Regulation of Human Neurotropic JC Virus Replication by Alternative Splicing Factor SF2/ASF in Glial Cells

Background: The human neurotropic virus, JC virus (JCV), is the etiologic agent of the fatal demyelinating disease of the central nervous system, Progressive Multifocal Leukoencephlopathy (PML) that is seen primarily in immunodeficient individuals. Productive infection of JCV occurs only in glial cells, and this restriction is, to a great extent, due to the activation of the viral promoter that has cell type-specific characteristics. Earlier studies led to the hypothesis that glial-specific activation of the JCV promoter is mediated through positive and negative transcription factors that control reactivation of the JCV genome under normal physiological conditions and suppress its activation in non-glial cells. Methodololgy/Principal Findings: Using a variety of virological and molecular biological approaches, we demonstrate that the alternative splicing factor SF2/ASF has the capacity to exert a negative effect on transcription of the JCV promoter in glial cells through direct association with a specific DNA sequence within the viral enhancer/promoter region. Our results show that down-regulation of SF2/ASF in fetal and adult glial cells increases the level of JCV gene expression and its replication indicating that negative regulation of the JCV promoter by SF2/ASF may control reactivation of JCV replication in brain. Conclusions/Significance: Our results establish a new regulatory role for SF2/ASF in controlling gene expression at th

The agnoprotein of polyomavirus JC is released by infected cells: Evidence for Its cellular uptake by uninfected neighboring cells

AbstractPoliomavirus JC replicates in glial cells in the brain, and causes the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). PML is usually seen in patients with underlying immunocompromised conditions, notably among AIDS patients and those on chronic immunosuppressive regimens. The late leader sequence of JC virus contains an open reading frame encoding a small regulatory protein called agnoprotein. Agnoprotein contributes to progressive viral infection by playing significant roles in viral replication cycle. Here, we demonstrate that agnoprotein can be detected in cell-free fractions of glial cultures infected with JCV, transfected with expression plasmids or transduced with an adenovirus expression system. We also provide evidence that extracellular agnoprotein can be taken up by uninfected neighboring cells. These studies have revealed a novel phenomenon of agnoprotein during the viral life cycle with a potential of developing diagnostic and therapeutic interventions

Viruses and Human Cancers: a Long Road of Discovery of Molecular Paradigms

About a fifth of all human cancers worldwide are caused by infectious agents. In 12% of cancers, seven different viruses have been causally linked to human oncogenesis: Epstein-Barr virus, hepatitis B virus, human papillomavirus, human T-cell lymphotropic virus, hepatitis C virus, Kaposi's sarcoma herpesvirus, and Merkel cell polyomavirus. Here, we review the many molecular mechanisms of oncogenesis that have been discovered over the decades of study of these viruses. We discuss how viruses can act at different stages in the complex multistep process of carcinogenesis. Early events include their involvement in mutagenic events associated with tumor initiation such as viral integration and insertional mutagenesis as well as viral promotion of DNA damage. Also involved in tumor progression is the dysregulation of cellular processes by viral proteins, and we describe how this has been investigated by studies in cell culture and in experimental animals and by molecular cellular approaches. Also important are the molecular mechanisms whereby viruses interact with the immune system and the immune evasion strategies that have evolved

Transcriptional regulation of human polyomavirus JC: evidence for a functional interaction between RelA (p65) and the Y-box-binding protein, YB-1.

The transcriptional control region of the human neurotropic polyomavirus JC virus contains a consensus NF-kappa B site which has been shown to enhance both basal and extracellular stimulus-induced levels of transcription of JC promoters. Here, we show that the expression of JC late promoter constructs containing the NF-kappa B site is decreased by cotransfection with the NF-kappa B/rel subunits, p50 and p52, but enhanced by the p65 subunit. However, JC promoter constructs lacking the NF-kappa B site were activated by p52 and p50 and repressed by p65. This antithetical response of the JC promoter mapped specifically to the D domain, which is a target site for the cellular transcription factor, YB-1. Band shift studies indicated that YB-1 and p65 modulate each other's binding to DNA: YB-1 augments the affinity of p65 for the NF-kappa B site, while p65 reduces the binding of YB-1 to the D domain. Results from coimmunoprecipitation followed by Western blot (immunoblot) analysis suggest an in vivo interaction between p65 and YB-1 in glial cells. Functionally, YB-1 appears to act synergistically with p65 to control transcription from the NF-kappa B site. A converse pattern is seen with the D domain, in which YB-1 acts synergistically with p50 and p52 to regulate transcription. p50 and p52 may function as transcriptional activators on the D domain by removing the repressive effect of p65 on YB-1 binding to the D domain. On the basis of these data, we propose a model in which NF-kappa B/rel subunits functionally interact with consensus NF-kappa B sites or YB-1-binding sites, with disparate effects on eukaryotic gene expression

Neuroprotective Effects of IGF-I against TNFα-Induced Neuronal Damage in HIV-Associated Dementia

AbstractHuman immunodeficiency virus type 1 (HIV-1) infection often results in disorders of the central nervous system, including HIV-associated dementia (HAD). It is suspected that tumor necrosis factor-α (TNFα) released by activated and/or infected macrophages/microglia plays a role in the process of neuronal damage seen in AIDS patients. In light of earlier studies showing that the activation of the insulin-like growth factor I receptor (IGF-IR) exerts a strong neuroprotective effect, we investigated the ability of IGF-I to protect neuronal cells from HIV-infected macrophages. Our results demonstrate that the conditioned medium from HIV-1-infected macrophages, HIV/CM, causes loss of neuronal processes in differentiated PC12 and P19 neurons and that these neurodegenerative effects are associated with the presence of TNFα. Furthermore, we demonstrate that IGF-I rescues differentiated neurons from both HIV/CM and TNFα-induced damage and that IGF-I-mediated neuroprotection is strongly enhanced by overexpression of the wt IGF-IR cDNA and attenuated by the antisense IGF-IR cDNA. Finally, IGF-I-mediated antiapoptotic pathways are continuously functional in differentiated neurons exposed to HIV/CM and are likely supported by TNFα-mediated phosphorylation of IκB. All together these results suggest that the balance between TNFα and IGF-IR signaling pathways may control the extent of neuronal injury in this HIV-related experimental setting

The Multifunctional Protein BAG3: A Novel Therapeutic Target in Cardiovascular Disease

The B-cell lymphoma 2–associated anthanogene (BAG3) protein is expressed most prominently in the heart, the skeletal muscle, and in many forms of cancer. In the heart, it serves as a co-chaperone with heat shock proteins in facilitating autophagy; binds to B-cell lymphoma 2, resulting in inhibition of apoptosis; attaches actin to the Z disk, providing structural support for the sarcomere; and links the α-adrenergic receptor with the L-type Ca2+ channel. When BAG3 is overexpressed in cancer cells, it facilitates prosurvival pathways that lead to insensitivity to chemotherapy, metastasis, cell migration, and invasiveness. In contrast, in the heart, mutations in BAG3 have been associated with a variety of phenotypes, including both hypertrophic/restrictive and dilated cardiomyopathy. In murine skeletal muscle and vasculature, a mutation in BAG3 leads to critical limb ischemia after femoral artery ligation. An understanding of the biology of BAG3 is relevant because it may provide a therapeutic target in patients with both cardiac and skeletal muscle disease

Gene Editing of HIV-1 Co-receptors to Prevent and/or Cure Virus Infection

Antiretroviral therapy has prolonged the lives of people living with human immunodeficiency virus type 1 (HIV-1), transforming the disease into one that can be controlled with lifelong therapy. The search for an HIV-1 vaccine has plagued researchers for more than three decades with little to no success from clinical trials. Due to these failures, scientists have turned to alternative methods to develop next generation therapeutics that could allow patients to live with HIV-1 without the need for daily medication. One method that has been proposed has involved the use of a number of powerful gene editing tools; Zinc Finger Nucleases (ZFN), Transcription Activator–like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 to edit the co-receptors (CCR5 or CXCR4) required for HIV-1 to infect susceptible target cells efficiently. Initial safety studies in patients have shown that editing the CCR5 locus is safe. More in depth in vitro studies have shown that editing the CCR5 locus was able to inhibit infection from CCR5-utilizing virus, but CXCR4-utilizing virus was still able to infect cells. Additional research efforts were then aimed at editing the CXCR4 locus, but this came with other safety concerns. However, in vitro studies have since confirmed that CXCR4 can be edited without killing cells and can confer resistance to CXCR4-utilizing HIV-1. Utilizing these powerful new gene editing technologies in concert could confer cellular resistance to HIV-1. While the CD4, CCR5, CXCR4 axis for cell-free infection has been the most studied, there are a plethora of reports suggesting that the cell-to-cell transmission of HIV-1 is significantly more efficient. These reports also indicated that while broadly neutralizing antibodies are well suited with respect to blocking cell-free infection, cell-to-cell transmission remains refractile to this approach. In addition to stopping cell-free infection, gene editing of the HIV-1 co-receptors could block cell-to-cell transmission. This review aims to summarize what has been shown with regard to editing the co-receptors needed for HIV-1 entry and how they could impact the future of HIV-1 therapeutic and prevention strategies

Decreased levels of BAG3 in a family with a rare variant and in idiopathic dilated cardiomyopathy.

The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease; however, in a third of all patients the cause remains undefined and patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC). Recent studies suggest that many patients with IDC have a family history of HF and rare genetic variants in over 35 genes have been shown to be causative of disease. We employed whole-exome sequencing to identify the causative variant in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10-nucleotide deletion in the BCL2-associated athanogene 3 (BAG3) gene (Ch10:del 121436332_12143641: del. 1266_1275 [NM 004281]) that segregated with all affected individuals. The deletion predicted a shift in the reading frame with the resultant deletion of 135 amino acids from the C-terminal end of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins there was a considerable amount of genetic heterogeneity in the affected family members. Interestingly, we also found that the levels of BAG3 protein were significantly reduced in the hearts from unrelated patients with end-stage HF undergoing cardiac transplantation when compared with non-failing controls. Diminished levels of BAG3 protein may be associated with both familial and non-familial forms of dilated cardiomyopathy