HIV Permissive Cell Research Articles

Measuring the Inducible, Replication-Competent HIV Reservoir Using an Ultra-Sensitive p24 Readout, the Digital ELISA Viral Outgrowth Assay.

Quantifying the inducible HIV reservoir provides an estimate of the frequency of quiescent HIV-infected cells in humans as well as in animal models, and can help ascertain the efficacy of latency reversing agents (LRAs). The quantitative viral outgrowth assay (QVOA) is used to measure inducible, replication competent HIV and generate estimations of reservoir size. However, traditional QVOA is time and labor intensive and requires large amounts of lymphocytes. Given the importance of reproducible and accurate assessment of both reservoir size and LRA activity in cure strategies, efforts to streamline the QVOA are of high priority. We developed a modified QVOA, the Digital ELISA Viral Outgrowth or DEVO assay, with ultra-sensitive p24 readout, capable of femtogram detection of HIV p24 protein in contrast to the picogram limitations of traditional ELISA. For each DEVO assay, 8–12 × 106 resting CD4 + T cells from aviremic, ART-treated HIV + participants are plated in limiting dilution and maximally stimulated with PHA, IL-2 and uninfected allogeneic irradiated PBMC. CD8-depleted PHA blasts from an uninfected donor or HIV-permissive cells (e.g., Molt4/CCR5) are added to the cultures and virus allowed to amplify for 8–12 days. HIV p24 from culture supernatant is measured at day 8 by Simoa (single molecule array, ultra-sensitive p24 assay) confirmed at day 12, and infectious units per million CD4 + T cells (IUPM) are calculated using the maximum likelihood method. In all DEVO assays performed, HIV p24 was detected in the supernatant of cultures as early as 8 days post stimulation. Importantly, DEVO IUPM values at day 8 were comparable or higher than traditional QVOA IUPM values obtained at day 15. Interestingly, DEVO IUPM values were similar with or without the addition of allogeneic CD8-depleted target PHA blasts or HIV permissive cells traditionally used to expand virus. The DEVO assay uses fewer resting CD4 + T cells and provides an assessment of reservoir size in less time than standard QVOA. This assay offers a new platform to quantify replication competent HIV during limited cell availability. Other potential applications include evaluating LRA activity, and measuring clearance of infected cells during latency clearance assays.

Single-cell analysis identifies cellular markers of the HIV permissive cell.

Cellular permissiveness to HIV infection is highly heterogeneous across individuals. Heterogeneity is also found across CD4+ T cells from the same individual, where only a fraction of cells gets infected. To explore the basis of permissiveness, we performed single-cell RNA-seq analysis of non-infected CD4+ T cells from high and low permissive individuals. Transcriptional heterogeneity translated in a continuum of cell states, driven by T-cell receptor-mediated cell activation and was strongly linked to permissiveness. Proteins expressed at the cell surface and displaying the highest correlation with T cell activation were tested as biomarkers of cellular permissiveness to HIV. FACS sorting using antibodies against several biomarkers of permissiveness led to an increase of HIV cellular infection rates. Top candidate biomarkers included CD25, a canonical activation marker. The combination of CD25 high expression with other candidate biomarkers led to the identification of CD298, CD63 and CD317 as the best biomarkers for permissiveness. CD25highCD298highCD63highCD317high cell population showed an enrichment of HIV-infection of up to 28 fold as compared to the unsorted cell population. The purified hyper-permissive cell subpopulation was characterized by a downregulation of interferon-induced genes and several known restriction factors. Single-cell RNA-seq analysis coupled with functional characterization of cell biomarkers provides signatures of the “HIV-permissive cell”.

Is gibbon ape leukaemia virus still a threat?

Abstract In the late 1960s and early 1970s, an outbreak of lymphoma and leukaemia in gibbons (Hylobatidae), attributed to the retrovirus gibbon ape leukaemia virus (GALV), was widely reported in the literature. The virus was identified in captive gibbon colonies in Thailand, the USA and Bermuda. The virus is a known cell culture contaminant and, in particular, research into HIV can be impeded by expression of GALV particles in HIV permissive cell lines. In this review, we bring together published work, laboratory records from early GALV research, correspondence about the transportation of gibbons during the 1960s and 1970s, phylogenetic analyses, laboratory screening and zoological records for the first time, to discover more about the origin and transmission of GALV. Based on this evidence, we suggest that GALV may have been transmitted to gibbons as an iatrogenic event and was never widespread. Instead, all infected gibbons were probably transported from the site of the original outbreak, housed with gibbons from this site or infected with material derived from gibbons from this site. We also propose that GALV is not an ongoing pathogen of captive gibbons.

Innate immune defects in HIV permissive cell lines

BackgroundPrimary CD4+ T cells and cell lines differ in their permissiveness to HIV infection. Impaired innate immunity may contribute to this different phenotype.FindingsWe used transcriptome profiling of 1503 innate immunity genes in primary CD4+ T cells and permissive cell lines. Two clusters of differentially expressed genes were identified: a set of 249 genes that were highly expressed in primary cells and minimally expressed in cell lines and a set of 110 genes with the opposite pattern. Specific to HIV, HEK293T, Jurkat, SupT1 and CEM cell lines displayed unique patterns of downregulation of genes involved in viral sensing and restriction. Activation of primary CD4+ T cells resulted in reversal of the pattern of expression of those sets of innate immunity genes. Functional analysis of prototypical innate immunity pathways of permissive cell lines confirmed impaired responses identified in transcriptome analyses.ConclusionIntegrity of innate immunity genes and pathways needs to be considered in designing gain/loss functional genomic screens of viral infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0275-8) contains supplementary material, which is available to authorized users.

Establishing Restricted Expression of Caveolin-1 in HIV Infected Cells and Inhibition of Virus Replication.

Background: Caveolin-1 (Cav-1) is the major protein of the caveolae and plays a role in multiple cellular functions and implicated to have anti-HIV activity. Regulated expression of Cav-1 is important for safe and effective use in order to exploit Cav-1 for HIV therapeutic applications.Methods: A series of Cav-1 and GFP expression vectors were constructed under the control of the HIV LTR for conditional expression or CMV promoter and the expression of Cav-1 was monitored in the presence or absence of Tat or HIV infection in order to establish the restricted expression of Cav-1 to HIV infected cells. Results: Cav-1 expression was evident under the control of the HIV LTR in the absence of Tat or HIV infection as demonstrated by immunoblot. Placing two internal ribosomal entry sequences (IRES) and a Rev response element, RRE (5’~ LTR-IRES-GFP-RRE-IRES-Cav-1~3’) resulted in no expression of Cav-1 in the absence of Tat with effective expression in the presence of Tat. Transduction of HIV permissive cells with this construct using a foamy virus vector show that Cav-1 was able to inhibit HIV replication by 82%. Cells that received LTR-IRES-GFP-RRE-IRES-Cav-1 remain healthy in the absence of Tat or HIV infection. Conclusion: These results taken together reveal the inclusion of two IRES establishes a significant reduction of leak through expression of Cav-1 in the absence of Tat or HIV infection. Such regulated expression will have therapeutic application of Cav-1 for HIV infection as well as broad applications which can be beneficial for other host-targeted interventions as therapeutics.

Replication of biotinylated human immunodeficiency viruses

Previous work demonstrated recently the adaptation of the Escherichia coli biotin ligase BirA – biotin acceptor sequence (BAS) labeling system to produce human immunodeficiency virus type 1 viruses with biotinylated integrase (NLXIN B) and matrix (NLXMA B) proteins ( Belshan et al., 2009). This report describes the construction of an HIV permissive cell line stably expressing BirA (SupT1.BirA). Consistent with the results in the previous report, NLXMA B replicated similar to wild-type levels and expressed biotinylated Gag and MA proteins in the SupT1.BirA cells, whereas the replication of NLXIN B was reduced severely. Three additional HIV type 2 (HIV-2) viruses were constructed with the BAS inserted into the vpx and vpr accessory genes. Two BAS insertions were made into the C-terminal half of the Vpx, including one internal insertion, and one at the N-terminus of Vpr. All three viruses were replication competent in the SupT1.BirA cells and their target proteins biotinylated efficiently and incorporated into virions. These results demonstrate the potential utility of the biotinylation system to label and capture HIV protein complexes in the context of replicating virus.

Activity of TAR in inducible inhibition of HIV replication by foamy virus vector expressing siRNAs under the control of HIV LTR

In this report we describe foamy virus vectors with conditional expression of short interfering RNAs (siRNAs) in HIV infected cells. Short hairpin RNAs (shRNAs) based on two targets in the 5′ end of the untranslated region and one in the rev gene flanked with 5′ and 3′ microRNA 30 (miR30) sequences were synthesized and placed under the control of an HIV promoter for Tat-mediated expression. HIV permissive cells were transduced with foamy virus vectors containing each hybrid shRNA expression cassette and tested for their efficacy on the inhibition of HIV replication. Effective Tat dependent expression of the shRNAs, as well as GFP placed downstream each shRNA was evident. In addition the results show inhibition of HIV replication by greater than 98%. Interestingly, transduction of cells with a vector lacking an shRNA also revealed GFP expression in the presence of Tat with similar levels of inhibition of virus replication. When the TAR region was removed from this vector there was neither reduction in virus replication nor Tat-induced GFP expression. These results suggest that TAR in the vector, which Tat interacts to promote expression of the shRNA, is a potent inhibitor of virus replication. Previous studies with TAR regulated expression of antiviral genes ignore the contribution of TAR in the repression of virus replication. Interpretation of effective inhibition of HIV replication by antiviral genes located downstream of TAR while neglecting the efficacy of a potent repression by TAR is misleading.

HIV-1 envelope glycoproteins-mediated apoptosis is regulated by CD4 dependent and independent mechanisms.

The progressive loss of CD4 T lymphocytes is one of the hallmarks of HIV infection. The reverse correlation observed in vivo, between plasmatic HIV levels and CD4 T lymphocyte counts, supports the concept that direct HIV-mediated cell death contributes to this depletion. Previously, we and others have demonstrated, in vitro, that interactions between membrane-expressed HIV-envelope glycoprotein complexes and CD4 ecto-molecules are critical to cell killing which occurs mainly by apoptosis. Here, by the use of a co-culture model, in which chronically HIV-1 infected cells trigger apoptosis in uninfected CD4+ target cells, we have investigated the role of different CD4 domains in HIV envelope-mediated apoptosis. Target cells were A201 lymphoblastoid cell lines expressing wild-type CD4 or mutant forms of CD4. We show that the cytoplasmic domain of CD4 was not required for apoptosis induction. In contrast, the HIV permissive cell line expressing a CD4/CD8 chimeric molecule which contains only the first 171 amino acids of CD4, appeared to be resistant to HIV-induced apoptosis; thus suggesting that the D3-D4 CD4 module plays somewhat a regulatory role. Pre-treatment of wild-type CD4 expressing target cells by the phorbol ester PMA which leads to down-regulation of CD4, completely abolished apoptosis. Interestingly, in cells expressing CD4 devoid of its cytoplasmic domain, PMA blocked partially cell death without affecting, as expected, the CD4 expression. Taken together, these results demonstrate that although CD4 expression is essential for HIV envelope induced apoptosis, the apoptotic signal could be delivered in the absence of its cytoplasmic domain. Consistent with this, we suggest that other membrane associated molecule(s) are recruited for the signalling to initiate apoptosis.

HIV-permissive cells from solid tissues: Cytokine induction and effects

HIV-1 is a polytropic virus that, apart from hematopoietic cells, can replicate in several cells from solid tissues. Virus yields vary widely, closely resembling differences in HIV binding to the cells; the latter phenomenon appears mainly related to the extent of CD4 expression, even though other molecules have been implied in virus-cell interactions, the expression of which on the cell membrane can be modulated by several stimuli. During the course of HIV replication, a series of cytokines is produced and released in parallel to virus growth, particularly IL-6 and TNF-α. In fact, exposure of fibroblasts or epithelial cells to these HIV-in-duced cytokines causes an increase of CD4 expression, HIV adsorption to uninfected cells, and release of infectious virions by infected cells. In cells exposed to interferon-γ, IL-6 and TNF-α allow the virus to bypass interferon inhibition of virus release. The fact that HIV-1 adsorption and spread can be mediated by HIV-induced cytokines may be relevant in AIDS pathogenesis, accelerating virus transmission both within the organ and to infiltrating cells, activating a self-maintaining mechanism of infection.

Ferritin downregulation in HIV-infected cells.

Levels of serum ferritin are increased in AIDS patients in relation to the progression of the disease. To establish whether or not this in vivo increase could be due to a direct effect of the virus on the infected cells, three HIV-permissive cell lines, the CD4-positive HeLa-T4-6c and C8166 cells and the CD4-negative RD cells, were infected with HIV-1 strains. The expression of ferritin was followed during the course of acute infection, in parallel to other cellular components. Unexpectedly, all three cell lines showed a phase of decrease in their ferritin content after infection by HIV-1, not justified by the modest and late increase of ferritin in the fluids, due to disruption of infected cells. Since ferritin is involved in the control of cell growth and DNA synthesis, its downregulation may be implied both in cell toxicity and DNA abnormalities due to HIV infection.

A binding assay to identify HIV (LAV) target cells

Summary As an approach to identifying HIV (LAV) target cells, viral particles were labelled with fluorescein isothiocyanate (FITC-HIV) and the binding of FITC-HIV to lymphocytes and HIV permissive cells was analysed by fluorescence microscopy and by flow cytometry. No B cells or T8 cells were stained by FITC-HIV in contrast to normal CD4 + lymphocytes and HIV permissive cells. Preincubation with either unlabelled HIV or anti-CD4 monoclonal antibodies blocked further FITC-HIV binding on these cells, indicating the specificity of this binding assay. Similarly, pre-incubation of T lymphocytes with FITC-HIV prevented a subsequent HIV infection. Ten per cent of normal CD4 + lymphocytes and CD4 − bone marrow immature T cells as well as 10 to 30 % of CD4 + cell lines were stained by FITC-HIV. These results provide an experimental model for the further study of HIV (LAV) receptor(s) and for determining whether, in addition to CD4, other molecules are involved in HIV functional receptors.