Extracellular vesicles from HTLV-1 infected cells modulate target cells and viral spread.

BACKGROUND: The Human T-cell Lymphotropic Virus Type-1 (HTLV-1) is a blood-borne pathogen and etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 has currently infected up to 10 million globally with highly endemic areas in Japan, Africa, the Caribbean and South America. We have previously shown that Extracellular Vesicles (EVs) enhance HTLV-1 transmission by promoting cell-cell contact. RESULTS: Here, we separated EVs into subpopulations using differential ultracentrifugation (DUC) at speeds of 2 k (2000×g), 10 k (10,000×g), and 100 k (100,000×g) from infected cell supernatants. Proteomic analysis revealed that EVs contain the highest viral/host protein abundance in the 2 k subpopulation (2 k > 10 k > 100 k). The 2 k and 10 k populations contained viral proteins (i.e., p19 and Tax), and autophagy proteins (i.e., LC3 and p62) suggesting presence of autophagosomes as well as core histones. Interestingly, the use of 2 k EVs in an angiogenesis assay (mesenchymal stem cells + endothelial cells) caused deterioration of vascular-like-tubules. Cells commonly associated with the neurovascular unit (i.e., astrocytes, neurons, and macrophages) in the blood-brain barrier (BBB) showed that HTLV-1 EVs may induce expression of cytokines involved in migration (i.e., IL-8; 100 k > 2 k > 10 k) from astrocytes and monocyte-derived macrophages (i.e., IL-8; 2 k > 10 k). Finally, we found that EVs were able to promote cell-cell contact and viral transmission in monocytic cell-derived dendritic cell. The EVs from both 2 k and 10 k increased HTLV-1 spread in a humanized mouse model, as evidenced by an increase in proviral DNA and RNA in the Blood, Lymph Node, and Spleen. CONCLUSIONS: Altogether, these data suggest that various EV subpopulations induce cytokine expression, tissue damage, and viral spread.

A comprehensive review of COVID-19 biology, diagnostics, therapeutics, and disease impacting the central nervous system.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a highly transmissible disease. SARS-CoV-2 is estimated to have infected over 153 million people and to have caused over 3.2 million global deaths since its emergence in December 2019. SARS-CoV-2 is the seventh coronavirus known to infect humans, and like other coronaviruses, SARS-CoV-2 infection is characterized by a variety of symptoms including general flu-like symptoms such as a fever, sore throat, fatigue, and shortness of breath. Severe cases often display signs of pneumonia, lymphopenia, acute kidney injury, cardiac injury, cytokine storms, lung damage, acute respiratory distress syndrome (ARDS), multiple organ failure, sepsis, and death. There is evidence that around 30% of COVID-19 cases have central nervous system (CNS) or peripheral nervous system (PNS) symptoms along with or in the absence of the previously mentioned symptoms. In cases of CNS/PNS impairments, patients display dizziness, ataxia, seizure, nerve pain, and loss of taste and/or smell. This review highlights the neurological implications of SARS-CoV-2 and provides a comprehensive summary of the research done on SARS-CoV-2 pathology, diagnosis, therapeutics, and vaccines up to May 5.

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.

Retroviral b-Zip protein (HBZ) contributes to the release of soluble and exosomal immune checkpoint molecules in the context of neuroinflammation.

HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive, neuroinflammatory demyelinating condition of the spinal cord. We have previously shown that aberrant expression and activity of immune checkpoint (ICP) molecules such as PD-1 and PD-L1/PD-L2, negatively associates with the cytolytic potential of T cells in individuals with HAM/TSP. Interestingly, ICPs can exist in a soluble cell-free form and can be carried on extracellular vesicles (EVs) and exosomes (small EVs, <300nm) while maintaining their immunomodulatory activity. Therefore, we investigated the role of soluble and exosomal ICPs in HTLV-1 associated neuroinflammation. For the very first time, we demonstrate a unique elevated presence of several stimulatory (CD27, CD28, 4-1BB) and inhibitory (BTLA, CTLA-4, LAG-3, PD-1, PD-L2) ICP receptors in HAM/TSP sera, and in purified exosomes from a HAM/TSP-derived HTLV-1-producing (OSP2) cells. These ICPs were found to be co-localized with the endosomal sorting complex required for transport (ESCRT) pathway proteins and exhibited functional binding with their respective ligands. Viral proteins and cytokines (primarily IFNγ) were found to be present in purified exosomes. IFNγ exposure enhanced the release of ICP molecules while antiretroviral drugs (Azidothymidine and Lopinavir) significantly inhibited this process. HTLV-1 b-Zip protein (HBZ) has been linked to factors that enhance EV release and concurrent knockdown here led to the reduced expression of ESCRT associated genes (eg. Hrs, Vsp4, Alix, Tsg101) as well as abrogated the release of ICP molecules, suggesting HBZ involvement in this process. Moreso, exosomes from OSP2 cells adversely affected CD8 T-cell functions by dimishing levels of cytokines and cytotoxic factors. Collectively, these findings highlight exosome-mediated immunmodulation of T-cell functions with HBZ and ESCRT pathways as an underlying mechanism in the context of HTLV-1-induced neuroinflammation.

Army liposome formulation containing QS-21 render human monocyte-derived macrophages less permissive to HIV-1 infection by upregulating ABOBEC3A.

Monocyte-derived macrophages (MDM) are highly permissive to HIV-1 infection potentially due to the downregulation of innate factors during the differentiation process. The environmental milieu and innate anti-viral factors which are modulated during macrophage differentiation, have been associated with their increased permissiveness to HIV-1 infection. Here, we demonstrate that the Army Liposome Formulation containing MPLA, and QS-21 (ALFQ) activated MDM that are normally permissive to HIV-1 infection to generate a proinflammatory environment and upregulated anti-viral factors notably APOBEC3A. Induction of APOBEC3A by ALFQ decreased permissiveness to HIV-1 infection, while knockdown of APOBEC3A with APOBEC3AsiRNA resulted in a significant loss in the restriction of HIV-1 infectivity. The liposome formulation ALF55, with identical lipid composition but lacking QS-21 had no effect. Furthermore, the capacity of ALFQ to modulate MDM permissiveness to HIV-1 infection was predominantly mediated by large ALFQ liposomes. Our findings highlight a relationship between innate immune activation, proinflammatory milieu, and upregulation of anti-HIV proteins. Induction of these responses can switch the HIV-1 permissive MDM into a more refractory phenotype.

Extracellular Vesicles from Infected Cells Are Released Prior to Virion Release.

Here, we have attempted to address the timing of EV and virion release from virally infected cells. Uninfected (CEM), HIV-1-infected (J1.1), and human T cell leukemia virus-1 (HTLV-1)-infected (HUT102) cells were synchronized in G0. Viral latency was reversed by increasing gene expression with the addition of serum-rich media and inducers. Supernatants and cell pellets were collected post-induction at different timepoints and assayed for extracellular vesicle (EV) and autophagy markers; and for viral proteins and RNAs. Tetraspanins and autophagy-related proteins were found to be differentially secreted in HIV-1- and HTLV-1-infected cells when compared with uninfected controls. HIV-1 proteins were present at 6 h and their production increased up to 24 h. HTLV-1 proteins peaked at 6 h and plateaued. HIV-1 and HTLV-1 RNA production correlated with viral protein expression. Nanoparticle tracking analysis (NTA) showed increase of EV concentration over time in both uninfected and infected samples. Finally, the HIV-1 supernatant from the 6-h samples was found not to be infectious; however, the virus from the 24-h samples was successfully rescued and infectious. Overall, our data indicate that EV release may occur prior to viral release from infected cells, thereby implicating a potentially significant effect of EVs on uninfected recipient cells prior to subsequent viral infection and spread.

Extracellular Vesicles in HTLV-1 Communication: The Story of an Invisible Messenger.

Human T-cell lymphotropic virus type 1 (HTLV-1) infects 5-10 million people worldwide and is the causative agent of adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) as well as other inflammatory diseases. A major concern is that the most majority of individuals with HTLV-1 are asymptomatic carriers and that there is limited global attention by health care officials, setting up potential conditions for increased viral spread. HTLV-1 transmission occurs primarily through sexual intercourse, blood transfusion, intravenous drug usage, and breast feeding. Currently, there is no cure for HTLV-1 infection and only limited treatment options exist, such as class I interferons (IFN) and Zidovudine (AZT), with poor prognosis. Recently, small membrane-bound structures, known as extracellular vesicles (EVs), have received increased attention due to their potential to carry viral cargo (RNA and proteins) in multiple pathogenic infections (i.e., human immunodeficiency virus type I (HIV-1), Zika virus, and HTLV-1). In the case of HTLV-1, EVs isolated from the peripheral blood and cerebral spinal fluid (CSF) of HAM/TSP patients contained the viral transactivator protein Tax. Additionally, EVs derived from HTLV-1-infected cells (HTLV-1 EVs) promote functional effects such as cell aggregation which enhance viral spread. In this review, we present current knowledge surrounding EVs and their potential role as immune-modulating agents in cancer and other infectious diseases such as HTLV-1 and HIV-1. We discuss various features of EVs that make them prime targets for possible vehicles of future diagnostics and therapies.

Extracellular Vesicle Activation of Latent HIV-1 Is Driven by EV-Associated c-Src and Cellular SRC-1 via the PI3K/AKT/mTOR Pathway.

HIV-1 is a global health crisis that has infected more than 37 million people. Latent reservoirs throughout the body are a major hurdle when it comes to eradicating the virus. In our previous study, we found that exosomes, a type of extracellular vesicle (EV), from uninfected cells activate the transcription of HIV-1 in latent infected cells, regardless of combination antiretroviral therapy (cART). In this study, we investigated the specific mechanism behind the EV activation of latent HIV-1. We found that phosphorylated c-Src is present in EVs of various cell lines and has the ability to activate downstream proteins such as EGFR, initiating a signal cascade. EGFR is then able to activate the PI3K/AKT/mTOR pathway, resulting in the activation of STAT3 and SRC-1, culminating in the reversal of HIV-1 latency. This was verified by examining levels of HIV-1 TAR, genomic RNA and HIV-1 Gag p24 protein in cell lines and primary cells. We found that EVs containing c-Src rescued HIV-1 despite the presence of inhibitors, validating the importance of EV-associated c-Src in latent HIV-1 activation. Lastly, we discovered an increased recruitment of p300 and NF-κB in the nucleus of EV-treated infected cells. Collectively, our data suggest that EV-associated c-Src is able to activate latent HIV-1 via the PI3K/AKT/mTOR pathway and SRC-1/p300-driven chromatin remodeling. These findings could aid in designing new strategies to prevent the reactivation of latent HIV-1 in patients under cART.

Stem Cell Extracellular Vesicles and their Potential to Contribute to the Repair of Damaged CNS Cells.

Neurological diseases and disorders are leading causes of death and disability worldwide. Many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. As the global burden of these pathologies continues to rise there is a significant need for the development of novel therapeutics. Due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. It is now believed that paracrine factors, such as extracellular vesicles (EVs), play a critical role in the functionality associated with stem cells. The diverse biological cargo contained within EVs are proposed to mediate these effects and, to date, the reparative and regenerative effects of stem cell EVs have been demonstrated in a wide range of cell types. While a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the CNS. Here, we report the isolation, characterization, and functional assessment of EVs from two sources of human stem cells, mesenchymal stem cells and induced pluripotent stem cells. We demonstrate the ability of these EVs to enhance the processes of cellular migration and angiogenesis, which are critical for both normal cellular development as well as cellular repair. Furthermore, we investigate their reparative effects on damaged cells, specifically those with relevance to the central nervous system. Collectively, our data highlight the similarities and differences among these EV populations and support the view that stem cells EV can be used to repair or partially reverse cellular damage. Graphical Abstract Stem cell-derived Extracellular Vesicles (EVs) for repair of damaged cells. EVs isolated from human induced pluripotent stem cells and mesenchymal stem cells contribute to the partial reversal of phenotypes induced by different sources of cellular damage.

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.

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.

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.

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.