scholarly journals Akt Interacts with Usutu Virus Polymerase, and Its Activity Modulates Viral Replication

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 244
Author(s):  
Laura Albentosa-González ◽  
Rosario Sabariegos ◽  
Armando Arias ◽  
Pilar Clemente-Casares ◽  
Antonio Mas
Keyword(s):  
Public Health ◽  
Confocal Microscopy ◽  
Viral Replication ◽  
Insufficient Data ◽  
Health Concerns ◽  
Usutu Virus ◽  
A Cell ◽  
Infected Cells ◽  
Specific Inhibitors

Usutu virus (USUV) is a flavivirus that mainly infects wild birds through the bite of Culex mosquitoes. Recent outbreaks have been associated with an increased number of cases in humans. Despite being a growing source of public health concerns, there is yet insufficient data on the virus or host cell targets for infection control. In this work we have investigated whether the cellular kinase Akt and USUV polymerase NS5 interact and co-localize in a cell. To this aim, we performed co-immunoprecipitation (Co-IP) assays, followed by confocal microscopy analyses. We further tested whether NS5 is a phosphorylation substrate of Akt in vitro. Finally, to examine its role in viral replication, we chemically silenced Akt with three inhibitors (MK-2206, honokiol and ipatasertib). We found that both proteins are localized (confocal) and pulled down (Co-IP) together when expressed in different cell lines, supporting the fact that they are interacting partners. This possibility was further sustained by data showing that NS5 is phosphorylated by Akt. Treatment of USUV-infected cells with Akt-specific inhibitors led to decreases in virus titers (>10-fold). Our results suggest an important role for Akt in virus replication and stimulate further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral target.

scholarly journals MERS-CoV Accessory ORFs Play Key Role for Infection and Pathogenesis

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Vineet D. Menachery ◽  
Hugh D. Mitchell ◽  
Adam S. Cockrell ◽  
Lisa E. Gralinski ◽  
Boyd L. Yount ◽  
...  
Keyword(s):  
Public Health ◽  
Viral Replication ◽  
Rapid Response ◽  
Mutant Virus ◽  
Global Public Health ◽  
Vaccine Platform ◽  
Zoonotic Pathogens ◽  
Related Group

ABSTRACT While dispensable for viral replication, coronavirus (CoV) accessory open reading frame (ORF) proteins often play critical roles during infection and pathogenesis. Utilizing a previously generated mutant, we demonstrate that the absence of all four Middle East respiratory syndrome CoV (MERS-CoV) accessory ORFs (deletion of ORF3, -4a, -4b, and -5 [dORF3-5]) has major implications for viral replication and pathogenesis. Importantly, attenuation of the dORF3-5 mutant is primarily driven by dysregulated host responses, including disrupted cell processes, augmented interferon (IFN) pathway activation, and robust inflammation. In vitro replication attenuation also extends to in vivo models, allowing use of dORF3-5 as a live attenuated vaccine platform. Finally, examination of ORF5 implicates a partial role in modulation of NF-κB-mediated inflammation. Together, the results demonstrate the importance of MERS-CoV accessory ORFs for pathogenesis and highlight them as potential targets for surveillance and therapeutic treatments moving forward. IMPORTANCE The initial emergence and periodic outbreaks of MERS-CoV highlight a continuing threat posed by zoonotic pathogens to global public health. In these studies, mutant virus generation demonstrates the necessity of accessory ORFs in regard to MERS-CoV infection and pathogenesis. With this in mind, accessory ORF functions can be targeted for both therapeutic and vaccine treatments in response to MERS-CoV and related group 2C coronaviruses. In addition, disruption of accessory ORFs in parallel may offer a rapid response platform to attenuation of future emergent strains based on both SARS- and MERS-CoV accessory ORF mutants. IMPORTANCE The initial emergence and periodic outbreaks of MERS-CoV highlight a continuing threat posed by zoonotic pathogens to global public health. In these studies, mutant virus generation demonstrates the necessity of accessory ORFs in regard to MERS-CoV infection and pathogenesis. With this in mind, accessory ORF functions can be targeted for both therapeutic and vaccine treatments in response to MERS-CoV and related group 2C coronaviruses. In addition, disruption of accessory ORFs in parallel may offer a rapid response platform to attenuation of future emergent strains based on both SARS- and MERS-CoV accessory ORF mutants.

scholarly journals Transcriptional role of p53 in interferon-mediated antiviral immunity

2008 ◽  
Vol 205 (8) ◽  
pp. 1929-1938 ◽  
Author(s):  
César Muñoz-Fontela ◽  
Salvador Macip ◽  
Luis Martínez-Sobrido ◽  
Lauren Brown ◽  
Joseph Ashour ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Viral Replication ◽  
Viral Infections ◽  
Suppressor Gene ◽  
Central Component ◽  
Type I ◽  
Infected Cells

Tumor suppressor p53 is activated by several stimuli, including DNA damage and oncogenic stress. Previous studies (Takaoka, A., S. Hayakawa, H. Yanai, D. Stoiber, H. Negishi, H. Kikuchi, S. Sasaki, K. Imai, T. Shibue, K. Honda, and T. Taniguchi. 2003. Nature. 424:516–523) have shown that p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Moreover, many viruses, including SV40, human papillomavirus, Kaposi's sarcoma herpesvirus, adenoviruses, and even RNA viruses such as polioviruses, have evolved mechanisms designated to abrogate p53 responses. We describe a novel p53 function in the activation of the IFN pathway. We observed that infected mouse and human cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling, specifically the induction of genes containing IFN-stimulated response elements. Of note, p53 also contributed to an increase in IFN release from infected cells. We established that this p53-dependent enhancement of IFN signaling is dependent to a great extent on the ability of p53 to activate the transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Our results demonstrate that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene.

Matrix metalloproteinases mediate the metastatic phenotype ofTheileria annulata-transformed cells

Parasitology ◽  
1996 ◽  
Vol 113 (5) ◽  
pp. 449-455 ◽  
Author(s):  
R. E. Adamson ◽  
F. R. Hall
Keyword(s):  
Marked Reduction ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Transformed Cells ◽  
Pathological Features ◽  
Metastatic Phenotype ◽  
Infected Cells ◽  
Metalloproteinase 9 ◽  
Metalloproteinase Activity ◽  
Specific Inhibitors

SUMMARYTheileria annulatainfects and reversibly transforms bovine leucocytes. The parasite-transformed cells are immortalized, metastatic and express a number of metalloproteinases including matrix metalloproteinase 9 which they secrete. All the metalloproteinases observed on substrate gels are inhibited by tissue inhibitor of metalloproteinase 1 and 4 synthetic inhibitors BB94, GM6001, BRL29808AI and Ro31–4724. We have adapted anin vitroassay for metastatic behaviour that measures the ability of parasitized cells to cross reconstituted basement membrane, Matrigel™. Using this we demonstrated that macroschizont-infected cells are invasivein vitroand that their invasive properties can be almost eliminated by the same specific inhibitors of metalloproteinases as used in the substrate gels. This demonstrates that the metastatic behaviour of the infected cells is due in part to metalloproteinase activity and strongly suggests a role for the metalloproteinases we observed on gels. This is further supported by the fact that an attenuated vaccine line which shows much reduced metalloproteinase activity also exhibits a marked reduction in metastatic behaviour. We suggest that these metalloproteinases are virulence factors mediating some pathological features of the disease and their loss in the vaccine line could provide an explanation for attenuation.

scholarly journals Differential Biophysical Properties of Infectious Intracellular and Secreted Hepatitis C Virus Particles

2006 ◽  
Vol 80 (22) ◽  
pp. 11074-11081 ◽  
Author(s):  
Pablo Gastaminza ◽  
Sharookh B. Kapadia ◽  
Francis V. Chisari
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Buoyant Density ◽  
Infection Model ◽  
Virus Particles ◽  
Viral Egress ◽  
Viral Particles ◽  
A Cell ◽  
Infected Cells

ABSTRACT The recent development of a cell culture infection model for hepatitis C virus (HCV) permits the production of infectious particles in vitro. In this report, we demonstrate that infectious particles are present both within the infected cells and in the supernatant. Kinetic analysis indicates that intracellular particles constitute precursors of the secreted infectious virus. Ultracentrifugation analyses indicate that intracellular infectious viral particles are similar in size (∼65 to 70 nm) but different in buoyant density (∼1.15 to 1.20 g/ml) from extracellular particles (∼1.03 to 1.16 g/ml). These results indicate that infectious HCV particles are assembled intracellularly and that their biochemical composition is altered during viral egress.

scholarly journals An Antiviral Peptide Inhibitor That Is Active against Picornavirus 2A Proteinases but Not Cellular Caspases

2006 ◽  
Vol 80 (19) ◽  
pp. 9619-9627 ◽  
Author(s):  
Luiza Deszcz ◽  
Regina Cencic ◽  
Carla Sousa ◽  
Ernst Kuechler ◽  
Tim Skern
Keyword(s):  
Initiation Factor ◽  
Caspase Inhibitor ◽  
Wild Type ◽  
Caspase 9 ◽  
General Caspase Inhibitor ◽  
Infected Cells ◽  
Antiviral Peptide ◽  
Specific Inhibitors

ABSTRACT The replication of many viruses is absolutely dependent on proteolytic cleavage. Infected cells also use this biological mechanism to induce programmed cell death in response to viral infection. Specific inhibitors for both viral and cellular proteases are therefore of vital importance. We have recently shown that the general caspase inhibitor zVAD.fmk inhibits not only caspases, but also the 2A pro of human rhinoviruses (HRVs) (L. Deszcz, J. Seipelt, E. Vassilieva, A. Roetzer, and E. Kuechler, FEBS Lett. 560:51-55, 2004). Here, we describe a derivative of zVAD.fmk that inhibits HRV2 2A pro but that has no effect on caspase 9. This gain in specificity was achieved by replacing the aspartic acid of zVAD.fmk with methionine to generate zVAM.fmk. Methionine was chosen because an oligopeptide with methionine at the P1 position was a much better substrate than an oligopeptide with an alanine residue, which is found at the P1 position of the wild-type HRV2 2A pro cleavage site. zVAM.fmk inhibits the replication of HRV type 2 (HRV2), HRV14, and HRV16. In contrast to zVAD.fmk, however, zVAM.fmk did not inhibit apoptosis induced by puromycin in HeLa cells. zVAM.fmk inhibited in vitro the intermolecular cleavage of eukaryotic initiation factor 4GI (eIF4GI) by HRV2 2A pro at nanomolar concentrations. However, much higher concentrations of zVAM.fmk were required to inhibit HRV14 2A pro cleavage of eIF4GI. In contrast, intramolecular self-processing of HRV14 2A pro was much more susceptible to inhibition by zVAM.fmk than that of HRV2 2A pro , suggesting that zVAM.fmk inhibits HRV2 and HRV14 replication by targeting different reactions of the same proteinase.

scholarly journals The aminobisphosphonate pamidronate controls influenza pathogenesis by expanding a γδ T cell population in humanized mice

2011 ◽  
Vol 208 (7) ◽  
pp. 1511-1522 ◽  
Author(s):  
Wenwei Tu ◽  
Jian Zheng ◽  
Yinping Liu ◽  
Sin Fun Sia ◽  
Ming Liu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
T Cell ◽  
Viral Replication ◽  
Mononuclear Cells ◽  
Immunodeficient Mice ◽  
H5n1 Influenza ◽  
Infected Cells ◽  
Peripheral Mononuclear Cells ◽  
Vγ9vδ2 T Cells

There are few antiviral drugs for treating influenza, and the emergence of antiviral resistance has further limited the available therapeutic options. Furthermore, antivirals are not invariably effective in severe influenza, such as that caused by H5N1 viruses. Thus, there is an urgent need to develop alternative therapeutic strategies. Here, we show that human Vγ9Vδ2 T cells expanded by the aminobisphosphonate pamidronate (PAM) kill influenza virus–infected cells and inhibit viral replication in vitro. In Rag2−/−γc−/− immunodeficient mice reconstituted with human peripheral mononuclear cells (huPBMCs), PAM reduces disease severity and mortality caused by human seasonal H1N1 and avian H5N1 influenza virus, and controls the lung inflammation and viral replication. PAM has no such effects in influenza virus–infected Rag2−/−γc−/− mice reconstituted with Vγ9Vδ2 T cell–depleted huPBMCs. Our study provides proof-of-concept of a novel therapeutic strategy for treating influenza by targeting the host rather than the virus, thereby reducing the opportunity for the emergence of drug-resistant viruses. As PAM has been commonly used to treat osteoporosis and Paget’s disease, this new application of an old drug potentially offers a safe and readily available option for treating influenza.

scholarly journals Incomplete inhibition of HIV infection results in more HIV infected lymph node cells by reducing cell death

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Laurelle Jackson ◽  
Jessica Hunter ◽  
Sandile Cele ◽  
Isabella Markham Ferreira ◽  
Andrew C Young ◽  
...  
Keyword(s):  
Cell Death ◽  
Lymph Node ◽  
Hiv Transmission ◽  
Neutralizing Antibody ◽  
Force Of Infection ◽  
Infected Lymph Node ◽  
A Cell ◽  
Lymph Node Cells ◽  
Infected Cells

HIV has been reported to be cytotoxic in vitro and in lymph node infection models. Using a computational approach, we found that partial inhibition of transmissions of multiple virions per cell could lead to increased numbers of live infected cells. If the number of viral DNA copies remains above one after inhibition, then eliminating the surplus viral copies reduces cell death. Using a cell line, we observed increased numbers of live infected cells when infection was partially inhibited with the antiretroviral efavirenz or neutralizing antibody. We then used efavirenz at concentrations reported in lymph nodes to inhibit lymph node infection by partially resistant HIV mutants. We observed more live infected lymph node cells, but with fewer HIV DNA copies per cell, relative to no drug. Hence, counterintuitively, limited attenuation of HIV transmission per cell may increase live infected cell numbers in environments where the force of infection is high.

scholarly journals A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection

2008 ◽  
Vol 82 (15) ◽  
pp. 7640-7652 ◽  
Author(s):  
Andrew N. Blackford ◽  
Rachel K. Bruton ◽  
Orkide Dirlik ◽  
Grant S. Stewart ◽  
A. Malcolm R. Taylor ◽  
...  
Keyword(s):  
Dna Damage ◽  
Viral Replication ◽  
Signaling Pathways ◽  
Protein A ◽  
Interacting Protein ◽  
Dna Damage Signaling ◽  
Infected Cells ◽  
Cellular Dna ◽  
Nuclear Ribonucleoprotein

ABSTRACT E1B-55K-associated protein 5 (E1B-AP5) is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus (Ad) E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Here, we show a novel role for E1B-AP5 as a key regulator of ATR signaling pathways activated during Ad infection. E1B-AP5 is recruited to viral replication centers during infection, where it colocalizes with ATR-interacting protein (ATRIP) and the ATR substrate replication protein A 32 (RPA32). Indeed, E1B-AP5 associates with ATRIP and RPA complex component RPA70 in both uninfected and Ad-infected cells. Additionally, glutathione S-transferase pull-downs show that E1B-AP5 associates with RPA components RPA70 and RPA32 directly in vitro. E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX. In this regard, it is interesting that Ad5 and Ad12 differentially promote the phosphorylation of RPA32, Rad9, and Smc1 during infection such that Ad12 promotes a significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly promotes RPA32 phosphorylation and does not induce Rad9 phosphorylation. These data suggest that Ad5 and Ad12 have evolved different strategies to regulate DNA damage signaling pathways during infection in order to promote viral replication. Taken together, our results define a role for E1B-AP5 in ATR signaling pathways activated during infection. This might have broader implications for the regulation of ATR activity during cellular DNA replication or in response to DNA damage.

Aptamers targeting rabies virus-infected cells inhibit viral replication both in vitro and in vivo

2013 ◽  
Vol 173 (2) ◽  
pp. 398-403 ◽  
Author(s):  
Hong-Ru Liang ◽  
Quan Liu ◽  
Xue-Xing Zheng ◽  
Wei-Wei Gai ◽  
Xiang-hong Xue ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rabies Virus ◽  
Infected Cells

scholarly journals Active Escape of Orientia tsutsugamushi from Cellular Autophagy

2012 ◽  
Vol 81 (2) ◽  
pp. 552-559 ◽  
Author(s):  
Youngho Ko ◽  
Ji-Hye Choi ◽  
Na-Young Ha ◽  
Ik-Sang Kim ◽  
Nam-Hyuk Cho ◽  
...  
Keyword(s):  
Defense Mechanism ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Orientia Tsutsugamushi ◽  
Content Type ◽  
Cellular Autophagy ◽  
A Cell ◽  
Infected Cells ◽  
Obligate Intracellular Pathogen

ABSTRACTOrientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen. After entry into host cells, the bacterium rapidly escapes from the endosomal pathway and replicates in the cytosol of eukaryotic host cells. Here we show thatO. tsutsugamushiinfection efficiently promotes cellular autophagy, a cell-autonomous defense mechanism of innate immunity. However, most of the internalized bacteria barely colocalized with the induced autophagosomes, even when stimulated with rapamycin, a chemical inducer of autophagy. Treatment of infected cells with tetracycline suppressed bacterial evasion from autophagy and facilitatedO. tsutsugamushitargeting to autophagosomes, suggesting that the intracellular pathogen may be equipped with a bacterial factor or factors that block autophagic recognition. Finally, we also found that chemical modulators of cellular autophagy or genetic knockout of theatg3gene does not significantly affect the intracellular growth ofO. tsutsugamushiin vitro. These results suggest thatO. tsutsugamushihas evolved to block autophagic microbicidal defense by evading autophagic recognition even though it activates the autophagy pathway during the early phase of infection.

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