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

The use of nanotrap particles technology in capturing HIV-1 virions and viral proteins from infected cells

HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients\u27 serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1 detection by concentrating viral proteins and infectious virions from infected samples

Modulation of GSK-3β activity in Venezuelan equine encephalitis virus infection

Alphaviruses, including Venezuelan Equine Encephalitis Virus (VEEV), cause disease in both equine and humans that exhibit overt encephalitis in a significant percentage of cases. Features of the host immune response and tissue-specific responses may contribute to fatal outcomes as well as the development of encephalitis. It has previously been shown that VEEV infection of mice induces transcription of pro-inflammatory cytokines genes (e.g., IFN-γ, IL-6, IL-12, iNOS and TNF-α) within 6 h. GSK-3β is a host protein that is known to modulate pro-inflammatory gene expression and has been a therapeutic target in neurodegenerative disorders such as Alzheimer\u27s. Hence inhibition of GSK-3β in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3β inhibitors and GSK-3β siRNA experiments indicated that GSK-3β was important for VEEV replication. Thirty-eight second generation BIO derivatives were tested and BIOder was found to be the most potent inhibitor, with an IC50 of ~0.5 µM and a CC50 of \u3e100 µM. BIOder was a more potent inhibitor of GSK-3β than BIO, as demonstrated through in vitro kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3β is found in three distinct complexes in VEEV infected cells, whereas GSK-3β is only present in one complex in uninfected cells. Cells treated with BIOder demonstrated an increase in the anti-apoptotic gene, survivin, and a decrease in the pro-apoptotic gene, BID, suggesting that modulation of pro- and anti-apoptotic genes contributes to the protective effect of BIOder treatment. Finally, BIOder partially protected mice from VEEV induced mortality. Our studies demonstrate the utility of GSK-3β inhibitors for modulating VEEV infection

Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA.

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

Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo

Long-lasting, latently infected resting CD4+ T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir1. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect2–9. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow–liver–thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4+ T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal—in combination with appropriate tools for systemic clearance of persistent HIV infection—greatly increases opportunities for HIV eradication

Identification of Modulators of HIV-1 Proviral Transcription from a Library of FDA-Approved Pharmaceuticals

Human immunodeficiency virus 1 (HIV-1) is the most prevalent human retrovirus. Recent data show that 34 million people are living with HIV-1 worldwide. HIV-1 infections can lead to AIDS which still causes nearly 20,000 deaths annually in the USA alone. As this retrovirus leads to high morbidity and mortality conditions, more effective therapeutic regimens must be developed to treat these viral infections. A key target for intervention for which there are no current FDA-approved modulators is at the point of proviral transcription. One successful method for identifying novel therapeutics for treating infectious diseases is the repurposing of pharmaceuticals that are approved by the FDA for alternate indications. Major benefits of using FDA-approved drugs include the fact that the compounds have well established toxicity profiles, approved manufacturing processes, and immediate commercial availability to the patients. Here, we demonstrate that pharmaceuticals previously approved for other indications can be utilized to either activate or inhibit HIV-1 proviral transcription. Specifically, we found febuxostat, eltrombopag, and resveratrol to be activators of HIV-1 transcription, while mycophenolate was our lead inhibitor of HIV-1 transcription. Additionally, we observed that the infected cells of lymphoid and myeloid lineage responded differently to our lead transcriptional modulators. Finally, we demonstrated that the use of a multi-dose regimen allowed for enhanced activation with our transcriptional activators

Retrovirus infected cells contain viral microRNAs

<p>Abstract</p> <p>The encoding of microRNAs in retroviral genomes has remained a controversial hypothesis despite significant supporting evidence in recent years. A recent publication demonstrating the production of functional miRNAs from the retrovirus bovine leukemia virus adds further credence to the fact that retroviruses do indeed encode their own miRNAs. Here we comment on the importance of this paper to the field, as well as examine the other known examples of miRNAs encoded by RNA viruses.</p

Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

Ebola virus (EBOV) is an enveloped, ssRNA virus from the family Filoviridae capable of causing severe hemorrhagic fever with up to 80–90% mortality rates. The most recent outbreak of EBOV in West Africa starting in 2014 resulted in over 11,300 deaths; however, long-lasting persistence and recurrence in survivors has been documented, potentially leading to further transmission of the virus. We have previously shown that exosomes from cells infected with HIV-1, HTLV-1 and Rift Valley Fever virus are able to transfer viral proteins and non-coding RNAs to naïve recipient cells, resulting in an altered cellular activity. In the current manuscript, we examined the effect of Ebola structural proteins VP40, GP, NP and VLPs on recipient immune cells, as well as the effect of exosomes containing these proteins on naïve immune cells. We found that VP40-transfected cells packaged VP40 into exosomes, and that these exosomes were capable of inducing apoptosis in recipient immune cells. Additionally, we show that presence of VP40 within parental cells or in exosomes delivered to naïve cells could result in the regulation of RNAi machinery including Dicer, Drosha, and Ago 1, which may play a role in the induction of cell death in recipient immune cells. Exosome biogenesis was regulated by VP40 in transfected cells by increasing levels of ESCRT-II proteins EAP20 and EAP45, and exosomal marker proteins CD63 and Alix. VP40 was phosphorylated by Cdk2/Cyclin complexes at Serine 233 which could be reversed with r-Roscovitine treatment. The level of VP40-containing exosomes could also be regulated by treated cells with FDA-approved Oxytetracycline. Additionally, we utilized novel nanoparticles to safely capture VP40 and other viral proteins from Ebola VLPs spiked into human samples using SDS/reducing agents, thus minimizing the need for BSL-4 conditions for most downstream assays. Collectively, our data indicates that VP40 packaged into exosomes may be responsible for the deregulation and eventual destruction of the T-cell and myeloid arms of the immune system (bystander lymphocyte apoptosis), allowing the virus to replicate to high titers in the immunocompromised host. Moreover, our results suggest that the use of drugs such as Oxytetracycline to modulate the levels of exosomes exiting EBOV-infected cells may be able to prevent the devastation of the adaptive immune system and allow for an improved rate of survival

Complex role of microRNAs in HTLV-1 infections

Human T-lymphotropic virus 1 (HTLV-1) was the first human retrovirus to be discovered and is the causative agent of adult T-cell leukemia/lymphoma (ATL) and the neurodegenerative disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The importance of microRNA (miRNA) in the replicative cycle of several other viruses, as well as in the progression of associated pathologies, has been well established in the past decade. Moreover, involvement of miRNA alteration in the HTLV-1 life cycle, and in the progression of its related oncogenic and neurodegenerative diseases, has recently come to light. Several HTLV-1 derived proteins alter transcription factor functionalities, interact with chromatin remodelers, or manipulate components of the RNA interference (RNAi) machinery, thereby establishing various routes by which miRNA expression can be up- or down-regulated in the host cell. Furthermore, the mechanism of action through which dysregulation of host miRNAs affects HTLV-1 infected cells can vary substantially and include mRNA silencing via the RNA-induced silencing complex (RISC), transcriptional gene silencing, inhibition of RNAi components, and chromatin remodeling. These miRNA induced changes can lead to increased cell survival, invasiveness, proliferation, and differentiation, as well as allow for viral latency. While many recent studies have successfully implicated miRNAs in the life cycle and pathogenesis of HTLV-1 infections, there are still significant outstanding questions to be addressed. Here we will review recent discoveries elucidating HTLV-1 mediated manipulation of host cell miRNA profiles and examine the impact on pathogenesis, as well as explore future lines of inquiry that could increase understanding in this field of study