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

JC Virus T-Antigen Regulates Glucose Metabolic Pathways in Brain Tumor Cells

Recent studies have reported the detection of the human neurotropic virus, JCV, in a significant population of brain tumors, including medulloblastomas. Accordingly, expression of the JCV early protein, T-antigen, which has transforming activity in cell culture and in transgenic mice, results in the development of a broad range of tumors of neural crest and glial origin. Evidently, the association of T-antigen with a range of tumor-suppressor proteins, including p53 and pRb, and signaling molecules, such as β-catenin and IRS-1, plays a role in the oncogenic function of JCV T-antigen. We demonstrate that T-antigen expression is suppressed by glucose deprivation in medulloblastoma cells and in glioblastoma xenografts that both endogenously express T-antigen. Mechanistic studies indicate that glucose deprivation-mediated suppression of T-antigen is partly influenced by 5′-activated AMP kinase (AMPK), an important sensor of the AMP/ATP ratio in cells. In addition, glucose deprivation-induced cell cycle arrest in the G1 phase is blocked with AMPK inhibition, which also prevents T-antigen downregulation. Furthermore, T-antigen prevents G1 arrest and sustains cells in the G2 phase during glucose deprivation. On a functional level, T-antigen downregulation is partially dependent on reactive oxygen species (ROS) production during glucose deprivation, and T-antigen prevents ROS induction, loss of ATP production, and cytotoxicity induced by glucose deprivation. Additionally, we have found that T-antigen is downregulated by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the pentose phosphate inhibitors, 6-aminonicotinamide and oxythiamine, and that T-antigen modulates expression of the glycolytic enzyme, hexokinase 2 (HK2), and the pentose phosphate enzyme, transaldolase-1 (TALDO1), indicating a potential link between T-antigen and metabolic regulation. These studies point to the possible involvement of JCV T-antigen in medulloblastoma proliferation and the metabolic phenotype and may enhance our understanding of the role of viral proteins in glycolytic tumor metabolism, thus providing useful targets for the treatment of virus-induced tumors

Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells.

OBJECTIVE:PML is a rare and fatal demyelinating disease of the CNS caused by the human polyomavirus, JC virus (JCV), which occurs in AIDS patients and those on immunosuppressive monoclonal antibody therapies (mAbs). We sought to identify mechanisms that could stimulate reactivation of JCV in a cell culture model system and targeted pathways which could affect early gene transcription and JCV T-antigen production, which are key steps of the viral life cycle for blocking reactivation of JCV. Two important regulatory partners we have previously identified for T-antigen include Pur-alpha and SRSF1 (SF2/ASF). SRSF1, an alternative splicing factor, is a potential regulator of JCV whose overexpression in glial cells strongly suppresses viral gene expression and replication. Pur-alpha has been most extensively characterized as a sequence-specific DNA- and RNA-binding protein which directs both viral gene transcription and mRNA translation, and is a potent inducer of the JCV early promoter through binding to T-antigen. METHODS AND RESULTS:Pur-alpha and SRSF1 both act directly as transcriptional regulators of the JCV promoter and here we have observed that Pur-alpha is capable of ameliorating SRSF1-mediated suppression of JCV gene expression and viral replication. Interestingly, Pur-alpha exerted its effect by suppressing SRSF1 at both the protein and mRNA levels in glial cells suggesting this effect can occur independent of T-antigen. Pur-alpha and SRSF1 were both localized to oligodendrocyte inclusion bodies by immunohistochemistry in brain sections from patients with HIV-1 associated PML. Interestingly, inclusion bodies were typically positive for either Pur-alpha or SRSF1, though some cells appeared to be positive for both proteins. CONCLUSIONS:Taken together, these results indicate the presence of an antagonistic interaction between these two proteins in regulating of JCV gene expression and viral replication and suggests that they play an important role during viral reactivation leading to development of PML

Inverse correlation of Pur-alpha and SRSF1 protein levels in Pur-alpha (-/-) MEF cells.

<p><b>A.</b> Lysates were prepared from mouse embryonic fibroblast (MEF) cultures obtained from wild type mice (MEF+/+), mice heterozygous for targeted disruption of the PURA gene (MEF+/-), or mice with homozygous deletion in PURA (MEF-/-). Whole cell protein extracts were analyzed by Western blotting for the expression of Pur-alpha, SRSF1, SRSF2, and SRSF3. Tubulin was probed in the same blots as an internal loading control. B. Band intensities of Pur-alpha, SRSF1, SRSF2, and SRSF3 in panel A were determined, normalized to tubulin, and are shown as a bar graph. C. Pur-alpha MEF-/- cells were transfected with an expression plasmid encoding GFP-Pur-alpha in increasing concentrations (1X and 3X). Whole cell protein lysates were prepared at 48 hrs post-transfection and examined by Western blotting for the detection of SRSF1 and Pur-alpha levels. Tubulin was probed in the same blots as an internal loading control. Band intensities of SRSF1 levels in panel C were quantified, normalized to tubulin, and are shown as a bar graph.</p

SRSF1-mediated suppression of JCV T-antigen is ameliorated by Pur-alpha.

<p>A. T98G cells were transiently transfected with expression plasmids encoding JCV T-antigen, T7-tagged SRSF1 and GFP-tagged Pur-alpha as indicated. Expression of the proteins were detected by Western blotting using anti-T-antigen, anti-SRSF1, and anti-GFP antibodies. Tubulin was probed in the same blots as an internal loading control. B. Bar graph presentation T-antigen expression levels based on signal intensity of T-antigen normalized to levels of the tubulin internal loading control in panel A.</p

Extinction of Tumor Antigen Expression by SF2/ASF in JCV-Transformed Cells

The human neurotropic polyomavirus JC (JCV) induces a broad range of neural-origin tumors in experimental animals and has been repeatedly detected in several human cancers, most notably neural crest–origin tumors including medulloblastomas and glioblastomas. The oncogenic activity of JCV is attributed to the viral early gene products, large T and small t antigens, as evident by results from in vitro cell culture and in vivo animal studies. Recently, we have shown that alternative splicing factor, SF2/ASF, has the capacity to exert a negative effect on transcription and splicing of JCV genes in glial cells through direct association with a specific DNA motif within the viral promoter region. Here, we demonstrate that SF2/ASF suppresses large T antigen expression in JCV-transformed tumor cell lines, and the expression of SF2/ASF in such tumor cells thereby inhibits the transforming capacity of the viral tumor antigens. Moreover, down-regulation of SF2/ASF in viral-transformed tumor cell lines induces growth and proliferation of the tumor cells. Mapping analysis of the minimal peptide domain of SF2/ASF responsible for JCV promoter silencing and tumor suppressor activity suggests that amino acid residues 76 to 100 of SF2/ASF are functionally sufficient to suppress the growth of the tumor cells. These observations demonstrate a role for SF2/ASF in JCV-mediated cellular transformation and provide a new avenue of research to pathogenic mechanisms of JCV-induced tumors

IFN-Gamma Inhibits JC Virus Replication in Glial Cells by Suppressing T-Antigen Expression.

Patients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), an often lethal disease of the brain characterized by lytic infection of oligodendrocytes in the central nervous system (CNS) with JC virus (JCV). The immune system plays an important regulatory role in controlling JCV reactivation from latent sites by limiting viral gene expression and replication. However, little is known regarding the molecular mechanisms responsible for this regulation.Here, we investigated the impact of soluble immune mediators secreted by activated PBMCs on viral replication and gene expression by cell culture models and molecular virology techniques. Our data revealed that viral gene expression and viral replication were suppressed by soluble immune mediators. Further studies demonstrated that soluble immune mediators secreted by activated PBMCs inhibit viral replication induced by T-antigen, the major viral regulatory protein, by suppressing its expression in glial cells. This unexpected suppression of T-antigen was mainly associated with the suppression of translational initiation. Cytokine/chemokine array studies using conditioned media from activated PBMCs revealed several candidate cytokines with possible roles in this regulation. Among them, only IFN-γ showed a robust inhibition of T-antigen expression. While potential roles for IFN-β, and to a lesser extent IFN-α have been described for JCV, IFN-γ has not been previously implicated. Further analysis of IFN-γ signaling pathway revealed a novel role of Jak1 signaling in control of viral T-antigen expression. Furthermore, IFN-γ suppressed JCV replication and viral propagation in primary human fetal glial cells, and showed a strong anti-JCV activity.Our results suggest a novel role for IFN-γ in the regulation of JCV gene expression via downregulation of the major viral regulatory protein, T-antigen, and provide a new avenue of research to understand molecular mechanisms for downregulation of viral reactivation that may lead to development of novel strategies for the treatment of PML

T-antigen prevents glucose deprivation-induced ROS production, reduction in ATP levels, and cytotoxicity.

<p>A. BsB8 cells were incubated with 24 mM N-acetylcysteine (NAC), 1 mM pyruvate, or were untreated and then exposed to glucose deprivation or control medium for 24 hours. Pyruvate was dissolved in a small volume of water and a negligible volume was added to the medium in each well. The expression of T-antigen was assessed in whole-cell extracts. B. Bs1a, Bs1f, or BsB8 cells were exposed to glucose deprivation or control medium for 16 hours, and ROS production was measured using the fluorescent dye, 25 µM carboxy-H₂-DCFDA. Hoechst staining was also performed to label nuclei. C. ROS levels were quantified by calculating the mean pixel intensity in triplicate images acquired. (* = control compared to GD, p<0.05; # = BsB8 compared to Bs1f, p<0.05). D. Bs1a, Bs1f, or BsB8 cells were exposed to glucose deprivation for 16 hours, and ATP levels were then measured. The levels of ATP per cell were measured and were normalized to the total protein present in each sample. E. Bs1a, Bs1f, or BsB8 cells were exposed to glucose deprivation or control medium for 24 hours, and cell viability was measured using Guava ViaCount reagent. (* = control compared to GD, p<0.05; # = BsB8 compared to Bs1a and Bs1f cells, p<0.05). F. Phase-contrast images of cells treated with glucose deprivation in D. C, control; GD, glucose deprivation.</p