ABSTRACTp63, the major regulator of epithelial development/differentiation, is mutated in human ectodermal dysplasias, such as ankyloblepharon, ectodermal dysplasia and clefting (AEC). We recently identified that p63α physically associated with mRNA processing/ splicing proteins. We previously showed that p63 mutations mapped to the sterile α-motif led to disruption of these interactions and modulated an aberrant splicing of keratinocyte growth factor receptor contributing into molecular mechanism underlying AEC phenotype.To further investigate the molecular mechanisms associated with AEC syndrome we established the cellular model for this disorder by stable introduction of mutated allele [L514F] of p63α into immortalized keratinocyte cells. We showed that mutated ∆Np63α mediated an aberrant splicing of its own p63 mRNA transcript, which in turn led to accumulation of proteasome-resistant C-terminal truncated p63. The truncated p63 failed to associate with the C-terminal domain of RNA polymerase II through SRA4 protein and, therefore affected keratinocyte proliferation, differentiation and survival and may strongly contribute to AEC phenotype.

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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.

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.

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.