Extracellular Vesicle Refractive Index Derivation Utilizing Orthogonal Characterization.

The analysis of small particles, including extracellular vesicles and viruses, is contingent on their ability to scatter sufficient light to be detected. These detection methods include flow cytometry, nanoparticle tracking analysis, and single particle reflective image sensing. To standardize measurements and enable orthogonal comparisons between platforms, a quantifiable limit of detection is required. The main parameters that dictate the amount of light scattered by particles include size, morphology, and refractive index. To date, there has been a lack of accessible techniques for measuring the refractive index of nanoparticles at a single-particle level. Here, we demonstrate two methods of deriving a small particle refractive index using orthogonal measurements with commercially available platforms. These methods can be applied at either a single-particle or population level, enabling the integration of diameter and scattering cross section values to derive the refractive index using Mie theory.

Effect of transcription inhibition and generation of suppressive viral non-coding RNAs.

BACKGROUND: HIV-1 patients receiving combination antiretroviral therapy (cART) survive infection but require life-long adherence at high expense. In chronic cART-treated patients with undetectable viral titers, cell-associated viral RNA is still detectable, pointing to low-level viral transcriptional leakiness. To date, there are no FDA-approved drugs against HIV-1 transcription. We have previously shown that F07#13, a third generation Tat peptide mimetic with competitive activity against Cdk9/T1-Tat binding sites, inhibits HIV-1 transcription in vitro and in vivo. RESULTS: Here, we demonstrate that increasing concentrations of F07#13 (0.01, 0.1, 1 µM) cause a decrease in Tat levels in a dose-dependent manner by inhibiting the Cdk9/T1-Tat complex formation and subsequent ubiquitin-mediated Tat sequestration and degradation. Our data indicate that complexes I and IV contain distinct patterns of ubiquitinated Tat and that transcriptional inhibition induced by F07#13 causes an overall reduction in Tat levels. This reduction may be triggered by F07#13 but ultimately is mediated by TAR-gag viral RNAs that bind suppressive transcription factors (similar to 7SK, NRON, HOTAIR, and Xist lncRNAs) to enhance transcriptional gene silencing and latency. These RNAs complex with PRC2, Sin3A, and Cul4B, resulting in epigenetic modifications. Finally, we observed an F07#13-mediated decrease of viral burden by targeting the R region of the long terminal repeat (HIV-1 promoter region, LTR), promoting both paused polymerases and increased efficiency of CRISPR/Cas9 editing in infected cells. This implies that gene editing may be best performed under a repressed transcriptional state. CONCLUSIONS: Collectively, our results indicate that F07#13, which can terminate RNA Polymerase II at distinct sites, can generate scaffold RNAs, which may assemble into specific sets of "RNA Machines" that contribute to gene regulation. It remains to be seen whether these effects can also be seen in various clades that have varying promoter strength, mutant LTRs, and in patient samples.

Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles.

Background: Ebola virus (EBOV) mainly targets myeloid cells; however, extensive death of T cells is often observed in lethal infections. We have previously shown that EBOV VP40 in exosomes causes recipient immune cell death. Methods: Using VP40-producing clones, we analyzed donor cell cycle, extracellular vesicle (EV) biogenesis, and recipient immune cell death. Transcription of cyclin D1 and nuclear localization of VP40 were examined via kinase and chromatin immunoprecipitation assays. Extracellular vesicle contents were characterized by mass spectrometry, cytokine array, and western blot. Biosafety level-4 facilities were used for wild-type Ebola virus infection studies. Results: VP40 EVs induced apoptosis in recipient T cells and monocytes. VP40 clones were accelerated in growth due to cyclin D1 upregulation, and nuclear VP40 was found bound to the cyclin D1 promoter. Accelerated cell cycling was related to EV biogenesis, resulting in fewer but larger EVs. VP40 EV contents were enriched in ribonucleic acid-binding proteins and cytokines (interleukin-15, transforming growth factor-ß1, and interferon-γ). Finally, EBOV-infected cell and animal EVs contained VP40, nucleoprotein, and glycoprotein. Conclusions: Nuclear VP40 upregulates cyclin D1 levels, resulting in dysregulated cell cycle and EV biogenesis. Packaging of cytokines and EBOV proteins into EVs from infected cells may be responsible for the decimation of immune cells during EBOV pathogenesis.

Autophagy Deregulation in HIV-1-Infected Cells Increases Extracellular Vesicle Release and Contributes to TLR3 Activation.

Human immunodeficiency virus type 1 (HIV-1) infection can result in HIV-associated neurocognitive disorder (HAND), a spectrum of disorders characterized by neurological impairment and chronic inflammation. Combined antiretroviral therapy (cART) has elicited a marked reduction in the number of individuals diagnosed with HAND. However, there is continual, low-level viral transcription due to the lack of a transcription inhibitor in cART regimens, which results in the accumulation of viral products within infected cells. To alleviate stress, infected cells can release accumulated products, such as TAR RNA, in extracellular vesicles (EVs), which can contribute to pathogenesis in neighboring cells. Here, we demonstrate that cART can contribute to autophagy deregulation in infected cells and increased EV release. The impact of EVs released from HIV-1 infected myeloid cells was found to contribute to CNS pathogenesis, potentially through EV-mediated TLR3 (Toll-like receptor 3) activation, suggesting the need for therapeutics to target this mechanism. Three HIV-1 TAR-binding compounds, 103FA, 111FA, and Ral HCl, were identified that recognize TAR RNA and reduce TLR activation. These data indicate that packaging of viral products into EVs, potentially exacerbated by antiretroviral therapeutics, may induce chronic inflammation of the CNS observed in cART-treated patients, and novel therapeutic strategies may be exploited to mitigate morbidity.

Emerging role of extracellular vesicles in multiple sclerosis: From cellular surrogates to pathogenic mediators and beyond.

Multiple Sclerosis (MS) is a chronic, inflammatory demyelinating disease of the central nervous system (CNS) driven by a complex interplay of genetic and environmental factors. While the therapeutic arsenal has expanded significantly for management of relapsing forms of MS, treatment of individuals with progressive MS is suboptimal. This treatment inequality is in part due to an incomplete understanding of pathomechanisms at different stages of the disease-underscoring the critical need for new biomarkers. Extracellular vesicles (EVs) and their bioactive cargo have emerged as endogenous nanoparticles with great theranostic potential-as diagnostic and prognostic biomarkers and ultimately as therapeutic candidates for precision nanotherapeutics. The goals of this review are to: 1) summarize the current data investigating the role of EVs and their bioactive cargo in MS pathogenesis, 2) provide a high level overview of advances and challenges in EV isolation and characterization for translational studies, and 3) conclude with future perspectives on this evolving field.

Viral Immune signatures from cerebrospinal fluid extracellular vesicles and particles in HAM and other chronic neurological diseases.

Background and objectives: Extracellular vesicles and particles (EVPs) are released from virtually all cell types, and may package many inflammatory factors and, in the case of infection, viral components. As such, EVPs can play not only a direct role in the development and progression of disease but can also be used as biomarkers. Here, we characterized immune signatures of EVPs from the cerebrospinal fluid (CSF) of individuals with HTLV-1-associated myelopathy (HAM), other chronic neurologic diseases, and healthy volunteers (HVs) to determine potential indicators of viral involvement and mechanisms of disease. Methods: We analyzed the EVPs from the CSF of HVs, individuals with HAM, HTLV-1-infected asymptomatic carriers (ACs), and from patients with a variety of chronic neurologic diseases of both known viral and non-viral etiologies to investigate the surface repertoires of CSF EVPs during disease. Results: Significant increases in CD8+ and CD2+ EVPs were found in HAM patient CSF samples compared to other clinical groups (p = 0.0002 and p = 0.0003 compared to HVs, respectively, and p = 0.001 and p = 0.0228 compared to MS, respectively), consistent with the immunopathologically-mediated disease associated with CD8+ T-cells in the central nervous system (CNS) of HAM patients. Furthermore, CD8+ (p < 0.0001), CD2+ (p < 0.0001), CD44+ (p = 0.0176), and CD40+ (p = 0.0413) EVP signals were significantly increased in the CSF from individuals with viral infections compared to those without. Discussion: These data suggest that CD8+ and CD2+ CSF EVPs may be important as: 1) potential biomarkers and indicators of disease pathways for viral-mediated neurological diseases, particularly HAM, and 2) as possible meditators of the disease process in infected individuals.

MPAPASS software enables stitched multiplex, multidimensional EV repertoire analysis and a standard framework for reporting bead-based assays.

Extracellular vesicles (EVs) of various types are released or shed from all cells. EVs carry proteins and contain additional protein and nucleic acid cargo that relates to their biogenesis and cell of origin. EV cargo in liquid biopsies is of widespread interest owing to its ability to provide a retrospective snapshot of cell state at the time of EV release. For the purposes of EV cargo analysis and repertoire profiling, multiplex assays are an essential tool in multiparametric analyte studies but are still being developed for high-parameter EV protein detection. Although bead-based EV multiplex analyses offer EV profiling capabilities with conventional flow cytometers, the utilization of EV multiplex assays has been limited by the lack of software analysis tools for such assays. To facilitate robust EV repertoire studies, we developed multiplex analysis post-acquisition analysis (MPAPASS) open-source software for stitched multiplex analysis, EV database-compatible reporting, and visualization of EV repertoires.

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.

Low-Level Ionizing Radiation Induces Selective Killing of HIV-1-Infected Cells with Reversal of Cytokine Induction Using mTOR Inhibitors.

HIV-1 infects 39.5 million people worldwide, and cART is effective in preventing viral spread by reducing HIV-1 plasma viral loads to undetectable levels. However, viral reservoirs persist by mechanisms, including the inhibition of autophagy by HIV-1 proteins (i.e., Nef and Tat). HIV-1 reservoirs can be targeted by the "shock and kill" strategy, which utilizes latency-reversing agents (LRAs) to activate latent proviruses and immunotarget the virus-producing cells. Yet, limitations include reduced LRA permeability across anatomical barriers and immune hyper-activation. Ionizing radiation (IR) induces effective viral activation across anatomical barriers. Like other LRAs, IR may cause inflammation and modulate the secretion of extracellular vesicles (EVs). We and others have shown that cells may secrete cytokines and viral proteins in EVs and, therefore, LRAs may contribute to inflammatory EVs. In the present study, we mitigated the effects of IR-induced inflammatory EVs (i.e., TNF-α), through the use of mTOR inhibitors (mTORi; Rapamycin and INK128). Further, mTORi were found to enhance the selective killing of HIV-1-infected myeloid and T-cell reservoirs at the exclusion of uninfected cells, potentially via inhibition of viral transcription/translation and induction of autophagy. Collectively, the proposed regimen using cART, IR, and mTORi presents a novel approach allowing for the targeting of viral reservoirs, prevention of immune hyper-activation, and selectively killing latently infected HIV-1 cells.

Differences in Transcriptional Dynamics Between T-cells and Macrophages as Determined by a Three-State Mathematical Model.

HIV-1 viral transcription persists in patients despite antiretroviral treatment, potentially due to intermittent HIV-1 LTR activation. While several mathematical models have been explored in the context of LTR-protein interactions, in this work for the first time HIV-1 LTR model featuring repressed, intermediate, and activated LTR states is integrated with generation of long (env) and short (TAR) RNAs and proteins (Tat, Pr55, and p24) in T-cells and macrophages using both cell lines and infected primary cells. This type of extended modeling framework allows us to compare and contrast behavior of these two cell types. We demonstrate that they exhibit unique LTR dynamics, which ultimately results in differences in the magnitude of viral products generated. One of the distinctive features of this work is that it relies on experimental data in reaction rate computations. Two RNA transcription rates from the activated promoter states are fit by comparison of experimental data to model predictions. Fitting to the data also provides estimates for the degradation/exit rates for long and short viral RNA. Our experimentally generated data is in reasonable agreement for the T-cell as well macrophage population and gives strong evidence in support of using the proposed integrated modeling paradigm. Sensitivity analysis performed using Latin hypercube sampling method confirms robustness of the model with respect to small parameter perturbations. Finally, incorporation of a transcription inhibitor (F07#13) into the governing equations demonstrates how the model can be used to assess drug efficacy. Collectively, our model indicates transcriptional differences between latently HIV-1 infected T-cells and macrophages and provides a novel platform to study various transcriptional dynamics leading to latency or activation in numerous cell types and physiological conditions.

Extracellular Vesicles and Ebola Virus: A New Mechanism of Immune Evasion.

Ebola virus (EBOV) disease can result in a range of symptoms anywhere from virtually asymptomatic to severe hemorrhagic fever during acute infection. Additionally, spans of asymptomatic persistence in recovering survivors is possible, during which transmission of the virus may occur. In acute infection, substantial cytokine storm and bystander lymphocyte apoptosis take place, resulting in uncontrolled, systemic inflammation in affected individuals. Recently, studies have demonstrated the presence of EBOV proteins VP40, glycoprotein (GP), and nucleoprotein (NP) packaged into extracellular vesicles (EVs) during infection. EVs containing EBOV proteins have been shown to induce apoptosis in recipient immune cells, as well as contain pro-inflammatory cytokines. In this manuscript, we review the current field of knowledge on EBOV EVs including the mechanisms of their biogenesis, their cargo and their effects in recipient cells. Furthermore, we discuss some of the effects that may be induced by EBOV EVs that have not yet been characterized and highlight the remaining questions and future directions.

HTLV-1 Extracellular Vesicles Promote Cell-to-Cell Contact.

Human T-cell leukemia virus-1 (HTLV-1) is a neglected and incurable retrovirus estimated to infect 5 to 10 million worldwide. Specific indigenous Australian populations report infection rates of more than 40%, suggesting a potential evolution of the virus with global implications. HTLV-1 causes adult T-cell leukemia/lymphoma (ATLL), and a neurological disease named HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Even though HTLV-1 transmission primarily occurs from cell-to-cell, there is still a gap of knowledge regarding the mechanisms of viral spread and disease progression. We have recently shown that Extracellular Vesicles (EVs) ubiquitously produced by cells may be used by HTLV-1 to transport viral proteins and RNA, and elicit adverse effects on recipient uninfected cells. The viral proteins Tax and HBZ are involved in disease progression and impairment of autophagy in infected cells. Here, we show that activation of HTLV-1 via ionizing radiation (IR) causes a significant increase of intracellular Tax, but not EV-associated Tax. Also, lower density EVs from HTLV-1-infected cells, separated by an Iodixanol density gradient, are positive for gp61+++/Tax+++/HBZ+ proteins (HTLV-1 EVs). We found that HTLV-1 EVs are not infectious when tested in multiple cell lines. However, these EVs promote cell-to-cell contact of uninfected cells, a phenotype which was enhanced with IR, potentially promoting viral spread. We treated humanized NOG mice with HTLV-1 EVs prior to infection and observed an increase in viral RNA synthesis in mice compared to control (EVs from uninfected cells). Proviral DNA levels were also quantified in blood, lung, spleen, liver, and brain post-treatment with HTLV-1 EVs, and we observed a consistent increase in viral DNA levels across all tissues, especially the brain. Finally, we show direct implications of EVs in viral spread and disease progression and suggest a two-step model of infection including the release of EVs from donor cells and recruitment of recipient cells as well as an increase in recipient cell-to-cell contact promoting viral spread.

Purification of High Yield Extracellular Vesicle Preparations Away from Virus.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

Viral antigens detectable in CSF exosomes from patients with retrovirus associated neurologic disease: functional role of exosomes.

BACKGROUND: HTLV-1 infects over 20 million people worldwide and causes a progressive neuroinflammatory disorder in a subset of infected individuals called HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The detection of HTLV-1 specific T cells in the cerebrospinal fluid (CSF) suggests this disease is immunopathologically mediated and that it may be driven by viral antigens. Exosomes are microvesicles originating from the endosomal compartment that are shed into the extracellular space by various cell types. It is now understood that several viruses take advantage of this mode of intercellular communication for packaging of viral components as well. We sought to understand if this is the case in HTLV-1 infection, and specifically if HTLV-1 proteins can be found in the CSF of HAM/TSP patients where we know free virus is absent, and furthermore, if exosomes containing HTLV-1 Tax have functional consequences. RESULTS: Exosomes that were positive for HTLV-1 Tax by Western blot were isolated from HAM/TSP patient PBMCs (25/36) in ex vivo cultures by trapping exosomes from culture supernatants. HTLV-1 seronegative PBMCs did not have exosomes with Tax (0/12), (Fisher exact test, p = 0.0001). We were able to observe HAM/TSP patient CSF (12/20) containing Tax+ exosomes but not in HTLV-1 seronegative MS donors (0/5), despite the absence of viral detection in the CSF supernatant (Fisher exact test p = 0.0391). Furthermore, exosomes cultivated from HAM/TSP PBMCs were capable of sensitizing target cells for HTLV-1 specific CTL lysis. CONCLUSION: Cumulatively, these results show that there are HTLV-1 proteins present in exosomes found in virus-free CSF. HAM/TSP PBMCs, particularly CD4+CD25+ T cells, can excrete these exosomes containing HTLV-1 Tax and may be a source of the exosomes found in patient CSF. Importantly, these exosomes are capable of sensitizing an HTLV-1 specific immune response, suggesting that they may play a role in the immunopathology observed in HAM/TSP. Given the infiltration of HTLV-1 Tax-specific CTLs into the CNS of HAM/TSP patients, it is likely that exosomes may also contribute to the continuous activation and inflammation observed in HAM/TSP, and may suggest future targeted therapies in this disorder.

Autophagy, EVs, and Infections: A Perfect Question for a Perfect Time.

Autophagy, a highly conserved process, serves to maintain cellular homeostasis in response to an extensive variety of internal and external stimuli. The classic, or canonical, pathway of autophagy involves the coordinated degradation and recycling of intracellular components and pathogenic material. Proper regulation of autophagy is critical to maintain cellular health, as alterations in the autophagy pathway have been linked to the progression of a variety of physiological and pathological conditions in humans, namely in aging and in viral infection. In addition to its canonical role as a degradative pathway, a more unconventional and non-degradative role for autophagy has emerged as an area of increasing interest. This process, known as secretory autophagy, is gaining widespread attention as many viruses are believed to use this pathway as a means to release and spread viral particles. Moreover, secretory autophagy has been found to intersect with other intracellular pathways, such as the biogenesis and secretion of extracellular vesicles (EVs). Here, we provide a review of the current landscape surrounding both degradative autophagy and secretory autophagy in relation to both aging and viral infection. We discuss their key features, while describing their interplay with numerous different viruses (i.e. hepatitis B and C viruses, Epstein-Barr virus, SV40, herpesviruses, HIV, chikungunya virus, dengue virus, Zika virus, Ebola virus, HTLV, Rift Valley fever virus, poliovirus, and influenza A virus), and compare secretory autophagy to other pathways of extracellular vesicle release. Lastly, we highlight the need for, and emphasize the importance of, more thorough methods to study the underlying mechanisms of these pathways to better advance our understanding of disease progression.

Corrigendum: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction.

[This corrects the article on p. 1765 in vol. 7, PMID: 27872619.].

Antiretroviral Drugs Alter the Content of Extracellular Vesicles from HIV-1-Infected Cells.

To date, the most effective treatment of HIV-1 is a combination antiretroviral therapy (cART), which reduces viral replication and reverses pathology. We investigated the effect of cART (RT and protease inhibitors) on the content of extracellular vesicles (EVs) released from HIV-1-infected cells. We have previously shown that EVs contain non-coding HIV-1 RNA, which can elicit responses in recipient cells. In this manuscript, we show that TAR RNA levels demonstrate little change with the addition of cART treatment in cell lines, primary macrophages, and patient biofluids. We determined possible mechanisms involved in the selective packaging of HIV-1 RNA into EVs, specifically an increase in EV-associated hnRNP A2/B1. More recent experiments have shown that several other FDA-approved drugs have the ability to alter the content of exosomes released from HIV-1-infected cells. These findings on cART-altered EV content can also be applied to general viral inhibitors (interferons) which are used to treat other chronic infections. Additionally, we describe unique mechanisms of ESCRT pathway manipulation by antivirals, specifically the targeting of VPS4. Collectively, these data imply that, despite antiretroviral therapy, EVs containing viral products are continually released and may cause neurocognitive and immunological dysfunction.

Publisher Correction: Antiretroviral Drugs Alter the Content of Extracellular Vesicles from HIV-1-Infected Cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The Role of Exosomal VP40 in Ebola Virus Disease.

Ebola virus (EBOV) can cause a devastating hemorrhagic disease, leading to death in a short period of time. After infection, the resulting EBOV disease results in high levels of circulating cytokines, endothelial dysfunction, coagulopathy, and bystander lymphocyte apoptosis in humans and nonhuman primates. The VP40 matrix protein of EBOV is essential for viral assembly and budding from the host cell. Recent data have shown that VP40 exists in the extracellular environment, including in exosomes, and exosomal VP40 can impact the viability of recipient immune cells, including myeloid and T cells, through the regulation of the RNAi and endosomal sorting complexes required for transport pathways. In this study, we discuss the latest findings of the impact of exosomal VP40 on immune cells in vitro and its potential implications for pathogenesis in vivo.

Isolation of Exosomes from HTLV-Infected Cells.

Exosomes are small vesicles, approximately 30-100 nm in diameter, that transport various cargos, such as proteins and nucleic acids, between cells. It has been previously shown that exosomes can also transport viral proteins, such as the HTLV protein Tax, and viral RNAs, potentially contributing to disease pathogenesis. Therefore, it is important to understand their impact on recipient cells. Here, we describe methods of isolating and purifying exosomes from cell culture or tissue through ultracentrifugation, characterizing exosomes by surface biomarkers, and assays that evaluate the effect of exosomes on cells.