HIV-1 targets L-selectin for adhesion and induces its shedding for viral release.

CD4 and chemokine receptors mediate HIV-1 attachment and entry. They are, however, insufficient to explain the preferential viral infection of central memory T cells. Here, we identify L-selectin (CD62L) as a viral adhesion receptor on CD4+ T cells. The binding of viral envelope glycans to L-selectin facilitates HIV entry and infection, and L-selectin expression on central memory CD4+ T cells supports their preferential infection by HIV. Upon infection, the virus downregulates L-selectin expression through shedding, resulting in an apparent loss of central memory CD4+ T cells. Infected effector memory CD4+ T cells, however, remain competent in cytokine production. Surprisingly, inhibition of L-selectin shedding markedly reduces HIV-1 infection and suppresses viral release, suggesting that L-selectin shedding is required for HIV-1 release. These findings highlight a critical role for cell surface sheddase in HIV-1 pathogenesis and reveal new antiretroviral strategies based on small molecular inhibitors targeted at metalloproteinases for viral release.

The role of the glycosyl moiety of myricetin derivatives in anti-HIV-1 activity in vitro.

BACKGROUND: Plant extracts are sources of valuable compounds with biological activity, especially for the anti-proliferative activity against pathogens or tumor cells. Myricetin is a flavonoid found in several plants that has been described as an inhibitor of Human immunodeficiency virus type 1 (HIV-1) through its action against the HIV reverse transcriptase, but myricetin derivatives have not been fully studied. The aim of this study was to evaluate the anti-HIV-1 activity of glycosylated metabolites obtained from Marcetia taxifolia and derived from myricetin: myricetin rhamnoside and myricetin 3-(6-rhamnosylgalactoside). METHODS: Compounds were obtained from organic extracts by maceration of aerial parts of M. taxifolia. All biological assays were performed in the MT4 cell line. Antiviral activity was measured as inhibition of p24 and reverse transcriptase with a fluorescent assay. RESULTS: Both flavonoids have antiviral activity in vitro, with an EC50 of 120 µM for myricetin 3-rhamnoside (MR) and 45 µM for myricetin 3-(6-rhamnosylgalactoside) (MRG), both significantly lower than the EC50 of myricetin (230 µM). Although both compounds inhibited the reverse transcriptase activity, with an IC50 of 10.6 µM for MR and 13.8 µM for MRG, myricetin was the most potent, with an IC50 of 7.6 µM, and an inhibition greater than 80%. Molecular docking approach showed correlation between the free energy of binding with the assays of enzyme inhibition. CONCLUSIONS: The results suggest that glycosylated moiety might enhance the anti-HIV-1 activity of myricetin, probably by favoring the internalization of the flavonoid into the cell. The inhibition of the HIV-1 reverse transcriptase is likely responsible for the antiviral activity.

Combinatorial CRISPR-Cas9 and RNA Interference Attack on HIV-1 DNA and RNA Can Lead to Cross-Resistance.

Many potent antiviral drugs have been developed against HIV-1, and their combined action is usually successful in achieving durable virus suppression in infected individuals. This success is based on two effects: additive or even synergistic virus inhibition and an increase in the genetic threshold for development of drug resistance. More recently, several genetic approaches have been developed to attack the HIV-1 genome in a gene therapy setting. We set out to test the combinatorial possibilities for a therapy based on the CRISPR-Cas9 and RNA interference (RNAi) mechanisms that attack the viral DNA and RNA, respectively. When two different sites in the HIV-1 genome were targeted, either with dual CRISPR-Cas9 antivirals or with a combination of CRISPR-Cas9 and RNAi antivirals, we observed additive inhibition, much like what was reported for antiviral drugs. However, when the same or overlapping viral sequence was attacked by the antivirals, rapid escape from a CRISPR-Cas9 antiviral, assisted by the error-prone nonhomologous end joining (NHEJ) DNA repair machinery, accelerated the development of cross-resistance to the other CRISPR-Cas9 or RNAi antiviral. Thus, genetic antiviral approaches can be combined, but overlap should be avoided.

Development of pyridine dicoumarols as potent anti HIV-1 leads, targeting HIV-1 associated topoisomeraseIIß kinase.

AIM: A structural study of a series of pyridine dicoumarol derivatives with potential activity against a novel Topoisomerase IIß kinase which was identified in the HIV-1 viral lysate, compounds were designed and synthesized based on a 3D-QSAR study. MATERIALS & METHODS: Based on QSAR model we have designed and synthesized a series of pyridine dicoumarol derivatives and characterized by spectral studies, all the molecules are biologically evaluated by kinase assay, cytotoxicity assay, ELISA and PCR method. RESULT: We demonstrated the achievement of water soluble disodium pyridine dicoumarate derivatives showing high anti-HIV-1 activity (IC50 <25 nM) which provides a crucial point for further development of pyridine dicoumarol series as HIV-1-associated topoisomerase IIß kinase inhibitors for clinical application against AIDS. CONCLUSION: A new class of anti-HIV-1 lead compounds have been designed and tested. Further studies would result in development of  novel and potential drugs.

Influence of the loop size and nucleotide composition on AgoshRNA biogenesis and activity.

Short hairpin RNAs (shRNAs) are widely used for gene silencing by the RNA interference (RNAi) mechanism. The shRNA precursor is processed by the Dicer enzyme into active small interfering RNAs (siRNAs) that subsequently target a complementary mRNA for cleavage by the Argonaute 2 (Ago2) complex. Recent evidence indicates that shRNAs with a relatively short basepaired stem bypass Dicer and are instead processed by Ago2. We termed these molecules AgoshRNAs as both processing and silencing steps are mediated by Ago2 and proposed rules for the design of effective AgoshRNA molecules. Active and non-cytotoxic AgoshRNAs against HIV-1 RNA were generated, but their silencing activity was generally reduced compared with the matching shRNAs. Thus, further optimization of the AgoshRNA design is needed. In this study, we evaluated the importance of the single-stranded loop, in particular its size and nucleotide sequence, in AgoshRNA-mediated silencing. We document that the pyrimidine/purine content is important for AgoshRNA-mediated silencing activity.

Characterization and in vitro activity of a branched peptide boronic acid that interacts with HIV-1 RRE RNA.

A branched peptide containing multiple boronic acids was found to bind RRE IIB selectively and inhibit HIV-1 p24 capsid production in a dose-dependent manner. Structure-activity relationship studies revealed that branching in the peptide is crucial for the low micromolar binding towards RRE IIB, and the peptide demonstrates selectivity towards RRE IIB in the presence of tRNA. Footprinting studies suggest a binding site on the upper stem and internal loop regions of the RNA, which induces enzymatic cleavage of the internal loops of RRE IIB upon binding.

A Cell Internalizing Antibody Targeting Capsid Protein (p24) Inhibits the Replication of HIV-1 in T Cells Lines and PBMCs: A Proof of Concept Study.

There remains a need for newer therapeutic approaches to combat HIV/AIDS. Viral capsid protein p24 plays important roles in HIV pathogenesis. Peptides and small molecule inhibitors targeting p24 have shown to inhibit virus replication in treated cell. High specificity and biological stability of monoclonal antibodies (mAbs) make them an attractive contender for in vivo treatments. However, mAbs do not enter into cells, thus are restricted to target surface molecules. This also makes targeting intracellular HIV-1 p24 a challenge. A mAb specific to p24 that can internalize into the HIV-infected cells is hypothesized to inhibit the virus replication. We selected a mAb that has previously shown to inhibit p24 polymerization in an in vitro assay and chemically conjugated it with cell penetrating peptides (CPP) to generate cell internalizing anti-p24 mAbs. Out of 8 CPPs tested, κFGF-MTS -conjugated mAbs internalized T cells most efficiently. At nontoxic concentration, the κFGF-MTS-anti-p24-mAbs reduced the HIV-1 replication up to 73 and 49% in T-lymphocyte and PBMCs respectively. Marked inhibition of HIV-1 replication in relevant cells by κFGF-MTS-anti-p24-mAbs represents a viable strategy to target HIV proteins present inside the cells.

Gold nanoparticles to improve HIV drug delivery.

BACKGROUND: Antiretroviral therapy (ART) has improved lifespan and quality of life of patients infected with the HIV-1. However, ART has several potential limitations, including the development of drug resistance and suboptimal penetration to selected anatomic compartments. Improving the delivery of antiretroviral molecules could overcome several of the limitations of current ART. RESULTS & CONCLUSION: Two to ten nanometer diameter inorganic gold crystals serve as a base scaffold to combine molecules with an array of properties in its surface. We show entry into different cell types, antiviral activity of an HIV integrase inhibitor conjugated in a gold nanoparticle and penetration into the brain in vivo without toxicity. Herein, gold nanoparticles prove to be a promising tool to use in HIV therapy.

4-Quinolone alkaloids from Melochia odorata.

The methanol extract of Melochia odorata yielded three 4-quinolone alkaloids including waltherione A (1) and two new alkaloids, waltherione C (2) and waltherione D (3). Waltheriones A and C showed significant activities in an in vitro anti-HIV cytoprotection assay at concentrations of 56.2 and 0.84 µM and inhibition of HIV P24 formation of more than 50% at 1.7 and 0.95 µM, respectively. The structures of the alkaloids were established by spectroscopic data interpretation.

Measuring inhibition of HIV replication by ex vivo CD8⁺ T cells.

HIV replication is unrestrained in vivo in the vast majority of infected subjects, and the ability of some rare individuals to control this virus is poorly understood. Standard immunogenicity assays for detecting HIV-1-specific CD8(+) T-cell responses, such as IFN-γ ELISpot and intracellular cytokine staining, generally fail to correlate with in vivo inhibition of HIV replication. Several viral inhibition assays, which measure the effectiveness of CD8(+) T-cell responses in suppressing HIV replication in vitro, have been described; but most depend on in vitro expansion of CD8(+) T cells, and some show inhibitory activity in HIV-negative individuals. We have optimized an assay to assess the suppressive capability of CD8(+) T cells directly ex vivo, eliminating the potential for altering their function through activation or expansion prior to assay setup, and thereby enhancing the assay's sensitivity by avoiding non-specific inhibition. With this method, the ability of ex vivo CD8(+) T cells to control HIV-1 replication in vitro can be quantified over several orders of magnitude. Specifically, our assay can be used to better define the antiviral function of CD8(+) T cells induced by vaccination, and can provide insight into their ability to control viral replication if HIV infection occurs post-vaccination.

Molecular recognition in the human immunodeficiency virus capsid and antiviral design.

Many compounds able to interfere with HIV-1 infection have been identified; some 25 of them have been approved for clinical use. Current anti-HIV-1 therapy involves the use of drug cocktails, which reduces the probability of virus escape. However, many issues remain, including drug toxicity and the emergence of drug-resistant mutant viruses, even in treated patients. Therefore, there is a constant need for the development of new anti-HIV-1 agents targeting other molecules in the viral cycle. The capsid protein CA plays a key role in many molecular recognition events during HIV-1 morphogenesis and uncoating, and is eliciting increased interest as a promising target for antiviral intervention. This article provides a structure-based, integrated review on the CA-binding small molecules and peptides identified to date, and their effects on virus capsid assembly and stability, with emphasis on recent results not previously reviewed. As a complement, we present novel experimental results on the development and proof-of-concept application of a combinatorial approach to study molecular recognition in CA and its inhibition by peptide compounds.

7,8-secolignans from Schisandra wilsoniana and their anti-HIV-1 activities.

Two new 7,8-secolignans, marphenols A and B (1 and 2, resp.), together with a known related derivative, 7,8-secoholostylone B (3), were isolated from the stems of Schisandra wilsoniana. The structures of 1 and 2 were elucidated by spectroscopic methods, including extensive 1D- and 2D-NMR techniques. The anti-HIV-1 activities of 1-3 were evaluated. Compound 1 inhibited HIV-1(IIIB)-induced syncytia formation with an EC(50) value of 0.55 µg ml(-1). It reduced p24 antigen expression in acutely HIV-1(IIIB)-infected C8166 cells and primary isolate HIV-1(TC-2)-infected peripheral blood mononuclear cells (PBMCs), with EC(50) values of 3.34 and 0.52 µg ml(-1), respectively. It showed no effects on the HIV-1(IIIB) replication in chronically infected H9 cells as well as fusion inhibition.

Erythrocytes as carriers of antisense PNA addressed against HIV-1 gag-pol transframe domain.

PNA(PR2) is a peptide nucleic acid (PNA) complementary to a sequence of the viral protease-encoding gene, effective in blocking HIV release, when used at high doses. Erythrocytes (RBC) were used to target PNA(PR2) to the macrophage compartment. The antiviral activity was assessed in human HIV-infected macrophages both as inhibition of p24 production and reduction of HIV DNA content. PNA(PR2), either added to the medium at a concentration of 100 microM or loaded into RBC at about 40 microM, inhibited p24 production approximately 80% compared with infected samples and reduced HIV DNA content by 83% and 90%, respectively. The results show that (1) a stronger anti-HIV effect is achievable with higher doses of PNA(PR2), both when given free and encapsulated into RBC; (2) the antiviral effect obtained by free PNA(PR2) at a concentration of 100 microM is achievable by encapsulating it into RBC at a concentration of 40 microM, suggesting that RBC can be used as a delivery system to increase the antisense effect of PNA(PR2).

Preclinical development of the green tea catechin, epigallocatechin gallate, as an HIV-1 therapy.

BACKGROUND: Previously, we presented evidence that at physiologic concentrations the green tea catechin, epigallocatechin gallate (EGCG), inhibited attachment of HIV-1 glycoprotein 120 to the CD4 molecule on T cells, but the downstream effects of EGCG on HIV-1 infectivity were not determined. OBJECTIVE: To evaluate the inhibition of HIV-1 infectivity by EGCG and begin preclinical development of EGCG as a possible therapy. METHODS: PBMCs, CD4(+) T cells, and macrophages were isolated from blood of HIV-1-uninfected donors. HIV-1 infectivity was assessed by an HIV-1 p24 ELISA. Cell survival was assessed by cell viability by Trypan blue exclusion assay, cell growth by thymidine incorporation, and apoptosis by flow-cytometric analysis of annexin-V binding. RESULTS: Epigallocatechin gallate inhibited HIV-1 infectivity on human CD4(+) T cells and macrophages in a dose-dependent manner. At a physiologic concentration of 6 mumol/L, EGCG significantly inhibited HIV-1 p24 antigen production across a broad spectrum of both HIV-1 clinical isolates and laboratory-adapted subtypes (B [P < .001], C, D, and G [P < .01]). The specificity of the EGCG-induced inhibition was substantiated by the failure of EGCG derivatives lacking galloyl and/or pyrogallol side groups to alter HIV-1 p24 levels. EGCG-induced inhibition of HV-1 infectivity was not a result of cytotoxicity, cell growth inhibition, or apoptosis. CONCLUSION: We conclude that by preventing the attachment of HIV-1-glycoprotein 120 to the CD4 molecule, EGCG inhibits HIV-1 infectivity. Because this inhibition can be achieved at physiologic concentrations, the natural anti-HIV agent EGCG is a candidate as an alternative therapy in HIV-1 therapy.

HIV-1 capsid assembly inhibitor (CAI) peptide: structural preferences and delivery into human embryonic lung cells and lymphocytes.

The Human immunodeficiency virus 1 derived capsid assembly inhibitor peptide (HIV-1 CAI-peptide) is a promising lead candidate for anti-HIV drug development. Its drawback, however, is that it cannot permeate cells directly. Here we report the transport of the pharmacologically active CAI-peptide into human lymphocytes and Human Embryonic Lung cells (HEL) using the BioShuttle platform. Generally, the transfer of pharmacologically active substances across membranes, demonstrated by confocal laser scanning microscopy (CLSM), could lead to a loss of function by changing the molecule's structure. Molecular dynamics (MD) simulations and circular dichroism (CD) studies suggest that the CAI-peptide has an intrinsic capacity to form a helical structure, which seems to be critical for the pharmacological effect as revealed by intensive docking calculations and comparison with control peptides. This coupling of the CAI-peptide to a BioShuttle-molecule additionally improved its solubility. Under the conditions described, the HIV-1 CAI peptide was transported into living cells and could be localized in the vicinity of the mitochondria.

Three new derivatives of anti-HIV-1 polyphenols isolated from Salvia yunnanensis.

During the study of anti-HIV-1 active components of the aqueous extracts of the roots of Salvia yunnanensis, three new derivatives of polyphenols, namely: methyl salvianolate A (2), ethyl salvianolate A (3) and cis-lithospermic acid (5) were isolated along with two known polyphenols, salvianolic acid A (1) and lithospermic acid (4) their structures were elucidated on the basis of NMR and MS spectral analyses. The anti-HIV-1 activities of the 5 polyphenols were tested for the inhibition of P24 antigen in HIV-1 infected MT-4 cell cultures and HIV-1 replicative enzymes in vitro.

A potent anti-HIV polyphenol from Salvia yunnanensis.

A new polyphenol, designated as salvianolic acid N, was isolated from the aqueous extracts of the roots of Salvia yunnanensis. Its chemical structure was elucidated as 3-(3,4-dihydroxylphenyl)-2-[(E)-3-(1,8,9-trihydroxyl-dibenzo[b,f]oxpin-3-yl)acryloxloxy]propanoic acid (1) on the basis of NMR and MS spectral analyses. The new polyphenol inhibited both HIV-1 IN in vitro and also reduced HIV-1 p24 antigen in MT-4 cell lines.