SupT1 Cells Research Articles

An improved cell-based method for determining the γ-secretase enzyme activity against both Notch and APP substrates

γ-Secretase modulators (GSM), which reduce amyloidogenic Aβ42 production while maintaining total Aβ levels, and Notch-sparing γ-secretase inhibitors (GSIs) are promising therapies for the treatment of Alzheimer's Disease (AD). To have a safety margin for therapeutic use, GSMs and GSIs need to allow Notch intracellular domain (NICD) production, while preventing neurotoxic Aβ peptide production. Typically, GSI and GSM effects on these substrates are determined using two different cell lines, one for the measurement of enzyme activity against each substrate. However, predicting selectivity for different substrates across cell systems may reduce the reliability of such ratios such that the in vitro data are not useful for predicting in vivo safety margins. This is especially concerning since the IC50's of some GSIs vary depending upon the level of APP expression in a cell line. To circumvent this problem, we utilized the SUP-T1 cell line which expresses a truncated Notch receptor fragment that does not need sheddase cleavage to be a γ-secretase substrate. When combined with a sensitive method of measuring Aβ production, this assay system allows both substrates to be measured simultaneously, reducing the potential to calculate imprecise selectivity margins. To demonstrate the value of this system, known GSIs and GSMs were examined in the SUP-T1 dual substrate assay. IC50's were determined for both substrates and the in vitro selectivity margin was calculated. These data suggest using a single cell line is a more accurate prediction of the fold difference between NICD inhibition and Aβ42 lowering for therapeutically promising GSIs and GSMs.

Impact of sustained RNAi-mediated suppression of cellular cofactor Tat-SF1 on HIV-1 replication in CD4+ T cells

BackgroundConventional anti-HIV drug regimens targeting viral enzymes are plagued by the emergence of drug resistance. There is interest in targeting HIV-dependency factors (HDFs), host proteins that the virus requires for replication, as drugs targeting their function may prove protective. Reporter cell lines provide a rapid and convenient method of identifying putative HDFs, but this approach may lead to misleading results and a failure to detect subtle detrimental effects on cells that result from HDF suppression. Thus, alternative methods for HDF validation are required. Cellular Tat-SF1 has long been ascribed a cofactor role in Tat-dependent transactivation of viral transcription elongation. Here we employ sustained RNAi-mediated suppression of Tat-SF1 to validate its requirement for HIV-1 replication in a CD4+ T cell-derived line and its potential as a therapeutic target.ResultsshRNA-mediated suppression of Tat-SF1 reduced HIV-1 replication and infectious particle production from TZM-bl reporter cells. This effect was not a result of increased apoptosis, loss of cell viability or an immune response. To validate its requirement for HIV-1 replication in a more relevant cell line, CD4+ SupT1 cell populations were generated that stably expressed shRNAs. HIV-1 replication was significantly reduced for two weeks (~65%) in cells with depleted Tat-SF1, although the inhibition of viral replication was moderate when compared to SupT1 cells expressing a shRNA targeting the integration cofactor LEDGF/p75. Tat-SF1 suppression was attenuated over time, resulting from decreased shRNA guide strand expression, suggesting that there is a selective pressure to restore Tat-SF1 levels.ConclusionsThis study validates Tat-SF1 as an HDF in CD4+ T cell-derived SupT1 cells. However, our findings also suggest that Tat-SF1 is not a critical cofactor required for virus replication and its suppression may affect cell growth. Therefore, this study demonstrates the importance of examining HIV-1 replication kinetics and cytotoxicity in cells with sustained HDF suppression to validate their therapeutic potential as targets.

Virus-producing cells determine the host protein profiles of HIV-1 virion cores

BackgroundUpon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized.ResultsWe examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of incorporation of some RNA binding (RHA and HELIC2) and DNA binding proteins (MCM5 and Ku80) in the viral cores from T cells was higher than in the cores from both mMΦ and mMN and did not correlate with the abundance of these proteins in virus producing cells.ConclusionsProfiles of host proteins packaged in the cores of HIV-1 virions depend on the type of virus producing cell. The pool of proteins present in the cores of all virions is likely to contain factors important for viral functions. Incorporation ratio of certain RNA- and DNA-binding proteins suggests their more efficient, non-random packaging into virions in T cells than in mMΦ and mMN.

An Initial In Vitro Investigation into the Potential Therapeutic Use of SupT1 Cells to Prevent AIDS in HIV-Seropositive Individuals

HIV infection usually leads to a progressive decline in number and functionality of CD4+ T lymphocytes, resulting in AIDS development. In this study, I investigated the strategy of using inoculated SupT1 cells to move infection from HIV-1 X4 strains toward the inoculated cells, which should theoretically prevent infection and depletion of normal CD4+ T cells, preventing the development of AIDS-related pathologies. Interestingly, the persistent in vitro replication in SupT1 cells renders the virus less cytopathic and more sensitive to antibody-mediated neutralization, suggesting that replication of the virus in the inoculated SupT1 cells may have a vaccination effect in the long run. In order to mimic the scenario of a therapy in which SupT1 cells are inoculated in an HIV-seropositive patient, I used infected SupT1/PBMC cocultures and a series of control experiments. Infections were done with equal amounts of the wild type HIV-1 LAI virus. The SupT1 CD4+CD8+ T cell population was distinguished from the PBMC CD4+CD8− T cell population by FACS analysis. The results of this study show that the virus-mediated killing of primary CD4+ T cells in the SupT1/PBMC cocultures was significantly delayed, suggesting that the preferential infection of SupT1 cells can induce the virus to spare primary CD4+ T cells from infection and depletion. The preferential infection of SupT1 cells can be explained by the higher viral tropism for the SupT1 cell line. In conclusion, this study demonstrates that it's possible in an in vitro system to use SupT1 cells to prevent HIV infection of primary CD4+ T cells, suggesting that further exploration of the SupT1 cell line as a cell-based therapy against HIV-1 may prove worthwhile.

Quantitative proteomic analysis of HIV-1 infected CD4+ T cells reveals an early host response in important biological pathways: Protein synthesis, cell proliferation, and T-cell activation

Human immunodeficiency virus (HIV-1) depends upon host-encoded proteins to facilitate its replication while at the same time inhibiting critical components of innate and/or intrinsic immune response pathways. To characterize the host cell response on protein levels in CD4+ lymphoblastoid SUP-T1 cells after infection with HIV-1 strain LAI, we used mass spectrometry (MS)-based global quantitation with iTRAQ (isobaric tag for relative and absolute quantification). We found 266, 60 and 22 proteins differentially expressed (DE) (P-value≤0.05) at 4, 8, and 20hours post-infection (hpi), respectively, compared to time-matched mock-infected samples. The majority of changes in protein abundance occurred at an early stage of infection well before the de novo production of viral proteins. Functional analyses of these DE proteins showed enrichment in several biological pathways including protein synthesis, cell proliferation, and T-cell activation. Importantly, these early changes before the time of robust viral production have not been described before.

Abstract 5441: Anticancer effects of 3,3′-diindolylmethane (DIM) in multiple T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) cell lines

Abstract Previously, we reported that in utero exposure to the bioactive food chemical indole-3-carbinol (I3C) via the maternal diet reduced risk of T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) in a mouse model of transplacental chemical carcinogenesis. Subsequent in vitro experiments in human CCRF-CEM cells, a T-ALL cell line originating from precursor T-cells, showed that the I3C derivative compound 3,3′-diindolylmethane (DIM) was likely responsible for the anticancer effect of the parent chemical. Specifically, DIM treatment markedly reduced CCRF-CEM cell viability, blocked cell proliferation, increased the rate of apoptosis and induced a G1 cell cycle arrest in vitro at low micromolar concentrations, whereas I3C was only effective at high, supraphysiological concentrations. T-ALL is a heterogeneous disease resulting from the developmental arrest and abnormal proliferation of T-cells at different stages of maturation. Thus, the objective of the present study was to determine whether the anticancer effects observed for DIM in CCRF-CEM cells (CD3-/CD4+/CD8-) are consistent in other T-ALL cell lines, including CCRF-HSB2 (pre-T; CD3-/CD4-/CD8-), SUP-T1 (cortical T; CD3-/CD4+/CD8+) and Jurkat cells (mature T; CD3+/CD4+/CD8-). Each cell line was treated with 0 to 60 μM DIM (BioResponse formulation), 0 to 500 μM I3C or a 0.1% DMSO vehicle control for up to 48 hours, at which time cell viability, proliferation and apoptosis were determined by flow cytometry. In vitro treatment with DIM effectively reduced proliferation of CCRF-HSB2, SUP-T1 and Jurkat cells with calculated IC50 values of 7.5, 13 and 9.1 μM, respectively, whereas I3C was much less effective in these cell lines (IC50 values of 86, 284 and 228 μM, respectively). Cell viability was similarly impacted by DIM (IC50 values of 7.2, 14 and 15 μM for CCRF-HSB2, SUP-T1 and Jurkat cells, respectively) and I3C (IC50 values of 83, 262 and 222 μM, respectively). Interestingly, treatment with 15 μM DIM caused a significant increase in apoptosis in CCRF-HSB2 (52% apoptotic cells, DIM; 9.3%, control; p = 0.02) and SUP-T1 cells (29%, DIM; 8%, control; p < 0.001), but not in Jurkat cells (9.7%, DIM; 3%, control; p = 0.08). Overall, these observations are similar to those we previously reported for CCRF-CEM cells, particularly with respect to the large difference in effectiveness of DIM compared to I3C in vitro. Also of special interest is the finding that both DIM and I3C appear most effective in blocking the growth of CCRF-HSB2 cells, a cell line representing T-ALL derived from pre-T cells. Collectively, these data suggest that DIM (via dietary I3C) could be an effective anticancer agent against T-ALL cancers originating from T cells at different stages of maturation at concentrations that can be reasonably achieved in vivo in humans and in animal models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5441. doi:1538-7445.AM2012-5441

Therapeutic potential of HIV protease-activable CASP3

Development of a therapeutic application of CASP3/caspase 3/CPP32, an executor of apoptosis, has been challenging because regulation of its activation is complicated. This study aimed to inhibit cancer cell growth and human immunodeficiency virus type 1 (HIV-1) propagation through a CASP3 mutant, CASP3*, activable by HIV-1-encoded aspartate protease. Active CASP3* was delivered to leukemic cells using a protein transduction vehicle, the lentivirus-like nanoparticle (LENA), which should contain thousands of CASP3*-Gag protein molecules and release the activated CASP3* into the target cell cytoplasm. CASP3*-LENA induced apoptosis in various types of leukemic cells. In addition to being effective against leukemic cells, constitutive expression of CASP3* restricted HIV-1 propagation in SUP-T1 cells. The attenuation of HIV-1 replication in SUP-T1/CASP3* cells was attributed to the elimination of HIV-1-infected cells by apoptosis. These data suggest that CASP3* has therapeutic potential against both lymphoid malignancies and HIV-1 infection.

Molecular Cloning of a Novel PPEF-1 Gene Variant from a T-Cell Lymphoblastic Lymphoma Cell Line

To determine if there is a gene variant of protein serine-threonine phosphatase with EF hand (PPEF-1) in T-cell lymphoblastic lymphoma SUP-T1 cell line, both in silico and in vitro approaches were conducted. In silico, a cDNA clone showing similar sequence to PPEF-1 was isolated from the SUP-T1 cDNA library and named PPEF-1V. The full-length of the PPEF-1V cDNA clone is a 2135bp containing a 1503bp open reading frame extending from 188bp to 1690bp, which corresponds to an encoded protein of 501 amino acid residues with a predicted molecular mass of 57.8 kDa. Alignment on both PPEF-1V and PPEF-1 sequences showed that PPEF-1V is a 350bp deletion in the nucleotide sequence of PPEF-1 from 128-477bp and a 152-amino-acid N-terminal deletion in the amino acid sequence of PPEF-1. In vitro, PPEF-1V transcript fragment was only highly expressed in T-cell lymphoblastic lymphoma cell line. In conclusion, the present patent showed that PPEF-1V could be a potential target for diagnosis or treatment of T-cell lymphoblastic lymphoma.

Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

BackgroundAnkyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1.ResultsA phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named AnkGAG1D4 (16.5 kDa) was isolated. AnkGAG1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of Kd ~ 1 μM, and the AnkGAG1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing AnkGAG1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. AnkGAG1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The AnkGAG1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of AnkGAG1D4-CA with the Gag assembly and budding pathway.ConclusionsThe resistance of AnkGAG1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin AnkGAG1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules.

Efficient production of Hantaan and Puumala pseudovirions for viral tropism and neutralization studies

Puumala (PUUV) and Hantaan (HTNV) viruses are hantaviruses within the family Bunyaviridae and associated with Hemorrhagic Fever with Renal Syndrome (HFRS) in humans. Little is known about how these viruses interact with host cells, though pathogenic hantaviruses interact with α vβ 3 integrin. To study host cell interactions and rapidly test the ability of antibodies to prevent infection, we produced HTNV and PUUV pseudovirions on a vesicular stomatitis virus (VSV) core. Similar to replication-competent hantaviruses, infection was low-pH-dependent. Despite broad cell tropism, several human T cell lines were poorly permissive to hantavirus pseudovirions, compared to VSV, indicating a relative block to infection at the level of entry. Stable expression of α vβ 3 integrin in SupT1 cells did not restore infectivity. Finally, the pseudovirion system provided a rapid, quantitative, and specific method to screen for neutralizing antibodies in immune sera.

Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity

A human immunodeficiency virus type (HIV-1)-based lentiviral vector pseudotyped with the vesicular stomatitis virus envelope glycoprotein and encoding the GFP reporter gene was used to evaluate different methods of lentiviral vector titration. GFP expression, viral DNA quantification and the efficiency of vector DNA integration were assayed after infection of conventional HIV-1-permissive cell lines and human primary adult fibroblasts with the vector. We found that vector titers based on GFP expression determined by flow cytometry may vary by more than 50-fold depending on the cell type and the promoter-cell combination used. Interestingly, we observed that the viral integration process in primary HDFa cells was significantly more efficient compared to that in SupT1 or 293T cells. We propose that determination of the amount of integrated viral DNA by quantitative PCR be used in combination with the reporter gene expression assay.

HIV-1 splicing is modulated by RNA structure

Materials and methods The SD stem-loop structure in the HIV-1 leader RNA region was stabilized or destabilized through mutation in the context of the HIV-1 molecular clone pLAI. The effect of the SD hairpin mutation on viral gene expression was measured upon transfection of 293T cells and virus replication was measured upon infection of SupT1 cells. The mutations were also introduced into an LTRluciferase reporter plasmid that was designed to monitor SD splicing by causing reduced luciferase activity. Upon transfection of these constructs into 293T cells, the efficiency of splicing at the major SD site was analysed by luciferase activity assays and RNA analysis (RT-PCR). Results Further stabilization of the SD stem-loop structure significantly reduced HIV-1 gene expression and replication and splicing was inhibited in the LTR-luciferase assay. SD hairpin destabilization did not cause major virus replication effects, but LTR-luciferase experiments revealed a modest upregulation of the splicing efficiency. Conclusions The stability of the SD stem-loop structure modulates the frequency of splicing at the major SD site. These results suggest that the stability of this hairpin is finetuned to obtain the right balance between unspliced and spliced RNAs in HIV-infected cells.

Silencing of Notch3 Using shRNA Driven by Survivin Promoter Inhibits Growth and Promotes Apoptosis of Human T-Cell Acute Lymphoblastic Leukemia Cells

As a highly conserved system, the activation of the Notch pathway has been implicated in the tumorigenesis of various hematologic diseases, including leukemias, lymphomas, and multiple myeloma. The Notch3 receptor is frequently expressed in T-cell acute lymphoblastic leukemia (T-ALL). To explore its possibility as a therapeutic target for T-ALL, we investigated the effect of Notch3 silencing on Jurkat and SupT1 cells using a novel tumor-specific short hairpin RNA (shRNA) driven by survivin promoters. We found that downregulated expression of Notch3 correlated with significant apoptosis and inhibition of proliferation. These facts suggest that downregulating expression of Notch3 could attenuate the Notch signaling activity in T-ALL. All these results indicate that inhibition of Notch3 expression can result in potent antitumor activity in T-ALL.

High-throughput Screening Uncovers a Compound That Activates Latent HIV-1 and Acts Cooperatively with a Histone Deacetylase (HDAC) Inhibitor

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4(+) T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.

Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies.

Background:Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy.Results:Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC50 of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested.Conclusions:These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors.

Analysis of HIV-1 expression level and sense of transcription by high-throughput sequencing of the infected cell.

Next-generation sequencing offers an unprecedented opportunity to jointly analyze cellular and viral transcriptional activity without prerequisite knowledge of the nature of the transcripts. SupT1 cells were infected with a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped HIV vector. At 24 h postinfection, both cellular and viral transcriptomes were analyzed by serial analysis of gene expression followed by high-throughput sequencing (SAGE-Seq). Read mapping resulted in 33 to 44 million tags aligning with the human transcriptome and 0.23 to 0.25 million tags aligning with the genome of the HIV-1 vector. Thus, at peak infection, 1 transcript in 143 is of viral origin (0.7%), including a small component of antisense viral transcription. Of the detected cellular transcripts, 826 (2.3%) were differentially expressed between mock- and HIV-infected samples. The approach also assessed whether HIV-1 infection modulates the expression of repetitive elements or endogenous retroviruses. We observed very active transcription of these elements, with 1 transcript in 237 being of such origin, corresponding on average to 123,123 reads in mock-infected samples (0.40%) and 129,149 reads in HIV-1-infected samples (0.45%) mapping to the genomic Repbase repository. This analysis highlights key details in the generation and interpretation of high-throughput data in the setting of HIV-1 cellular infection.

CIITA enhances HIV-1 attachment to CD4+ T cells leading to enhanced infection and cell depletion (154.27)

Abstract Activated CD4+ T cells are more susceptible to HIV infection than resting T cells; the reason for this remains unresolved. Induction of CIITA and subsequent expression of the MHC class II isotype HLA-DR are hallmarks of CD4+ T cell activation; therefore, we investigated the role of CIITA expression in T cells during HIV infection. CIITA-expressing SupT1 cells display enhanced virion attachment in a gp160/CD4-dependent manner, resulting in increased HIV infection, virus release, and T cell depletion. Although increased attachment and infection of T cells correlated with HLA-DR surface expression, Ab blocking, transient expression of HLA-DR without CIITA, and short hairpin RNA knockdown demonstrate that HLA-DR does not directly enhance susceptibility of CIITA-expressing cells to HIV infection. Analysis of the remaining MHC class II isotypes, HLA-DP and HLA-DQ, MHC class I isotypes, HLA-A, HLA-B, and HLA-C, and the class II Ag presentation genes, invariant chain and HLA-DM, demonstrate that these proteins likely do not contribute to CIITA enhancement of HIV infection. Finally, we demonstrate that in activated primary CD4+ T cells as HLA-DR/CIITA expression increases there is a corresponding increase in virion attachment. Overall, this work suggests that induction of CIITA expression upon CD4+ T cell activation contributes to enhanced attachment, infection, virus release, and cell death through an undefined CIITA transcription product that may serve as a new antiviral target.

Evaluation of the Roche COBAS® TaqMan® HIV-1 test for quantifying HIV-1 RNA in infected cells and lymphoid tissue

Lymphoid tissue is the main reservoir of HIV-1 in infected individuals. In this study the COBAS® TaqMan® HIV-1 test was evaluated for use with the High Pure System (HPS), for quantifying HIV-1 RNA in infected cells and lymphoid tissue specimens. Serial dilutions of 8E5–LAV1 infected T-cells into SUP-T1 cells and 44 tonsil specimens were examined. Some modifications of the test were required, such as the removal of residual DNA and the HIV-1 RNA output copies were adjusted to the sample input and expressed as HIV-1 RNA copies/μg of total RNA. The Roche COBAS® TaqMan HIV-1® (HPS) test proved to be a robust, sensitive, specific and reproducible method for quantifying HIV-1 RNA in infected cells and lymphoid tissue. Linearity and reproducibility were observed in serial dilutions of 8E5–LAV1 infected T-cells (R2>0.86). High reproducibility was found in clinical tonsil specimens (Wilcoxon test p>0.05). rDNase I treatment was essential to avoid false positives caused by residual HIV-1 DNA, mainly in tonsil specimens obtained from infected patients receiving effective antiretroviral treatment. Probit analysis determined the limit of detection as 22HIV-1 RNA copies/μg of total RNA. The Roche COBAS® TaqMan® HIV-1 (HPS) test thereby proved to be a helpful tool for measuring the HIV-1 viral load in infected cells and lymphoid tissue reservoirs.

Differences in the mannose oligomer specificities of the closely related lectins from Galanthus nivalis and Zea maysstrongly determine their eventual anti-HIV activity

BackgroundIn a recent report, the carbohydrate-binding specificities of the plant lectins Galanthus nivalis (GNA) and the closely related lectin from Zea mays (GNAmaize) were determined by glycan array analysis and indicated that GNAmaize recognizes complex-type N-glycans whereas GNA has specificity towards high-mannose-type glycans. Both lectins are tetrameric proteins sharing 64% sequence similarity.ResultsGNAmaize appeared to be ~20- to 100-fold less inhibitory than GNA against HIV infection, syncytia formation between persistently HIV-1-infected HuT-78 cells and uninfected CD4+ T-lymphocyte SupT1 cells, HIV-1 capture by DC-SIGN and subsequent transmission of DC-SIGN-captured virions to uninfected CD4+ T-lymphocyte cells. In contrast to GNA, which preferentially selects for virus strains with deleted high-mannose-type glycans on gp120, prolonged exposure of HIV-1 to dose-escalating concentrations of GNAmaize selected for mutant virus strains in which one complex-type glycan of gp120 was deleted. Surface Plasmon Resonance (SPR) analysis revealed that GNA and GNAmaize interact with HIV IIIB gp120 with affinity constants (KD) of 0.33 nM and 34 nM, respectively. Whereas immobilized GNA specifically binds mannose oligomers, GNAmaize selectively binds complex-type GlcNAcβ1,2Man oligomers. Also, epitope mapping experiments revealed that GNA and the mannose-specific mAb 2G12 can independently bind from GNAmaize to gp120, whereas GNAmaize cannot efficiently bind to gp120 that contained prebound PHA-E (GlcNAcβ1,2man specific) or SNA (NeuAcα2,6X specific).ConclusionThe markedly reduced anti-HIV activity of GNAmaize compared to GNA can be explained by the profound shift in glycan recognition and the disappearance of carbohydrate-binding sites in GNAmaize that have high affinity for mannose oligomers. These findings underscore the need for mannose oligomer recognition of therapeutics to be endowed with anti-HIV activity and that mannose, but not complex-type glycan binding of chemotherapeutics to gp120, may result in a pronounced neutralizing activity against the virus.

The IFITM Proteins Inhibit HIV-1 Infection

Type I interferon protects cells from virus infection through the induction of a group of genes collectively named interferon-stimulated genes (ISGs). In this study, we utilized short hairpin RNA (shRNA) to deplete ISGs in SupT1 cells in order to identify ISGs that suppress the production of human immunodeficiency virus type 1 (HIV-1). Among the ISG candidates thus identified were interferon-induced transmembrane (IFITM) proteins, including IFITM1, IFITM2, and IFITM3, that potently inhibit HIV-1 replication at least partially through interfering with virus entry. Further mutagenesis analysis shows that the intracellular region, rather than the N- and C-terminal extracellular domains, is essential for the antiviral activity of IFITM1. Altogether, these data suggest that the IFITM proteins serve as important components of the innate immune system to restrict HIV-1 infection.