Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

ABSTRACTSevere fever with thrombocytopenia syndrome (SFTS) virus is a newly recognized member of the genusPhlebovirusin the familyBunyaviridae. The virus was isolated from patients presenting with hemorrhagic manifestations and an initial case fatality rate of 12 to 30% was reported. Due to the recent emergence of this pathogen, there is limited knowledge on the molecular virology of SFTS virus. Recently, we reported that the SFTS virus NSs protein inhibited the activation of the beta interferon (IFN-β) promoter. Furthermore, we also found that SFTS virus NSs relocalizes key components of the IFN response into NSs-induced cytoplasmic structures. Due to the important role these structures play during SFTS virus replication, we conducted live cell imaging studies to gain further insight into the role and trafficking of these cytoplasmic structures during virus infection. We found that some of the SFTS virus NSs-positive cytoplasmic structures were secreted to the extracellular space and endocytosed by neighboring cells. We also found that these secreted structures isolated from NSs-expressing cells and SFTS virus-infected cells were positive for the viral protein NSs and the host protein CD63, a protein associated with extracellular vesicles. Electron microscopy studies also revealed that the isolated CD63-immunoprecipitated extracellular vesicles produced during SFTS virus infection contained virions. The virions harbored within these structures were efficiently delivered to uninfected cells and were able to sustain SFTS virus replication. Altogether, these results suggest that SFTS virus exploits extracellular vesicles to mediate virus receptor-independent transmission to host cells and open the avenue for novel therapeutic strategies against SFTS virus and related pathogens.IMPORTANCESFTS virus is novel bunyavirus associated with hemorrhagic fever illness. Currently, limited information is available about SFTS virus. In the present study, we demonstrated that extracellular vesicles produced by SFTS virus-infected cells harbor infectious virions. We sought to determine whether these “infectious” extracellular vesicles can mediate transmission of the virus and confirmed that the SFTS virions were efficiently transported by these secreted structures into uninfected cells and were able to sustain efficient replication of SFTS virus. These results have significant impact on our understanding of how the novel tick-borne phleboviruses hijack cellular machineries to establish infection and point toward a novel mechanism for virus replication among arthropod-borne viruses.

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Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

International Journal of Molecular Sciences ◽

10.3390/ijms22094823 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4823

Author(s):

María Fernanda González ◽

Paula Díaz ◽

Alejandra Sandoval-Bórquez ◽

Daniela Herrera ◽

Andrew F. G. Quest

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Outer Membrane ◽

Extracellular Vesicles ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Gastric Epithelium ◽

Infected Cells ◽

H Pylori

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.

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Dengue virus sustains viability of infected cells by counteracting apoptosis-mediated DNA breakage

10.1101/2020.06.19.162479 ◽

2020 ◽

Author(s):

Himadri Nath ◽

Keya Basu ◽

Abhishek De ◽

Subhajit Biswas

Keyword(s):

Dengue Virus ◽

Virus Replication ◽

Host Cells ◽

Severe Dengue ◽

Dna Breaks ◽

Dna Breakage ◽

Major Player ◽

Infected Cells ◽

Dna Break ◽

Cellular Dna

AbstractDengue is the most important arboviral disease inflicting mankind. This mosquito-borne Flavivirus causes mild to severe dengue fever which in some cases leads to life-threatening conditions namely, dengue haemorrhagic fever and dengue shock syndrome. Annual infection is estimated at 390 million globally with 96 million manifesting clinically. So, ≥80% infections are asymptomatic and self-limiting. Dengue virus (DV) non-structural protein 1 (NS1) is a proven virotoxin abundantly present in the victim’s blood. We found that DV-infected or only NS1-expressing cells both can induce Cleaved Caspase3, due to antiviral response of host cells. NS1-transfected cells also showed nuclear damage and significant levels of DNA breaks suggestive of ensuing apoptosis. So, it was established that NS1 alone is capable of causing apoptosis. Surprisingly, despite secreting similar amount of soluble NS1, the DV-infected cells showed intact nuclear morphology and background levels of DNA nicks. These observations suggested that DV downregulates apoptosis of infected cells, which is a viral strategy against host defence. Furthermore, DV-infected cells counteracted Camptothecin-induced apoptotic DNA break. DV-infection was also found to keep the infected cells metabolically more active than only NS1 expressing cells. So, DV bypasses cellular defence against virus i.e. apoptosis by counteracting cellular DNA break and keeps the infected cells metabolically active to support virus replication for longer period which eventually results in high virus titer in circulation. Our findings reveal another level of intricacy involving dengue virus-host interactions and perhaps explain why ≥80% DV infections are asymptomatic/self-limiting despite the presence of NS1 virotoxin in infected cells.Author SummaryNS1, a virotoxin, abundantly present in Dengue patients blood, is a major player behind disease patho-biogenesis including plasma leakage. Despite the presence of NS1 in blood, Dengue is asymptomatic and self-limiting in more than 80% dengue virus (DV) infected people. We investigated this observation and found that plasmid-mediated NS1 expression and secretion in cells are sufficient to cause programmed cell death (apoptosis) and associated cellular DNA breakage. However, cells infected with dengue virus and secreting equivalent amounts of NS1 didn’t exhibit apoptotic DNA breakage. Consequently, DV-infected cells showed better survival than cells in which only NS1 was transiently expressed by transfection with expression plasmid. We also found that DV can even prevent chemical induced apoptotic DNA damage in infected host cells. So, DV bypasses host antiviral defence i.e. apoptosis by counteracting cellular DNA breakages and keeps the infected cells metabolically active to prolong virus replication.

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Safflomin A inhibits neuraminidase activity and influenza virus replication

RSC Advances ◽

10.1039/c5ra17336a ◽

2015 ◽

Vol 5 (114) ◽

pp. 94053-94066 ◽

Cited By ~ 10

Author(s):

Miao Yu ◽

Ye Wang ◽

Li Tian ◽

Yanyan Wang ◽

Xizhu Wang ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Virus Replication ◽

Neuraminidase Activity ◽

Viral Release ◽

Infected Cells ◽

Influenza Virus Replication

Neuraminidase (NA) is a glycoprotein on the surface of the influenza virus that plays an important role in the early processes of virus infection and viral release from the infected cells.

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Targeting cap-dependent translation to inhibit Chikungunya virus replication: selectivity of p38 MAPK inhibitors to virus-infected cells due to autophagy-mediated down regulation of phospho-ERK

Journal of General Virology ◽

10.1099/jgv.0.001629 ◽

2021 ◽

Vol 102 (7) ◽

Author(s):

Prashant Mudaliar ◽

Parvanendhu Pradeep ◽

Rachy Abraham ◽

Easwaran Sreekumar

Keyword(s):

Protein Expression ◽

P38 Mapk ◽

Virus Replication ◽

Chikungunya Virus ◽

Host Cells ◽

Hek293 Cells ◽

Viral Titre ◽

Mtor Inhibition ◽

Infected Cells ◽

Eif2 Alpha

The 5′ capped, message-sense RNA genome of Chikungunya virus (CHIKV) utilizes the host cell machinery for translation. Translation is regulated by eIF2 alpha at the initiation phase and by eIF4F at cap recognition. Translational suppression by eIF2 alpha phosphorylation occurs as an early event in many alphavirus infections. We observe that in CHIKV-infected HEK293 cells, this occurs as a late event, by which time the viral replication has reached an exponential phase, implying its minimal role in virus restriction. The regulation by eIF4F is mediated through the PI3K-Akt-mTOR, p38 MAPK and RAS-RAF-MEK-ERK pathways. A kinetic analysis revealed that CHIKV infection did not modulate AKT phosphorylation, but caused a significant reduction in p38 MAPK phosphorylation. It caused degradation of phospho-ERK 1/2 by increased autophagy, leaving the PI3K-Akt-mTOR and p38 MAPK pathways for pharmacological targeting. mTOR inhibition resulted in moderate reduction in viral titre, but had no effect on CHIKV E2 protein expression, indicating a minimal role of the mTOR complex in virus replication. Inhibition of p38 MAPK using SB202190 caused a significant reduction in viral titre and CHIKV E2 and nsP3 protein expression. Furthermore, inhibiting the two pathways together did not offer any synergism, indicating that inhibiting the p38 MAPK pathway alone is sufficient to cause restriction of CHIKV replication. Meanwhile, in uninfected cells the fully functional RAS-RAF-MEK-ERK pathway can circumvent the effect of p38 MAPK inhibition on cap-dependent translation. Thus, our results show that host-directed antiviral strategies targeting cellular p38 MAPK are worth exploring against Chikungunya as they could be selective against CHIKV-infected cells with minimal effects on uninfected host cells.

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Loss of IKK subunits limits NF-κB signaling in reovirus infected cells

10.1101/843680 ◽

2019 ◽

Author(s):

Andrew J. McNamara ◽

Pranav Danthi

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Virus Replication ◽

Nuclear Translocation ◽

Innate Immune ◽

Host Cells ◽

Reovirus Infection ◽

Cellular Environment ◽

Ikk Complex ◽

Infected Cells

ABSTRACTViruses commonly antagonize innate immune pathways that are primarily driven by Nuclear Factor-κB (NF-κB), Interferon Regulatory Factor (IRF) and Signal Transducer and Activator of Transcription (STAT) family of transcription factors. Such a strategy allows viruses to evade immune surveillance and maximize their replication. Using an unbiased RNA-seq based approach to measure gene expression induced by transfected viral genomic RNA (vgRNA) and reovirus infection, we discovered that mammalian reovirus inhibits host cell innate immune signaling. We found that while vgRNA and reovirus infection both induce a similar IRF dependent gene expression program, gene expression driven by the NF-κB family of transcription factors is lower in infected cells. Potent agonists of NF-κB, such as Tumor Necrosis Factor alpha (TNFα) and vgRNA, failed to induce NF-κB dependent gene expression in infected cells. We demonstrate that NF-κB signaling is blocked due to loss of critical members of the Inhibitor of KappaB Kinase (IKK) complex, NF-κB Essential MOdifier (NEMO) and IKKβ. The loss of the IKK complex components prevents nuclear translocation and phosphorylation of NF-κB, thereby preventing gene expression. Our studies demonstrate that reovirus infection selectively blocks NF-κB, likely to counteract its antiviral effects and promote efficient viral replication.IMPORTANCEHost cells mount a response to curb virus replication in infected cells and prevent infection of neighboring, as yet uninfected cells. The NF-κB family of proteins is important for the cell to mediate this response. In this study, we show that in cells infected with mammalian reovirus, NF-κB is inactive. Further, we demonstrate that NF-κB is rendered inactive because virus infection results in reduced levels of upstream intermediaries (called IKKs) that are needed for NF-κB function. Based on previous evidence that active NF-κB limits reovirus infection, we conclude that inactivating NF-κB is a viral strategy to produce a cellular environment that is favorable for virus replication.

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The secreted protein kinase CstK from Coxiella burnetii influences vacuole development and interacts with the GTPase-activating host protein TBC1D5

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010112 ◽

2020 ◽

Vol 295 (21) ◽

pp. 7391-7403 ◽

Cited By ~ 2

Author(s):

Eric Martinez ◽

Sylvaine Huc-Brandt ◽

Solène Brelle ◽

Julie Allombert ◽

Franck Cantet ◽

...

Keyword(s):

Protein Kinase ◽

Coxiella Burnetii ◽

Membrane Trafficking ◽

Q Fever ◽

Parasitophorous Vacuole ◽

Host Protein ◽

Host Cells ◽

Intracellular Replication ◽

Gtpase Activating Protein ◽

Infected Cells

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system–mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host–pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro. This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein–protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.

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Roles of Nonstructural Protein nsP2 and Alpha/Beta Interferons in Determining the Outcome of Sindbis Virus Infection

Journal of Virology ◽

10.1128/jvi.76.22.11254-11264.2002 ◽

2002 ◽

Vol 76 (22) ◽

pp. 11254-11264 ◽

Cited By ~ 160

Author(s):

Elena I. Frolova ◽

Rafik Z. Fayzulin ◽

Susan H. Cook ◽

Diane E. Griffin ◽

Charles M. Rice ◽

...

Keyword(s):

Virus Infection ◽

Host Cell ◽

Virus Replication ◽

Mammalian Cells ◽

Sindbis Virus ◽

Nonstructural Protein ◽

Virus Propagation ◽

Virus Growth ◽

Infected Cells ◽

Alpha Beta

ABSTRACT Alphaviruses productively infect a variety of vertebrate and insect cell lines. In vertebrate cells, Sindbis virus redirects cellular processes to meet the needs of virus propagation. At the same time, cells respond to virus replication by downregulating virus growth and preventing dissemination of the infection. The balance between these two mechanisms determines the outcome of infection at the cellular and organismal levels. In this report, we demonstrate that a viral nonstructural protein, nsP2, is a significant regulator of Sindbis virus-host cell interactions. This protein not only is a component of the replicative enzyme complex required for replication and transcription of viral RNAs but also plays a role in suppressing the antiviral response in Sindbis virus-infected cells. nsP2 most likely acts by decreasing interferon (IFN) production and minimizing virus visibility. Infection of murine cells with Sindbis virus expressing a mutant nsP2 leads to higher levels of IFN secretion and the activation of 170 cellular genes that are induced by IFN and/or virus replication. Secreted IFN protects naive cells against Sindbis virus infection and also stops viral replication in productively infected cells. Mutations in nsP2 can also attenuate Sindbis virus cytopathogenicity. Such mutants can persist in mammalian cells with defects in the alpha/beta IFN (IFN-α/β) system or when IFN activity is neutralized by anti-IFN-α/β antibodies. These findings provide new insight into the alphavirus-host cell interaction and have implications for the development of improved alphavirus expression systems with better antigen-presenting potential.

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Inhibition of Apoptosis in Chlamydia-infected Cells: Blockade of Mitochondrial Cytochrome c Release and Caspase Activation

Journal of Experimental Medicine ◽

10.1084/jem.187.4.487 ◽

1998 ◽

Vol 187 (4) ◽

pp. 487-496 ◽

Cited By ~ 406

Author(s):

Tao Fan ◽

Hang Lu ◽

He Hu ◽

Lianfa Shi ◽

Grant A. McClarty ◽

...

Keyword(s):

Cytochrome C ◽

Host Cell ◽

Kinase Inhibitor ◽

Host Protein ◽

Host Cells ◽

Mitochondrial Cytochrome ◽

Cytochrome C Release ◽

Infected Cells ◽

Apoptotic Pathways ◽

Mitochondrial Cytochrome C

We report that chlamydiae, which are obligate intracellular bacterial pathogens, possess a novel antiapoptotic mechanism. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by a wide spectrum of proapoptotic stimuli including the kinase inhibitor staurosporine, the DNA-damaging agent etoposide, and several immunological apoptosis-inducing molecules such as tumor necrosis factor-α, Fas antibody, and granzyme B/perforin. The antiapoptotic activity was dependent on chlamydial but not host protein synthesis. These observations suggest that chlamydia may encode factors that interrupt many different host cell apoptotic pathways. We found that activation of the downstream caspase 3 and cleavage of poly (ADP-ribose) polymerase were inhibited in chlamydia-infected cells. Mitochondrial cytochrome c release into the cytosol induced by proapoptotic factors was also prevented by chlamydial infection. These observations suggest that chlamydial proteins may interrupt diverse apoptotic pathways by blocking mitochondrial cytochrome c release, a central step proposed to convert the upstream private pathways into an effector apoptotic pathway for amplification of downstream caspases. Thus, we have identified a chlamydial antiapoptosis mechanism(s) that will help define chlamydial pathogenesis and may also provide information about the central mechanisms regulating host cell apoptosis.

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Influenza Virus Down-Modulates G6PD Expression and Activity to Induce Oxidative Stress and Promote Its Replication

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.804976 ◽

2022 ◽

Vol 11 ◽

Author(s):

Marta De Angelis ◽

Donatella Amatore ◽

Paola Checconi ◽

Alessandra Zevini ◽

Alessandra Fraternale ◽

...

Keyword(s):

Oxidative Stress ◽

Influenza Virus ◽

Virus Infection ◽

Viral Infection ◽

Virus Replication ◽

Influenza Virus Infection ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Host Cells ◽

Related Factor ◽

Expression And Activity

Influenza virus infection induces oxidative stress in host cells by decreasing the intracellular content of glutathione (GSH) and increasing reactive oxygen species (ROS) level. Glucose-6-phosphate dehydrogenase (G6PD) is responsible for the production of reducing equivalents of nicotinamide adenine dinucleotide phosphate (NADPH) that is used to regenerate the reduced form of GSH, thus restoring redox homeostasis. Cells deficient in G6PD display elevated levels of ROS and an increased susceptibility to viral infection, although the consequences of G6PD modulation during viral infection remain to be elucidated. In this study, we demonstrated that influenza virus infection decreases G6PD expression and activity, resulting in an increase in oxidative stress and virus replication. Moreover, the down regulation of G6PD correlated with a decrease in the expression of nuclear factor erythroid 2-related factor 2 (NRF2), a key transcription factor that regulates the expression of the antioxidant response gene network. Also down-regulated in influenza virus infected cells was sirtuin 2 (SIRT2), a NADPH-dependent deacetylase involved in the regulation of G6PD activity. Acetylation of G6PD increased during influenza virus infection in a manner that was strictly dependent on SIRT2 expression. Furthermore, the use of a pharmacological activator of SIRT2 rescued GSH production and NRF2 expression, leading to decreased influenza virus replication. Overall, these data identify a novel strategy used by influenza virus to induce oxidative stress and to favor its replication in host cells. These observations furthermore suggest that manipulation of metabolic and oxidative stress pathways could define new therapeutic strategies to interfere with influenza virus infection.

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Leishmania-infected macrophages release extracellular vesicles that can promote lesion development

Life Science Alliance ◽

10.26508/lsa.202000742 ◽

2020 ◽

Vol 3 (12) ◽

pp. e202000742

Author(s):

Anna Gioseffi ◽

Tim Hamerly ◽

Kha Van ◽

Naixin Zhang ◽

Rhoel R Dinglasan ◽

...

Keyword(s):

Endothelial Cells ◽

Extracellular Vesicles ◽

Leishmania Donovani ◽

Protein Profile ◽

Tube Formation ◽

Host Cells ◽

Western Blots ◽

Host Cell Proteins ◽

Transgenic Parasite ◽

Infected Cells

Leishmania donovani infection of macrophages results in quantitative and qualitative changes in the protein profile of extracellular vesicles (EVs) released by the infected host cells. We confirmed mass spectrometry results orthogonally by performing Western blots for several Leishmania-infected macrophage-enriched EVs (LieEVs) molecules. Several host cell proteins in LieEVs have been implicated in promoting vascular changes in other systems. We also identified 59 parasite-derived proteins in LieEVs, including a putative L. donovani homolog of mammalian vasohibins (LdVash), which in mammals promotes angiogenesis. We developed a transgenic parasite that expressed an endogenously tagged LdVash/mNeonGreen (mNG) and confirmed that LdVash/mNG is indeed expressed in infected macrophages and in LieEVs. We further observed that LieEVs induce endothelial cells to release angiogenesis promoting mediators including IL-8, G-CSF/CSF-3, and VEGF-A. In addition, LieEVs induce epithelial cell migration and tube formation by endothelial cells in surrogate angiogenesis assays. Taken together, these studies show that Leishmania infection alters the composition of EVs from infected cells and suggest that LieEVs may play a role in the promotion of vascularization of Leishmania infections.

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