KNOWN DRUG INDUCES NUCLEOPORINS Rae1/ mrnp41 AND Nup98: A PARADIGM SHIFT IN VSV ONCOLYTIC VIRUS MODULATION Paula A. Faria Waziry , Ana M. Castejon, and Luigi X. Cubeddu College of Pharmacy, Nova Southeastern University, Ft. Lauderdale, FL, USA P043_Nuclear Envelope Disease and Chromatin Organization. July 2011. Cambridge, UK INTRODUCTION An approach to the development of antiviral drugs is to target strategic antiviral host cell factors that are needed for viral replication. Drug A, which belongs to a class of drugs that are widely used, possesses antiviral actions against unrelated viruses (Fig. 1), supposedly by enhancing shared cellular antiviral pathways. In a search for common cellular factors, we have found that those unrelated viruses either (a) directly antagonize the Signal Transducer and Activator of Transcription 1 (STAT1) signaling pathway, and/or (b) antagonize/degrade components of the Nuclear Pore Complex (NPC). The NPC transport machinery has emerged as a key player in the discovery of experimental therapeutics that can modulate cellular genetic programs involved in antiviral and anticancer strategies. The vesicular stomatitis virus (VSV) is a (-)ssRNA non-transforming virus, and the prototype for NPC inhibition of mRNA export. VSV has a well- characterized mechanism of infection, which is STAT1-dependent and targets nucleoporins mRNA export factor 1 (Rae1) and Nucleoporin of 98KDa (Nup98). Rae1 assists mRNA export through the NPC via interactions with both mRNA export factors TAP/p15 and Nup98. Rae1 is the target of VSV’s matrix (M) protein, and is involved in vital cellular processes, such as mitosis and aging. Nup98 is a dynamic Nup, and shuttles between the nucleus and cytoplasm in a transcription-dependent manner. Nup98 is involved in the transport of proteins and export of RNA. Nup98 N-terminus when fused to C-termini of trascription factors, gives rise to several types of leukemia. Recently, both Nups were found to possess antiviral properties. Uncovering drugs capable of regulating Rae1 and Nup98 expression may enable modulation of immune response. Strategic and controlled replication of VSV will add safety and efficacy to live oncolytic viral therapy. Fig. 7 (A) Oligo-dT in situ hybridization of HeLa cells. Non- infected cells (mock-infections) show the effects of Drug A on distribution of poly(A) RNA (panels A-O). Control VSV infections show typical nuclear retention of poly(A) RNA (panels P-T). Cells pretreated with 0.125µM Drug A show partial recovery from mRNA block (panel U) and an increase in GFP Fig. 3 Drug A 24h pretreatment of HeLa cells followed by 16h VSV-GFP infection showed that 0.125µM Drug A enhanced the expression of viral proteins in both supernatants (A) and cell lysates (B). Increases in protein levels ranged from 140% to 175% relative to control. Increasing concentrations of Drug A pretreatment eliminated VSV protein levels in a dose-dependent manner in both (A) supernatants and (B) cell lysates. Levels of VSV proteins were lowered to undetectable amounts. Drug A Pretreatment Modulates Viral Titers Drug A Pretreatment Attenuates Expression of VSV Proteins Fig. 7 STAT1+/+ and STAT1-/- Mouse Embryo Fibroblasts (MEFs) were treated with different concentrations of Drug A as described, prior to IBs using anti-Nup98 and anti-Rae1 antibodies. In wild-type MEFs (STAT+/+), both Nups protein levels increased with increasing concentration of Drug A treatment. Contrary to STAT1+/+, STAT1-/- MEFs showed downregulation of both Nups upon Drug Atreatment. Shown are mean + SEM. Fig. 5 HeLa cells were pretreated with Drug A or Drug C, of the same class. (A) Drug A attenuates VSV replication (p<0.0001). (B) Drug C had no effect on viral titers (p=0.4644). Fig. 6 (A) HeLa cells were pretreated with Drug A 0.125 - 2 μM, then infected with VSV-GFP (closed circles), or pretreatment with Drug A, but not infected (open circles). (B) HeLa cells were pretreated with Drug C similarly to (A). Cell viability was similar for both drugs pretreatment followed (or not) by VSV infection. (C) Drug A or (D) Drug C pretreatment comparative graph of HeLa cell viability (open squares), titers (closed triangles) and VSV N/P protein levels (open circles). Effects of different pretreatment times with Drug A on VSV protein levels Fig. 4 HeLa cells were pre-treated with 1.0µM Drug A for time points ranging from 0 to 24 hrs prior to infection with VSV-GFP at MOI of 1.0 for 16 h. Viral proteins were measured by IB assays performed with anti-VSV-GFP antibodies. Plotted are the levels of VSV-M protein. Similar results were observed for the G and N/P proteins (data not shown). VSV infection Induces mRNA Export Block That is Reversed by Drug A Pretreatment SUMMARY 1) Drug A upregulates nucleoporins Rae1 and Nup98 in the presence and absence of VSV infection. 2) Low concentrations of Drug A enhances VSV replication, while higher concentrations inhibit the virus. 3) A similar drug, Drug C, does not modulate VSV replication. 4) Viability assays indicate that Drug A potentiates the VSV- killing of HeLa cells. 5) Upregulation of Rae1 and Nup98 by Drug A depends on STAT1 signaling. 6) mRNA cellular distribution is altered by Drug A treatment. 7) Drug A causes formation of vesicle-like structures on the cytoplasm of cells. These vesicle-like structures are mRNA- rich. 8) Drug A reverses VSV mRNA export block. 9) Here we show for the first time that a widely used drug upregulates Nups that are crucial for normal NPC function. 10) Our data support the use of Drug A as neoadjuvant and/or as attenuator of VSV oncolytic therapy. Drug A Upregulates NPC Antiviral Nucleoporins Rae1/ mrnp41 and Nup98 Fig.2 HeLa cells were exposed to different concentrations of Drug A for 24 hr prior to lysis and Immunoblot (IB). Control levels of both Nups were arbritrarily set to 100%. Protein levels of Nup98 increased to an average of 170% of control for both uninfected and infected cells (A and B). Protein levels of Rae1 increased to 370% in non-infected cells (A) and 160% in VSV-infected cells (B). Non- infected cells were more responsive to Drug A treatment than VSV-infected cells. ABSTRACT Viral infections persistently threaten human health. Development of broad-range antiviral drugs capable of enhancing cellular innate immunity could potentially avoid future pandemics. Similarly, cancer is a major health concern and leading cause of death worldwide. Innovative therapeutic approaches, such as the use of oncolytic viruses, are worth exploring. Such therapy requires drugs that would minimize viral infectivity to normal cells and/or enhance viral oncolytic potency. Drug “A” (concealed due to patent) is known to exert effects that include cancer- cell killing and inhibition of viruses such as HIV, HCV, polio and influenza A. Strikingly, viruses that are affected by Drug A have two mechanisms in common: blockage of antiviral STAT1 pathway and take-over of cellular NCP function. VSV is the prototype virus for take-over of NPC function via blockage of mRNA export. In this study, we investigated the effects of Drug A on the expression of nucleoporins Nup98 and Rae1/mrnp41, as well as on the infection/replication of oncolytic VSV. Here we show for the first time that Drug A up-regulates both Nup98 and Rae1 in a concentration-dependent and STAT1-dependent manner. Drug A also exerts biphasic effects on VSV protein expression and replication. Low concentrations of Drug A (0.125μM) increases VSV protein levels and viral titers, while higher concentrations (0.25-2.0μM) decrease viral protein levels and titers. Our findings lay the foundation for repositioning of Drug A as oncolytic virotherapy neoadjuvant. Grant: NSU Chancellor’s Research and Development Award 2010-11. Viruses Affected by Drug A Interfere with Nuclear Pore Complex Function Fig.1 Schematic representation of viruses that take over cell function by utilizing and/or degrading NPC components. The represented viruses also inhibit STAT1 signaling as a mechanistic strategy to suppress innate immune antiviral response. Abrev: Hepatitis B virus (HBV), Hepatitis C virus (HCV), human immunodeficiency virus (HIV), rous sarcoma virus (RSV), karyopherin (Kap), nucleoporin (Nup), importin (IMP), vesicular stomatitis virus (VSV), matrix protein of VSV (M). Chromosome Region Maintenance 1/Exportin 1 (CRM1) RESULTS Differential effects of Drugs A and C on VSV protein levels, VSV infectivity and HeLa cell viability  VSV protein levels and titers presented similar patterns of initial increase at low Drug A concentration, followed by a decrease at higher drug concentration pretreatments. Results were expressed as percentage of control values. Shown are mean values + SEM. (panel W). Cells pretreated with 2.0µM Drug A show near complete recovery from mRNA nuclear block (panels Z-γ), and normal distribution of poly(A) RNA between the nucleus and the cytoplasm (panel Z). Samples were examined by confocal microscopy. (B) Luciferase reporter gene expression assays show that VSV-mediated inhibition of gene expression is reversed by increasing concentrations of Drug A pretreatment. HeLa cells were co- transfected with reporter plasmids luciferase and β-gal, and pretreated with different concentrations of Drug A as described in (A). Cells were lysed and immediately assayed for luciferase protein expression, and normalized to β-gal. VSV infection inhibited luciferase expression, which was reversed by pretreatment of Drug A in a concentration-dependent manner.  Minimal pretreatment of 2 hrs is needed to observe Drug A’s effects on VSV proteins.  Titers profile of Drug A- treated cells corroborate the results of viral protein expression  Low concentration of Drug A enhances VSV protein expression, while higher concentrations inhibit VSV proteins. Drug A Upregulation of Nup98 and Rae1 is dependent on STAT1 signaling. B) A) A) B) Model for Drug A Reversal of M Protein-Mediated mRNA Export Block Fig. 8 (A) Normal mRNA export. (B) Rae1 mediates the interaction between M protein and Nup98, forming a complex that cannot function in mRNA export. (C) Drug A pretreatment increased expression of Rae1 and/or Nup98, which leads to sequestration of M protein, resulting in the formation of free Nup98-Rae1, which are competent for mRNA export. A B C rotected. F1000 Posters. Copyri s. Copyright protected. F1000 Posters. Copyright protected F1000 Posters. Copyright protected. F1000 Posters. Copyright protected. F1000 Poste ht protected. F1000 Posters. Copyright protected. F1000 Posters. Copyright protected. F1000 Posters. Copyright sters. Copyright protected. F1000 Posters. Copyright protected. F1000 Posters. Copyright protected. F1000 Posters. sters. Copyright protected. F1000 Posters. Copyright protected. F1000 Posters. Copyright protected. F1 ht protected. F1000 Posters. Copyright protected. F1000 Posters. Copyright F1000 Posters. Copyright protected. F1000 Poste s. Copyright protected