Elizabeth Jaworski

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA

Background: Exosomes are extracellular vesicles that have been implicated in intercellular communication. Results: Exosomes that originate from human cells infected with HIV-1 contain virus-derived small noncoding RNA. Conclusion: Virus-derived small RNA present in exosomes exert functional consequences in naive recipient cells. Significance: Viral RNA molecules present in exosomes may be critical mediators of intercellular viral spread in infected hosts. Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.

HTLV tax: a fascinating multifunctional co-regulator of viral and cellular pathways.

Human T-cell lymphotropic virus type 1 (HTLV-1) has been identified as the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The virus infects between 15 and 20 million people worldwide of which approximately 2–5% develop ATL. The past 35 years of research have yielded significant insight into the pathogenesis of HTLV-1, including the molecular characterization of Tax, the viral transactivator, and oncoprotein. In spite of these efforts, the mechanisms of oncogenesis of this pleiotropic protein remain to be fully elucidated. In this review, we illustrate the multiple oncogenic roles of Tax by summarizing a recent body of literature that refines our understanding of cellular transformation. A focused range of topics are discussed in this review including Tax-mediated regulation of the viral promoter and other cellular pathways, particularly the connection of the NF-κB pathway to both post-translational modifications (PTMs) of Tax and subcellular localization. Specifically, recent research on polyubiquitination of Tax as it relates to the activation of the IkappaB kinase (IKK) complex is highlighted. Regulation of the cell cycle and DNA damage responses due to Tax are also discussed, including Tax interaction with minichromosome maintenance proteins and the role of Tax in chromatin remodeling. The recent identification of HTLV-3 has amplified the importance of the characterization of emerging viral pathogens. The challenge of the molecular determination of pathogenicity and malignant disease of this virus lies in the comparison of the viral transactivators of HTLV-1, -2, and -3 in terms of transformation and immortalization. Consequently, differences between the three proteins are currently being studied to determine what factors are required for the differences in tumorogenesis.

Human T-lymphotropic Virus Type 1-infected Cells Secrete Exosomes That Contain Tax Protein * □

Background: Extracellular exosomes contain various functional elements. Results: Exosomal Tax protein causes phenotypic changes in uninfected cells. Conclusion: Exosomes may play critical roles in extracellular delivery of oncogenic material derived from HTLV-1-infected cells. Significance: Exosomal delivery of Tax and other putative oncogenic components produced during HTLV-1 infection potentially contributes to pathogenesis of adult T-cell leukemia, myelopathy, or tropical spastic paraparesis. Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells.

Effect of mimetic CDK9 inhibitors on HIV-1-activated transcription.

Potent anti-retroviral therapy has transformed HIV-1 infection into a chronic manageable disease; however, drug resistance remains a common problem that limits the effectiveness and clinical benefits of this type of treatment. The discovery of viral reservoirs in the body, in which HIV-1 may persist, has helped to explain why therapeutic eradication of HIV-1 has proved so difficult. In the current study, we utilized a combination of structure-based analysis of cyclin/CDK complexes with our previously published Tat peptide derivatives. We modeled the Tat peptide inhibitors with CDKs and found a particular pocket that showed the most stable binding site (Cavity 1) using in silico analysis. Furthermore, we were able to find peptide mimetics that bound to similar regions using in silico searches of a chemical library, followed by cell-based biological assays. Using these methods, we obtained the first-generation mimetic drugs and tested these compounds on HIV-1 long terminal repeat-activated transcription. Using biological assays followed by similar in silico analysis to find second-generation drugs resembling the original mimetic, we found the new targets of Cavity 1 and Cavity 2 regions on CDK9. We examined the second-generation mimetic against various viral isolates and observed a generalized suppression of most HIV-1 isolates. Finally, the drug inhibited viral replication in humanized mouse models of Rag2(-/-)γc(-/-) with no toxicity to the animals at tested concentrations. Our results suggest that it may be possible to model peptide inhibitors into available crystal structures and further find drug mimetics using in silico analysis.

Localization and sub-cellular shuttling of HTLV-1 tax with the miRNA machinery.

The innate ability of the human cell to silence endogenous retroviruses through RNA sequences encoding microRNAs, suggests that the cellular RNAi machinery is a major means by which the host mounts a defense response against present day retroviruses. Indeed, cellular miRNAs target and hybridize to specific sequences of both HTLV-1 and HIV-1 viral transcripts. However, much like the variety of host immune responses to retroviral infection, the virus itself contains mechanisms that assist in the evasion of viral inhibition through control of the cellular RNAi pathway. Retroviruses can hijack both the enzymatic and catalytic components of the RNAi pathway, in some cases to produce novel viral miRNAs that can either assist in active viral infection or promote a latent state. Here, we show that HTLV-1 Tax contributes to the dysregulation of the RNAi pathway by altering the expression of key components of this pathway. A survey of uninfected and HTLV-1 infected cells revealed that Drosha protein is present at lower levels in all HTLV-1 infected cell lines and in infected primary cells, while other components such as DGCR8 were not dramatically altered. We show colocalization of Tax and Drosha in the nucleus in vitro as well as coimmunoprecipitation in the presence of proteasome inhibitors, indicating that Tax interacts with Drosha and may target it to specific areas of the cell, namely, the proteasome. In the presence of Tax we observed a prevention of primary miRNA cleavage by Drosha. Finally, the changes in cellular miRNA expression in HTLV-1 infected cells can be mimicked by the add back of Drosha or the addition of antagomiRs against the cellular miRNAs which are downregulated by the virus.

Transcriptional Gene Silencing (TGS) via the RNAi Machinery in HIV-1 Infections

Gene silencing via non-coding RNA, such as siRNA and miRNA, can occur at the transcriptional, post-transcriptional, and translational stages of expression. Transcriptional gene silencing (TGS) involving the RNAi machinery generally occurs through DNA methylation, as well as histone post-translational modifications, and corresponding remodeling of chromatin around the target gene into a heterochromatic state. The mechanism by which mammalian TGS occurs includes the recruitment of RNA-induced initiation of transcriptional gene silencing (RITS) complexes, DNA methyltransferases (DNMTs), and other chromatin remodelers. Additionally, virally infected cells encoding miRNAs have also been shown to manipulate the host cell RNAi machinery to induce TGS at the viral genome, thereby establishing latency. Furthermore, the introduction of exogenous siRNA and shRNA into infected cells that target integrated viral promoters can greatly suppress viral transcription via TGS. Here we examine the latest findings regarding mammalian TGS, specifically focusing on HIV-1 infected cells, and discuss future avenues of exploration in this field.

Novel Neuroprotective GSK-3β Inhibitor Restricts Tat-Mediated HIV-1 Replication

ABSTRACT The implementation of new antiretroviral therapies targeting transcription of early viral proteins in postintegrated HIV-1 can aid in overcoming current therapy limitations. Using high-throughput screening assays, we have previously described a novel Tat-dependent HIV-1 transcriptional inhibitor named 6-bromoindirubin-3′-oxime (6BIO). The screening of 6BIO derivatives yielded unique compounds that show potent inhibition of HIV-1 transcription. We have identified a second-generation derivative called 18BIOder as an inhibitor of HIV-1 Tat-dependent transcription in TZM-bl cells and a potent inhibitor of GSK-3β kinase in vitro. Structurally, 18BIOder is half the molecular weight and structure of its parental compound, 6BIO. More importantly, we also have found a different GSK-3β complex present only in HIV-1-infected cells. 18BIOder preferentially inhibits this novel kinase complex from infected cells at nanomolar concentrations. Finally, we observed that neuronal cultures treated with Tat protein are protected from Tat-mediated cytotoxicity when treated with 18BIOder. Overall, our data suggest that HIV-1 Tat-dependent transcription is sensitive to small-molecule inhibition of GSK-3β.

Exosomes derived from HTLV-1 infected cells contain the viral protein Tax

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. The HTLV-1 transactivator protein Tax has been identified as a critical component in the proliferation and transformation of human primary T-cells. This 40 kDa phosphoprotein not only manipulates chromatin remodeling within the host, but also subverts host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Here we utilized a combination of filtration and ultracentrifugation methods to enrich for exosomes from culture supernatants of HTLV-1 infected cells. We then employed western blots, mass spectrometry, and cytokine arrays to proteomically characterize the host and viral components in these exosomes. Additionally, RT-PCR was used to determine the presence of viral transcripts in these exosomes. Our results demonstrate that exosomes derived from HTLV-1 infected cells contain traditional exosome proteins. Furthermore, our proteomics studies revealed that these exosomes contain viral components such as gp46 and Tax, as well as inflammatory mediators including IL-6 and IL-10. We also investigated the presence of HTLV-1 mRNA transcripts of Env, Tax, HBZ, and 5’LTR contained within exosomes. Moreover, we evaluated the functional impacts of treating naive recipient cells with exosomes secreted from HTLV-1 infected cells, and determined that the exosomes were able to induce a response in reactive oxygen species production. Our observation that the viral protein Tax is contained within exosomes and may be transmitted in an extracellular capacity raises important implications to pathogenesis associated with HTLV-1 infections.

Localization and sub-cellular shuttling of HTLV-1 Tax with the microRNA machinery

The innate ability of the human cell to silence endogenous retroviruses through RNA sequences encoding microRNAs suggests that the cellular RNAi machinery is a major means by which the host mounts a defense response against retroviruses. Indeed, cellular miRNAs target and hybridize to specific sequences of both HTLV-1 and HIV-1 viral transcripts. However, the virus itself contains various mechanisms that assist in the evasion of viral inhibition through control of the cellular RNAi pathway. Retroviruses can hijack components of the RNAi pathway, in some cases to produce novel viral miRNAs that can either assist in active infection or promote a latent state. Here, we show that HTLV-1 Tax contributes to the dysregulation of the RNAi pathway by altering the expression of key components. A survey of uninfected and HTLV-1 infected cells revealed that Drosha is present at lower levels in all HTLV-1 infected cell lines and infected primary cells, while other components such as DGCR8 were not dramatically altered. We show co-localization of Tax and Drosha in the nucleus in vitro as well as co-immunoprecipitation in the presence of proteasome inhibitors, indicating that Tax interacts with Drosha and may target it to specific areas of the cell, namely, the proteasome. In the presence of Tax we observed a prevention of primary miRNA cleavage by Drosha. Finally, the changes in cellular miRNA expression in HTLV-1 infected cells can be mimicked by the add back of Drosha or the addition of antagomiRs against the cellular miRNAs which are downregulated by the virus.

Exosomes derived from HTLV-1 infected cells contain viral proteins and mRNA

The HTLV-1 transactivator protein Tax controls many critical cellular pathways including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Recently, exosomes have been shown to play critical roles during pathogenic viral infections as delivery vehicles for host and viral components including proteins, mRNA and miRNA. We hypothesized that exosomes derived from HTLV-1 infected cells contain unique host and viral proteins that may contribute to pathogenesis. We have characterized exosomes released from uninfected and HTLV-1 infected cell lines, as well as ATL and HAM/TSP material. The functional impact of exosomes derived from HTLV-1 infected cells on naive recipient cells was evaluated by utilizing transcription and reactive oxygen species (ROS) assays. Exosomes from HTLV-1 infected cells displayed unique proteomic profiles distinct from exosomes derived from uninfected cells. For instance, proinflammatory mediators are contained within the exosomes, as well as viral mRNA transcripts including Tax, HBZ, and Env. We found that exosomes from infected cells deliver functional Tax to naA¯ve recipient cells as well as cytokines. The release of factors was through Calcium and Calcium channels. We observed that exosomes released from HTLV-1 infected, Tax-expressing cells contributed to enhanced survival of target cells treated with α-FAS. Two other critical proteins were found in these exosomes that could contribute to overall activation of neighboring cells including Tax1BP1 and PrP. Both proteins are critical for activation of NFkB patway in recipient cells. Collectively, our results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. Furthermore, exosomes derived from infected cells are capable of inducing an ROS response in naA¯ve cells and contribute to an anti-apoptotic phenotype in cells treated with α-FAS.