Papers by Velpandi Ayyavoo
Additional file 6: Fig. S5. Primary resting CD4+Â T cells were treated with multiple concentratio... more Additional file 6: Fig. S5. Primary resting CD4+Â T cells were treated with multiple concentrations of small molecules inhibiting specific cellular signaling pathway. Cell viability was evaluated after 3Â days by flow cytometry and trypan blue staining. Results from two independent donors tested in duplicates that are normalized to DMSO only control are included.
Additional file 2: Table S1 Fold change in expression of NT5C3 transcripts at 18 hours in ACH-2 c... more Additional file 2: Table S1 Fold change in expression of NT5C3 transcripts at 18 hours in ACH-2 cells following treatment with SAHA, prostratin or TNF-Îą.
Additional file 5: Fig. S4: J-Lat cells FL10.6, TGA1 and TGA2 were treated with multiple doses of... more Additional file 5: Fig. S4: J-Lat cells FL10.6, TGA1 and TGA2 were treated with multiple doses of (A) Rottlerin and (B) WP1066 or vehicle control (DMSO), and 16 h post-treatment, the live cells were evaluated by Trypan blue staining. The percentage of viable cells was calculated by subtracting the dead cells from total cells divided by total cell count. Viability of cells in the vehicle control was considered as 100 % for comparison across three independent experiments. Error bars represent standard deviation (N = 3).
Additional file 3: Fig. S2 Reversal of HIV-1 latency in ACH-2 cells is associated with induction ... more Additional file 3: Fig. S2 Reversal of HIV-1 latency in ACH-2 cells is associated with induction of NT5C3 gene expression. (A) Schematic representation of organization of genes on Chromosome 7 between nucleotides 32916815 to 33606068. (B) ACH-2 cells were treated with prostratin, SAHA or TNF-α and the average fold change in expression of NT5C3 transcripts over time relative to time 0 as evaluated by illumina HT-12 V4 array bead chips was included (N = 2). (C) Western blot analysis of NT5C3 in ACH-2 cells or A3.01 cells post-treatment with SAHA, prostratin or TNF-α. At indicated time points, the cells were washed and lysed. Equal amounts of cell lysate were analyzed by immunoblotting them with anti-NT5C3 or anti-tubulin antibodies. NT5C3 antibody also detects NT5C3L protein.
Additional file 4: Fig. S3: ACH-2 cells were pretreated with multiple doses of (A) Rottlerin and ... more Additional file 4: Fig. S3: ACH-2 cells were pretreated with multiple doses of (A) Rottlerin and (B) WP1066 or vehicle control (DMSO), and four hours later activated with SAHA (1 µM, white bars), prostratin (1 µM, grey bars) or TNF-α (0.1 ng/ml, bars with diagonal line upwards). HIV-1 reactivation was estimated at 12 h following treatment, by intracellular p24 Gag staining by flow cytometry. For comparison of results across samples from multiple experiments, HIV-1 reactivation observed in vehicle control pretreatment was considered as 100 % and the background (no reactivating agent) as 0 %. Error bars represent standard deviation (N = 3).
Journal of neuroinflammation, Jan 27, 2018
Human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorder (HAND) is a common... more Human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorder (HAND) is a common outcome of a majority of HIV-1-infected subjects and is associated with synaptodendritic damage. Neurogranin (Ng), a postsynaptic protein, and calmodulin (CaM) are two important players of synaptic integrity/functions. The biological role of Ng in the context of HAND is unknown. We compared the expression of Ng in frontal cortex (FC) tissues from control and HIV-1-positive subjects with and without HAND by immunohistochemistry, western blot, and qRT-PCR. The interaction between Ng and CaM was analyzed by co-immunoprecipitation. Ng, microtubule-associated protein 2 (MAP2), CaM, CaM-dependent protein kinase II (CaMKII), CREB, synaptophysin (Syp), and synapsin I (Syn I) expressions were evaluated by western blot using FC tissue lysates and differentiated SH-SY5Y (dSH-SY5Y) cells. Identification of inflammatory factors related to Ng loss was accomplished by exposing dSH-SY5Y cells to HIV-1 ...
AIDS (London, England), Mar 21, 2016
HIV-1 viral proteins and host inflammatory factors have a direct role in neuronal toxicity in in ... more HIV-1 viral proteins and host inflammatory factors have a direct role in neuronal toxicity in in vitro, however, the contribution of these factors in vivo in HIV-1 associated neurocognitive disorder (HAND) is not fully understood. We applied novel Systems Biology approaches to identify specific cellular and viral factors and their related pathways that are associated with different stages of HAND. A cross-sectional study of individuals enrolled in the Multicenter AIDS Cohort Study (MACS) including HIV-1 seronegative (N = 36) and HIV-1 seropositive individuals without neurocognitive symptoms (N = 16), or with mild neurocognitive disorder (MND) (N = 8) or HIV-associated dementia (HAD) (N = 16). A systematic evaluation of global transcriptome of peripheral blood mononuclear cells (PBMCs) obtained from HIV-1 seronegative individuals and from HIV-1 positive men without neurocognitive symptoms, or MND or HAD was performed. MND and HAD were associated with specific changes in mRNA transcri...
The Journal of Immunology, May 1, 2013
The ability of dendritic cells to mediate CD40L-dependent CD4+ T cell help for the induction and ... more The ability of dendritic cells to mediate CD40L-dependent CD4+ T cell help for the induction and support of effector CD8+ cytotoxic T lymphocyte responses greatly depends on activation signals they received during maturation. In addition to being 'programmed' to produce high levels of IL-12p70, we found that DC matured under type 1 polarizing conditions also form intricately branched tunneling nanotube-like structures upon CD40L stimulation. These formations can create extensive interconnected networks between cells, a process we termed 'reticulation'. Importantly, this response was found to occur exclusively in mature type 1 polarized DC and was uniquely induced by recombinant CD40L or CD40L-expressing cells. Using live-cell time-lapse confocal imaging, we were able to determine that these structures can support the transfer of endosomes, HIV-1 virus-like particles, and nano-beads representing antigen from 'donor' to 'recipient' type 1 polarized DC. This induction of the process of DC reticulation potentially represents a novel helper function of CD40L, as it likely enhances intercellular communication and contributes to the superior capacity of type 1 polarized DC to induce cellular immune responses. This active immune cell process may also be utilized by viruses such as HIV-1 for cell-to-cell spread.
Bioinformatics (Oxford, England), Jun 15, 2016
Most methods for reconstructing response networks from high throughput data generate static model... more Most methods for reconstructing response networks from high throughput data generate static models which cannot distinguish between early and late response stages. We present TimePath, a new method that integrates time series and static datasets to reconstruct dynamic models of host response to stimulus. TimePath uses an Integer Programming formulation to select a subset of pathways that, together, explain the observed dynamic responses. Applying TimePath to study human response to HIV-1 led to accurate reconstruction of several known regulatory and signaling pathways and to novel mechanistic insights. We experimentally validated several of TimePaths' predictions highlighting the usefulness of temporal models. Data, Supplementary text and the TimePath software are available from http://sb.cs.cmu.edu/timepath [email protected] Supplementary data are available at Bioinformatics online.
The Journal of Immunology, May 1, 2014
The ability of DC to mediate CD4+ T cell help for initiating cellular immunity greatly depends on... more The ability of DC to mediate CD4+ T cell help for initiating cellular immunity greatly depends on instructive signals that they receive during maturation. Here we report that, in addition to producing high levels of IL-12p70, DC matured in the presence of type-1 associated inflammatory factors were also uniquely programmed to ‘reticulate’, or develop networks of tunneling nanotube-like structures in response to the CD4+ T helper cell derived signal, CD40L. Using high resolution live-cell imaging techniques, we demonstrated that tunneling nanotube networks are capable of facilitating the direct intercellular trafficking of endosome-associated vesicles, antigens, and HIV-1-like particles. Importantly, DC matured under non-type-1 polarizing conditions, such as those exposed to prostaglandin E2, failed to develop these membrane extensions. Moreover, we found that the T helper 1 associated cytokine IFN-γ plays a critical role in enhancing DC reticulation, while the T helper 2 cytokine IL-4 has a substantial inhibitory effect on this process. These data indicate that subsets of T helper cells have divergent abilities to modulate DC reticulation and subsequent intercellular communication. The induction of the reticulation process in DC represents a novel immunologic helper function of CD40L-expressing T cells, which can also be exploited by pathogens such as HIV-1 to promote direct cell-to-cell spread.
Journal of Cellular Biochemistry, 2016
Both CD4+ T lymphocytes and macrophages are the major targets of human immunodeficiency virus typ... more Both CD4+ T lymphocytes and macrophages are the major targets of human immunodeficiency virus type 1 (HIV-1); however, they respond differently to HIV-1 infection. We hypothesized that HIV-1 infection alters gene expression in CD4+ T cells and monocyte-derived macrophages (MDMs) in a cell specific manner and microRNAs (miRNAs) in part play a role in cell-specific gene expression. Results indicate that 183 and 31 genes were differentially regulated in HIV-1 infected CD4+ T cells and MDMs, respectively, compared to their mock-infected counterparts. Among the differentially expressed genes, cell cycle regulatory gene, p21 (CDKN1A) was upregulated in virus infected CD4+ T cells both at the mRNA and protein level in CD4+ T cells, whereas no consistent change was observed in MDMs. Productively infected CD4+ T cells express higher amount of p21 compared to bystander cells. In determining the mechanism(s) of cell type specific regulation of p21, we found that the miRNAs miR-106b and miR-20a that target p21 were specifically downregulated in HIV-1 infected CD4+ T cells. Overexpression of these two miRNAs reduced p21 expression significantly in HIV-1 infected CD4+ T cells. These findings provide a potential mechanism, by which, HIV-1 could exploit host cellular machineries to regulate selective gene expression in target cells. J. Cell. Biochem. 117: 1902-1912, 2016. © 2016 Wiley Periodicals, Inc.
Genetic Manipulation of DNA and Protein - Examples from Current Research, 2013
Although the most common route of HIV infection is via sexual contact, the use of contaminated dr... more Although the most common route of HIV infection is via sexual contact, the use of contaminated drug paraphernalia, mother-child transmission via pregnancy or breastfeeding, and tainted blood transfusions comprise other means of infection. The symptomatic outcome of infection is AIDS, usually occurring ~10 years after initial infection. CD4+ T-cell counts drop below 200, and subsequent severe immune dysfunction results. This eventually leads to fatal coinfection by opportunistic pathogens. The advent of highly active retroviral therapy (HAART) in the 1990s led to a drastically improved prognosis for AIDS patients (Peters and Conway, 2011). This triple-drug cocktail controls viremia and allows immune function to recover to nearly uninfected levels, with the caveat of near-perfect patient adherence to a difficult combination of drug regimens. The extremely rigid treatment schedules have resulted in low compliance, leading to the emergence of viruses that exhibit resistance to drugs.
PLoS ONE, 2012
Host cells respond to exogenous infectious agents such as viruses, including HIV-1. Studies have ... more Host cells respond to exogenous infectious agents such as viruses, including HIV-1. Studies have evaluated the changes associated with virus infection at the transcriptional and translational levels of the cellular genes involved in specific pathways. While this approach is useful, in our view it provides only a partial view of genome-wide changes. Recently, technological advances in the expression profiling at the microRNA (miRNA) and mRNA levels have made it possible to evaluate the changes in the components of multiple pathways. To understand the role of miRNA and its interplay with host cellular gene expression (mRNA) during HIV-1 infection, we performed a comparative global miRNA and mRNA microarray using human PBMCs infected with HIV-1. The PBMCs were derived from multiple donors and were infected with virus generated from the molecular clone pNL4-3. The results showed that HIV-1 infection led to altered regulation of 21 miRNAs and 444 mRNA more than 2-fold, with a statistical significance of p,0.05. Furthermore, the differentially regulated miRNA and mRNA were shown to be associated with host cellular pathways involved in cell cycle/proliferation, apoptosis, T-cell signaling, and immune activation. We also observed a number of inverse correlations of miRNA and mRNA expression in infected PBMCs, further confirming the interrelationship between miRNA and mRNA regulation during HIV-1 infection. These results for the first time provide evidence that the miRNA profile could be an early indicator of host cellular dysfunction induced by HIV-1.
Journal of Molecular Biology, 2007
Activation of Src family kinases by HIV-1 Nef may play an important role in the pathogenesis of H... more Activation of Src family kinases by HIV-1 Nef may play an important role in the pathogenesis of HIV/AIDS. Here we investigated whether diverse Nef sequences universally activate Hck, a Src family member expressed in macrophages and other HIV-1 target cells. In general, we observed that Hck activation is a highly conserved Nef function. However, we identified an unusual Nef variant from an HIV-positive individual that did not develop AIDS which failed to activate Hck despite the presence of conserved residues linked to Hck SH3 domain binding and kinase activation. Amino acid sequence alignment with active Nef proteins revealed differences in regions not previously implicated in Hck activation, including a large internal flexible loop absent from available Nef structures. Substitution of these residues in active Nef compromised Hck activation without affecting SH3 domain binding. These findings show that residues at a distance from the SH3 domain binding site allosterically influence Nef interactions with a key effector protein linked to AIDS progression.
The Journal of Immunology, 2007
HLA-A2-restricted CTL responses to immunodominant HIV-1 epitopes do not appear to be very effecti... more HLA-A2-restricted CTL responses to immunodominant HIV-1 epitopes do not appear to be very effective in the control of viral replication in vivo. In this study, we studied human CD8+ T cell responses to the subdominant HLA-A2-restricted epitope TV9 (Gag p2419–27, TLNAWVKVV) to explore the possibility of increasing its immune recognition. We confirmed in a cohort of 313 patients, infected by clade B or clade C viruses, that TV9 is rarely recognized. Of interest, the functional sensitivity of the TV9 response can be relatively high. The potential T cell repertoires for TV9 and the characteristics of constituent clonotypes were assessed by ex vivo priming of circulating CD8+ T cells from healthy seronegative donors. TV9-specific CTLs capable of suppressing viral replication in vitro were readily generated, suggesting that the cognate T cell repertoire is not limiting. However, these cultures contained multiple discrete populations with a range of binding avidities for the TV9 tetramer a...
HIV-1 Vpr, a nonstructural viral protein associated with virus particles, has a positive role in ... more HIV-1 Vpr, a nonstructural viral protein associated with virus particles, has a positive role in the efficient transport of PIC into the nucleus of non-dividing target cells and enhances virus replication in primary T cells. Vpr is a 96 amino acid protein and the structure by NMR shows three helical domains. Vpr has been shown to exist as dimers and higher order oligomers. Considering the multifunctional nature of Vpr, the contribution of distinct helical domains to the dimer/oligomer structure of Vpr and the relevance of this feature to ...
Scientific Reports, 2020
HIV-1 associated neurocognitive disorder (HAND) is characterized by neuroinflammation and glial a... more HIV-1 associated neurocognitive disorder (HAND) is characterized by neuroinflammation and glial activation that, together with the release of viral proteins, trigger a pathogenic cascade resulting in synaptodendritic damage and neurodegeneration that lead to cognitive impairment. However, the molecular events underlying HIV neuropathogenesis remain elusive, mainly due to lack of brain-representative experimental systems to study HIV-CNS pathology. To fill this gap, we developed a three-dimensional (3D) human brain organoid (hBORG) model containing major cell types important for HIV-1 neuropathogenesis; neurons and astrocytes along with incorporation of HIV-infected microglia. Both infected and uninfected microglia infiltrated into hBORGs resulting in a triculture system (MG-hBORG) that mirrors the multicellular network observed in HIV-infected human brain. Moreover, the MG-hBORG model supported productive viral infection and exhibited increased inflammatory response by HIV-infected ...
Journal of Leukocyte Biology, 2000
The HIV‐1 vpr gene encodes a 14‐kDa virion‐packaged protein that has been implicated in viral pat... more The HIV‐1 vpr gene encodes a 14‐kDa virion‐packaged protein that has been implicated in viral pathogenesis. Vpr exhibits profound effects on human primary cells influencing proliferation, differentiation, apoptosis, and cytokine production, in part through NF‐κB‐mediated transcription. NF‐κB, a potent transcription factor, activates many proinflammatory cytokines/chemokines upon infection. Here, we analyzed the effect of extracellular Vpr as well as the virion‐associated Vpr on β chemokines (MIP‐1α, MIP‐1β, and RANTES) production in human macrophages and primary lymphocytes (PBLs). Macrophages and PBLs exposed to HIV‐1 vpr+ viruses or to recombinant Vpr protein produced significantly less β chemokines compared with cells infected with HIV‐1 vpr− viruses or irrelevant control protein (Gag)‐exposed cells. These results suggest that a Vpr‐mediated increase in virus replication could be in part through down‐regulation of chemokine production.
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Papers by Velpandi Ayyavoo