable insect cell lines are emerging as an alternative to the insect cell-baculovirus expression vector system (IC-BEVS) for protein expression, benefiting from being a virus-free, nonlytic system. Still, the titers achieved are considerably lower. In this study, stable insect (Sf-9 and High Five) cells producing Gag virus-like particles (VLPs) were first adapted to grow under hypothermic culture conditions (22°C instead of standard 27°C), and then pseudotyped with a model membrane protein (influenza hemagglutinin [HA]) for expression of Gag-HA VLPs. Adaptation to lower temperature led to an increase in protein titers of up to 12-fold for p24 (as proxy for Gag-VLP) and sixfold for HA, with adapted Sf-9 cells outperforming High Five cells. Resulting Gag-HA VLPs producer Sf-9 cells were cultured to high cell densities, that is, 100 × 106 cell/ml, using perfusion (ATF® 2) in 1 L stirred-tank bioreactors. Specific p24 and HA production rates were similar to those of batch culture, enabling to increase volumetric titers by 7-8-fold without compromising the assembly of Gag-HA VLPs. Importantly, the antigen (HA) quantity in VLPs generated using stable adapted cells in perfusion was ≈5-fold higher than that from IC-BEVS, with the added benefit of being a baculovirus-free system. This study demonstrates the potential of combining stable expression in insect cells adapted to hypothermic culture conditions with perfusion for improving Gag-HA VLPs production.

Ex vivo rectal explant model reveals potential opposing roles of Natural Killer cells and Marginal Zone-like B cells in HIV-1 infection

Our understanding of innate immune responses in human rectal mucosal tissues (RM) and their contributions to promoting or restricting HIV transmission is limited. We defined the RM composition of innate and innate-like cell subsets, including plasmacytoid dendritic cells; CD1c + myeloid DCs; neutrophils; macrophages; natural killer cells (NK); Marginal Zone-like B cells (MZB); γδ T cells; and mucosal-associated invariant T cells in RM from 69 HIV-negative men by flow cytometry. Associations between these cell subsets and HIV-1 replication in ex vivo RM explant challenge experiments revealed an inverse correlation between RM-NK and p24 production, in contrast to a positive association between RM-MZB and HIV replication. Comparison of RM and blood-derived MZB and NK illustrated qualitative and quantitative differences between tissue compartments. Additionally, 22 soluble molecules were measured in a subset of explant cultures (n = 26). Higher production of IL-17A, IFN-γ, IL-10, IP-10, GM-CSF, sFasL, Granzyme A, Granzyme B, Granulysin, and Perforin following infection positively correlated with HIV replication. These data show novel associations between MZB and NK cells and p24 production in RM and underscore the importance of inflammatory cytokines in mucosal HIV infection, demonstrating the likely critical role these innate immune responses play in early mucosal HIV replication in humans.

Betamethasone induces potent immunosuppression and reduces HIV infection in a PBMC in vitro model

Genital inflammation is an established risk factor for increased HIV acquisition risk. Certain HIV-exposed seronegative populations, who are naturally resistant to HIV infection, have an immune quiescent phenotype defined by...

Identification of Combinations of Protein Kinase C Activators and Histone Deacetylase Inhibitors That Potently Reactivate Latent HIV.

Combination antiretroviral therapy (cART) is successful in maintaining undetectable levels of HIV in the blood; however, the persistence of latent HIV reservoirs has become the major barrier for a HIV cure. Substantial efforts are underway in finding the best latency-reversing agents (LRAs) to purge the latent viruses from the reservoirs. We hypothesize that identifying the right combination of LRAs will be the key to accomplishing that goal. In this study, we evaluated the effect of combinations of three protein kinase C activators (prostratin, (-)-indolactam V, and TPPB) with four histone deacetylase inhibitors (AR-42, PCI-24781, givinostat, and belinostat) on reversing HIV latency in different cell lines including in a primary CD4+ T-cell model. Combinations including indolactam and TPPB with AR-42 and PCI produced a strong synergistic effect in reactivating latent virus as indicated by higher p24 production and envelope gp120 expression. Furthermore, treatment with TPPB and indolactam greatly downregulated the cellular receptor CD4. Indolactam/AR-42 combination emerged from this study as the best combination that showed a strong synergistic effect in reactivating latent virus. Although AR-42 alone did not downregulate CD4 expression, indolactam/AR-42 showed the most efficient downregulation. Our results suggest that indolactam/AR-42 is the most effective combination, showing a strong synergistic effect in reversing HIV latency combined with the most efficient CD4 downregulation.

HIV-1 protease and reverse transcriptase inhibition by tiger milk mushroom (Lignosus rhinocerus) sclerotium extracts: In vitro and in silico studies

Background and aimLignosus rhinocerus (LR) is an edible mushroom with a variety of medicinal properties such as neurostimulation, immunomodulation, anti-inflammation, anti-oxidation, anti-proliferation, anti-diabetes and especially antiviral activity. Human immunodeficiency virus type-1 (HIV-1) needs the HIV-1 protease (PR) and reverse transcriptase (RT) for its replication. Therefore, both HIV-1 PR and RT are important targets for antiretroviral drug development. Experimental procedureThe crude hexane (LRH), ethanol (LRE) and water (LRW) extracts of LR were in vitro screened for inhibitory activity against HIV-1 PR and RT, then anti-HIV-1 activity on the infected MOLT-4 cells were determined. Chemical constituents of the extracts were identified by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC)-MS. The identified compounds were in silico analysed for drug-likeness property and molecular modelling. Results and conclusionAccording to our screening assays, LRE and LRW significantly inhibited both enzymes (25–55%), while LRH suppressed only the HIV-1 PR activity (88.97%). At 0.5 mg/ml of LRW showed significant inhibition of HIV-1 induced syncytial formation and p24 production in the infected MOLT-4 cells. Investigation of chemical analysis revealed that major groups of identified constituents found in the extracts were fatty acids, peptides and terpenoids. In silico analysis showed that heliantriol F and 6 alpha-fluoroprogesterone displayed great binding energies with HIV-1 PR and HIV-1 RT, respectively. These findings suggest that LR could be a potential source of compounds to inhibit HIV-1 PR and/or RT activities in vitro. Furthermore, our results provide beneficial data for the development of novel HIV-1 PR and RT inhibitors.

Different Patterns of HIV-1 Replication in MACROPHAGES is Led by Co-Receptor Usage.

Background and objectives: To enter the target cell, HIV-1 binds not only CD4 but also a co-receptor β-chemokine receptor 5 (CCR5) or α chemokine receptor 4 (CXCR4). Limited information is available on the impact of co-receptor usage on HIV-1 replication in monocyte-derived macrophages (MDM) and on the homeostasis of this important cellular reservoir. Materials and Methods: Replication (measured by p24 production) of the CCR5-tropic 81A strain increased up to 10 days post-infection and then reached a plateau. Conversely, the replication of the CXCR4-tropic NL4.3 strain (after an initial increase up to day 7) underwent a drastic decrease becoming almost undetectable after 10 days post-infection. The ability of CCR5-tropic and CXCR4-tropic strains to induce cell death in MDM was then evaluated. While for CCR5-tropic 81A the rate of apoptosis in MDM was comparable to uninfected MDM, the infection of CXCR4-tropic NL4.3 in MDM was associated with a rate of 14.3% of apoptotic cells at day 6 reaching a peak of 43.5% at day 10 post-infection. Results: This suggests that the decrease in CXCR4-tropic strain replication in MDM can be due to their ability to induce cell death in MDM. The increase in apoptosis was paralleled with a 2-fold increase in the phosphorylated form of p38 compared to WT. Furthermore, microarray analysis showed modulation of proapoptotic and cancer-related genes induced by CXCR4-tropic strains starting from 24 h after infection, whereas CCR5 viruses modulated the expression of genes not correlated with apoptotic-pathways. Conclusions: In conclusion, CXCR4-tropic strains can induce a remarkable depletion of MDM. Conversely, MDM can represent an important cellular reservoir for CCR5-tropic strains supporting the role of CCR5-usage in HIV-1 pathogenesis and as a pharmacological target to contribute to an HIV-1 cure.

The Membrane-Active Phytopeptide Cycloviolacin O2 Simultaneously Targets HIV-1-infected Cells and Infectious Viral Particles to Potentiate the Efficacy of Antiretroviral Drugs.

Background: Novel strategies to increase the efficacy of antiretroviral (ARV) drugs will be of crucial importance. We hypothesize that membranes of HIV-1-infected cells and enveloped HIV-1 particles may be preferentially targeted by the phytopeptide, cycloviolacin O2 (CyO2) to significantly enhance ARV efficacy. Methods: Physiologically safe concentrations of CyO2 were determined via red blood cell (RBC) hemolysis. SYTOX-green dye-uptake and radiolabeled saquinavir (3H-SQV) uptake assays were used to measure pore-formation and drug uptake, respectively. ELISA, reporter assays and ultracentrifugation were conducted to analyze the antiviral efficacy of HIV-1 protease and fusion inhibitors alone and co-exposed to CyO2. Results: CyO2 concentrations below 0.5 μM did not show substantial hemolytic activity, yet these concentrations enabled rapid pore-formation in HIV-infected T-cells and monocytes and increased drug uptake. ELISA for HIV-1 p24 indicated that CyO2 enhances the antiviral efficacy of both SQV and nelfinavir. CyO2 (< 0.5 μM) alone decreases HIV-1 p24 production, but it did not affect the transcription regulatory function of the HIV-1 long terminal repeat (LTR). Ultracentrifugation studies clearly showed that CyO2 exposure disrupted viral integrity and decreased the p24 content of viral particles. Furthermore, direct HIV-1 inactivation by CyO2 enhanced the efficacy of enfuvirtide. Conclusions: The membrane-active properties of CyO2 may help suppress viral load and augment antiretroviral drug efficacy.

Effect of Amino Acid Substitutions on Biological Activity of Antimicrobial Peptide: Design, Recombinant Production, and Biological Activity.

Recently, antimicrobial peptides have been introduced as potent antibiotics with a wide range of antimicrobial activities. They have also exhibited other biological activities, including anti-inflammatory, growth stimulating, and anti-cancer activities. In this study, an analog of Magainin II was designed and produced as a recombinant fusion protein. The designed sequence contained 24 amino acid residues (P24), in which Lys, His, Ser residues were substituted with Arg and also, hydrophobic Phe was replaced with Trp. Recombinant production of P24 in Escherichia coli (E. coli) BL21 using pTYB21, containing chitin binding domain and intein sequence at the N-terminus of the peptide gene, resulted in 1 μg mL-1 product from culture. Chitin column chromatography, followed by online peptide cleavage with thiol reducing agent was applied to purify the peptide. Antimicrobial activity was evaluated using five bacteria strains including Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumonia, E. coli, and Pseudomonas aeruginosa. Designed AMP exhibited promising antimicrobial activities with low minimum inhibitory concentration, in the range of 64-256 µg/mL. P24 showed potent antimicrobial activity preferably against Gram-positive bacteria, and more potent than pexiganan as a successful Magainin II analog for topical infections. In general, further modification can be applied to improve its therapeutic index.

Interleukin 21 (IL-21)/microRNA-29 (miR-29) axis is associated with natural resistance to HIV-1 infection.

Interleukin-21 (IL-21) modulates HIV-1 infection through the elicitation of different antiviral mechanisms, including Th17 lineage commitment and induction of microRNA (miR)-29, a miRNA endowed with anti-HIV activity. As miR-29 expression is significantly increased in HIV-1-exposed seronegative individuals (HESN), we investigated the role of miR-29/IL21 axis in the natural control of HIV-1 infection. Peripheral blood mononuclear cells (PBMCs) isolated from 15 Italian sexually exposed HESN and 15 HIV-unexposed healthy controls were in-vitro infected with an R5-tropic HIV-1Ba-L strain. Seven days post HIV-1 infection we evaluated: 1) p24 production (ELISA); 2) CD4/IL-21 and CD4/IL-17 T lymphocytes (FACS); 3) IL-17 concentration in supernatants (ELISA); and 4) IL-6, IL-17, IL-21, and miR-29a,b,c expression by CD4 T lymphocytes as well as perforin and granzyme by peripheral blood mononuclear cells (qPCR). The same analyses were performed on the 15 HIV-positive partners. At baseline IL-6 expression alone was increased in HESN compared to healthy controls. Seven days after in-vitro HIV-1 infection, nevertheless, differences emerged. Thus, CD4/IL21 and CD4/IL17 T lymphocytes, as well as IL-21 and IL-17 expression and production were significantly augmented in HESN compared to healthy controls. Interestingly, IL-21 upregulation correlated with a significantly increased expression of miR-29a,b,c and a reduced susceptibility to in-vitro HIV-1 infection in HESN alone. No differences were observed in perforin and granzyme expression. The IL-21/miR-29 axis is upregulated by HIV-1 infection in HESN suggesting its involvement in the natural resistance to HIV-1 infection in HESN. Approaches that exogenously increase IL-21 production or prompt preexisting cellular IL-21 reservoir could confine the magnitude of the initial HIV-1 infection.

Different kinetics of viral replication and DNA integration in the main HIV-1 cellular reservoirs in the presence and absence of integrase inhibitors

To compare the kinetics of integration, p24 production and equilibrium of the different HIV-DNA forms in human primary cells in the presence/absence of integrase-inhibitors (INIs) in vitro.Monocyte-derived-macrophages (MDMs), CD4+ T-cells and peripheral blood mononuclear cells (PBMCs) were infected with HIV-1 in the presence/absence of raltegravir and dolutegravir. HIV-DNA levels and p24 production were measured by qPCR and ELISA assays, respectively.In the absence of INIs, levels of HIV-DNA forms were initially very low, with an increase in the integration process starting at 3 dpi. HIV-DNA increased more slowly in MDMs than it did in CD4+ T-cells and PMBCs peaking at 21 dpi with a mean of 1580 (±890) and 615 (±37) copies/103 cells for proviral and unintegrated HIV-DNA, and 455,972 (±213,255) pg/mL of p24 at the same time point. In CD4+ T-cells the proviral HIV-DNA increased together with unintegrated HIV-DNA peaking at 7 dpi (583 ± 261 and 338 ± 254 copies/103 cells) when the p24 was 218,000 (±75,600) pg/mL. A similar trend was observed in PBMCs (494 ± 361 and 350 ± 123 copies/103 cells for proviral and unintegrated HIV-DNA, and p24 production of 149,400 ± 131,800 pg/mL). Both INIs inhibited viral replication and integration in all the cell types that were tested, especially starting at 3 dpi. However, a small but measurable amount of HIV-DNA (<5 copies/103 cells) was still observed in treated-MDMs up to 30 dpi.In conclusion, our study showed differences in HIV-DNA kinetic integration between CD4+ T-cells and MDMs, which could explain the divergent kinetics of viral-replication. Both INIs inhibited HIV-1 integration and replication with no difference found between CD4+ T-cells and MDMs. However, residual HIV-DNA remained detectable up to 30 dpi in INI-treated MDMs although complete inhibition of HIV replication was achieved. The clinical significance of this minor DNA persistence deserves further investigation considering the role of macrophages as reservoirs.

Mesenchymal stem cells are attracted to latent HIV-1-infected cells and enable virus reactivation via a non-canonical PI3K-NF\u03baB signaling pathway

Persistence of latent HIV-1 in macrophages (MACs) and T-helper lymphocytes (THLs) remain a major therapeutic challenge. Currently available latency reversing agents (LRAs) are not very effective in vivo. Therefore, understanding of physiologic mechanisms that dictate HIV-1 latency/reactivation in reservoirs is clearly needed. Mesenchymal stromal/stem cells (MSCs) regulate the function of immune cells; however, their role in regulating virus production from latently-infected MACs & THLs is not known. We documented that exposure to MSCs or their conditioned media (MSC-CM) rapidly increased HIV-1 p24 production from the latently-infected U1 (MAC) & ACH2 (THL) cell lines. Exposure to MSCs also increased HIV-1 long terminal repeat (LTR) directed gene expression in the MAC and THL reporter lines, U937-VRX and J-Lat (9.2), respectively. MSCs exposed to CM from U1 cells (U1-CM) showed enhanced migratory ability towards latently-infected cells and retained their latency-reactivation potential. Molecular studies showed that MSC-mediated latency-reactivation was dependent upon both the phosphatidyl inositol-3-kinase (PI3K) and nuclear factor-κB (NFκB) signaling pathways. The pre-clinically tested inhibitors of PI3K (PX-866) and NFκB (CDDO-Me) suppressed MSC-mediated HIV-1 reactivation. Furthermore, coexposure to MSC-CM enhanced the latency-reactivation efficacy of the approved LRAs, vorinostat and panobinostat. Our findings on MSC-mediated latency-reactivation may provide novel strategies against persistent HIV-1 reservoirs.

CYTOTOXIC PROPERTIES OF TRITERPENE SAPONIN TAUROSID SX1 AND ITS EFFECT ON HUMAN IMMUNODEFICIENCY VIRUS AND INFLUENZA VIRUS INFECTION IN MICE.

Triterpene saponin Taurosid Sx1 purified from leaves of the plant Crimean Ivy Hedera taurica Carr. (Araliaceae) was evaluated for its cytotoxic activity against lymphoblastoid cell lines MT-4, Jurkat-tat, U937, and human peripheral blood monocytes. The ability of saponin to influence HIV-1 replication was studied as well. In addition, the ability of Taurosid Sx1 to increase survival of mice infected with influenza virus А/WSN/1/33(H1N1) and its capacity for strengthening the immune responses in mice immunized with the influenza vaccine Grippol® have been studied. Taurosid Sx1 has been shown to inhibit MT-4 cell line at 25 μg ml-1 concentration, IC50 33,3 μmol l-1 (MTT assay). The saponin concentration of 5 μg ml-1 was non-toxic for all the cell lines studied and demonstrated a moderate inhibitory effect on HIV p24 production in Jurkat-tat cells. In the lower concentrations Taurosid Sx1 did not stimulate HIV p24 production. It was shown that oral administration of 200 μg Taurosid Sx1 to the influenza virus infected mice caused 1.5-fold increase in their survival. Taurosid Sx1 given orally amplified immunopotentiating ability of an intramusculary administered subunit influenza vaccine. Antibody production was significantly higher in animals fed Taurosid Sx1 after primary or secondary immunizatuion. In mice given 2 doses of vaccine, from 1 to 3 weeks apart, feeding 200 μg saponin resulted in 2 to 10-fold enhancement in production of anti-H1, anti-H3, and anti-inluenza type B hemagglutinin antibodies. Thus, Taurosid Sx1 can be considered as low-toxic promising immunopotentiating agent uncapable of enhancing HIV-1 replication.