scholarly journals Multiple Pathways Involved in Porcine Parvovirus Cellular Entry and Trafficking toward the Nucleus

2010 ◽  
Vol 84 (15) ◽  
pp. 7782-7792 ◽  
Author(s):  
Maude Boisvert ◽  
Sandra Fernandes ◽  
Peter Tijssen
Keyword(s):  
Sialic Acids ◽  
Porcine Parvovirus ◽  
Actin Network ◽  
Cellular Mechanisms ◽  
Microtubule Network ◽  
Late Endosomes ◽  
Chemical Inhibitors ◽  
Endosomal Acidification ◽  
Infected Cells ◽  
Gain Access

ABSTRACT Porcine parvovirus (PPV) is a major cause of reproductive failure in swine. The mechanisms implicated in the first steps of infection that lead to the delivery of the PPV genome to the nucleus are poorly understood. In the present work, a panel of chemical inhibitors was used to dissect the cellular mechanisms involved in establishing a PPV infection. The results demonstrated that following binding to sialic acids on cell surface glycoproteins, the virus used both clathrin-mediated endocytosis and macropinocytosis pathways to gain access into cells. Virus obtained from infected cells was present either as isolated particles or as aggregates, and these two forms could be separated by low-speed centrifugation. Isolated and purified particles strongly preferred entry by clathrin-mediated endocytosis, whereas aggregates clearly favored macropinocytosis. Subsequent endosomal acidification and traffic to the late endosomes were also shown to be essential for infection. The microtubule network was found to be important during the first 10 h of infection, whereas an intact actin network was required for almost the whole viral cycle. Proteasome processing was found to be essential, and capsid proteins were ubiquitinated relatively early during infection. Taken together, these results provided new insights into the first steps of PPV infection, including the use of alternative entry pathways, unique among members of this viral family.

scholarly journals Glycoprotein B switches conformation during murid herpesvirus 4 entry

2008 ◽  
Vol 89 (6) ◽  
pp. 1352-1363 ◽  
Author(s):  
Laurent Gillet ◽  
Susanna Colaco ◽  
Philip G. Stevenson
Keyword(s):  
Neutralizing Antibodies ◽  
Structural Changes ◽  
Glycoprotein B ◽  
Cell Binding ◽  
Glycoprotein G ◽  
Late Endosomes ◽  
Endosomal Acidification ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Herpesvirus 4

Herpesviruses are ancient pathogens that infect all vertebrates. The most conserved component of their entry machinery is glycoprotein B (gB), yet how gB functions is unclear. A striking feature of the murid herpesvirus 4 (MuHV-4) gB is its resistance to neutralization. Here, we show by direct visualization of infected cells that the MuHV-4 gB changes its conformation between extracellular virions and those in late endosomes, where capsids are released. Specifically, epitopes on its N-terminal cell-binding domain become inaccessible, whilst non-N-terminal epitopes are revealed, consistent with structural changes reported for the vesicular stomatitis virus glycoprotein G. Inhibitors of endosomal acidification blocked the gB conformation switch. They also blocked capsid release and the establishment of infection, implying that the gB switch is a key step in entry. Neutralizing antibodies could only partially inhibit the switch. Their need to engage a less vulnerable, upstream form of gB, because its fusion form is revealed only in endosomes, helps to explain why gB-directed MuHV-4 neutralization is so difficult.

scholarly journals Overexpression of quality control proteins reduces prion conversion in prion-infected cells

2018 ◽  
Vol 293 (41) ◽  
pp. 16069-16082 ◽  
Author(s):  
Simrika Thapa ◽  
Basant Abdulrahman ◽  
Dalia H. Abdelaziz ◽  
Li Lu ◽  
Manel Ben Aissa ◽  
...  
Keyword(s):  
Quality Control ◽  
De Novo ◽  
Prion Diseases ◽  
Neuroblastoma Cells ◽  
Cellular Prion Protein ◽  
Control Mechanisms ◽  
Cellular Mechanisms ◽  
Prion Propagation ◽  
Infected Cells

Prion diseases are fatal infectious neurodegenerative disorders in humans and other animals and are caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc. These diseases have the potential to transmit within or between species, including zoonotic transmission to humans. Elucidating the molecular and cellular mechanisms underlying prion propagation and transmission is therefore critical for developing molecular strategies for disease intervention. We have shown previously that impaired quality control mechanisms directly influence prion propagation. In this study, we manipulated cellular quality control pathways in vitro by stably and transiently overexpressing selected quality control folding (ERp57) and cargo (VIP36) proteins and investigated the effects of this overexpression on prion propagation. We found that ERp57 or VIP36 overexpression in persistently prion-infected neuroblastoma cells significantly reduces the amount of PrPSc in immunoblots and prion-seeding activity in the real-time quaking-induced conversion (RT-QuIC) assay. Using different cell lines infected with various prion strains confirmed that this effect is not cell type– or prion strain–specific. Moreover, de novo prion infection revealed that the overexpression significantly reduced newly formed PrPSc in acutely infected cells. ERp57-overexpressing cells significantly overcame endoplasmic reticulum stress, as revealed by expression of lower levels of the stress markers BiP and CHOP, accompanied by a decrease in PrP aggregates. Furthermore, application of ERp57-expressing lentiviruses prolonged the survival of prion-infected mice. Taken together, improved cellular quality control via ERp57 or VIP36 overexpression impairs prion propagation and could be utilized as a potential therapeutic strategy.

scholarly journals Targeting the sAC-Dependent cAMP Pool to Prevent SARS-Cov-2 Infection

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1962
Author(s):  
Muhammad Aslam ◽  
Yury Ladilov
Keyword(s):  
Endocytic Pathway ◽  
Host Cells ◽  
The Novel ◽  
Cellular Mechanisms ◽  
Late Endosomes ◽  
Proven Efficacy ◽  
Potential Impact ◽  
Potential Strategy ◽  
Novel Coronavirus ◽  
High Level

An outbreak of the novel coronavirus (CoV) SARS-CoV-2, the causative agent of COVID-19 respiratory disease, infected millions of people since the end of 2019, led to high-level morbidity and mortality and caused worldwide social and economic disruption. There are currently no antiviral drugs available with proven efficacy or vaccines for its prevention. An understanding of the underlying cellular mechanisms involved in virus replication is essential for repurposing the existing drugs and/or the discovery of new ones. Endocytosis is the important mechanism of entry of CoVs into host cells. Endosomal maturation followed by the fusion with lysosomes are crucial events in endocytosis. Late endosomes and lysosomes are characterized by their acidic pH, which is generated by a proton transporter V-ATPase and required for virus entry via endocytic pathway. The cytoplasmic cAMP pool produced by soluble adenylyl cyclase (sAC) promotes V-ATPase recruitment to endosomes/lysosomes and thus their acidification. In this review, we discuss targeting the sAC-specific cAMP pool as a potential strategy to impair the endocytic entry of the SARS-CoV-2 into the host cell. Furthermore, we consider the potential impact of sAC inhibition on CoV-induced disease via modulation of autophagy and apoptosis.

scholarly journals Hepatitis C Virus Entry Depends on Clathrin-Mediated Endocytosis

2006 ◽  
Vol 80 (14) ◽  
pp. 6964-6972 ◽  
Author(s):  
Emmanuelle Blanchard ◽  
Sandrine Belouzard ◽  
Lucie Goueslain ◽  
Takaji Wakita ◽  
Jean Dubuisson ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Immunoblot Analysis ◽  
Target Cells ◽  
Cellular Mechanisms ◽  
Pit Formation ◽  
Infected Cells ◽  
Interfering Rna ◽  
Hcvcc Infection

ABSTRACT Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.

scholarly journals Photoswitchable microtubule stabilisers optically control tubulin cytoskeleton structure and function

2019 ◽  
Author(s):  
Adrian Müller-Deku ◽  
Kristina Loy ◽  
Yvonne Kraus ◽  
Constanze Heise ◽  
Rebekkah Bingham ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Biological Response ◽  
Structure And Function ◽  
Live Cells ◽  
Microtubule Cytoskeleton ◽  
Microtubule Network ◽  
Chemical Inhibitors ◽  
Functional Control ◽  
And Function ◽  
Dependent Processes

ABSTRACTSmall molecule inhibitors provide a versatile method for studies in microtubule cytoskeleton research, since tubulin is not readily amenable to functional control using genetics. However, traditional chemical inhibitors do not allow spatiotemporally precise applications on the length and time scales appropriate for selectively modulating microtubule-dependent processes. We have synthesised a panel of taxane-based light-responsive microtubule stabilisers, whose tubulin hyperpolymerisation activity can be induced by photoisomerisation to their thermodynamically metastable state. These reagents can be isomerised in live cells, optically controlling microtubule network integrity, cell cycle repartition, and cell survival, and offering biological response on the timescale of seconds and spatial precision to the level of individual cells. These azobenzene-based microtubule stabilisers offer the possibility of noninvasive, highly spatiotemporally precise modulation of the microtubule cytoskeleton in live cells, and can prove powerful reagents for studies of intracellular transport, cell motility, and neurodegeneration.Abstract Figure

scholarly journals Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Shashank Shekhar ◽  
Johnson Chung ◽  
Jane Kondev ◽  
Jeff Gelles ◽  
Bruce L. Goode
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Binding Protein ◽  
Actin Dynamics ◽  
Actin Network ◽  
Cellular Mechanisms ◽  
Actin Networks ◽  
Binding Event

AbstractCellular actin networks can be rapidly disassembled and remodeled in a few seconds, yet in vitro actin filaments depolymerize slowly over minutes. The cellular mechanisms enabling actin to depolymerize this fast have so far remained obscure. Using microfluidics-assisted TIRF, we show that Cyclase-associated protein (CAP) and Cofilin synergize to processively depolymerize actin filament pointed ends at a rate 330-fold faster than spontaneous depolymerization. Single molecule imaging further reveals that hexameric CAP molecules interact with the pointed ends of Cofilin-decorated filaments for several seconds at a time, removing approximately 100 actin subunits per binding event. These findings establish a paradigm, in which a filament end-binding protein and a side-binding protein work in concert to control actin dynamics, and help explain how rapid actin network depolymerization is achieved in cells.

scholarly journals Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Adrian Müller-Deku ◽  
Joyce C. M. Meiring ◽  
Kristina Loy ◽  
Yvonne Kraus ◽  
Constanze Heise ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Biological Response ◽  
Living Cells ◽  
Optical Control ◽  
Microtubule Cytoskeleton ◽  
Microtubule Network ◽  
Cellular Processes ◽  
Non Invasive ◽  
Chemical Inhibitors ◽  
Neuronal Physiology

Abstract Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.

scholarly journals Autophagy Promotes Porcine Parvovirus Replication and Induces Non-Apoptotic Cell Death in Porcine Placental Trophoblasts

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 15
Author(s):  
Xiujuan Zhang ◽  
Yingli Xiong ◽  
Jie Zhang ◽  
Ting Shao ◽  
Songbiao Chen ◽  
...  
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Porcine Parvovirus ◽  
Caspase Inhibitor ◽  
Autophagy Flux ◽  
Autophagy Induction ◽  
Cellular Apoptosis ◽  
Infected Cells ◽  
Later Phase

Autophagy plays important roles in the infection and pathogenesis of many viruses, yet the regulatory roles of autophagy in the process of porcine parvovirus (PPV) infection remain unclear. Herein, we show that PPV infection induces autophagy in porcine placental trophoblasts (PTCs). Induction of autophagy by rapamycin (RAPA) inhibited the occurrence of apoptotic cell death, yet promoted viral replication in PPV-infected cells; inhibition of autophagy by 3-MA or ATG5 knockdown increased cellular apoptosis and reduced PPV replication. Interestingly, we found that in the presence of caspase-inhibitor zVAD-fmk, PPV induces non-apoptotic cell death that was characterized by lysosomal damage and associated with autophagy. Induction of complete autophagy flux by RAPA markedly promoted PPV replication compared with incomplete autophagy induced by RAPA plus bafilomycin (RAPA/BAF) in the early phase of PPV infection (24 h.p.i.). Meanwhile, induction of complete autophagy with RAPA increased lysosomal damage and non-apoptotic cell death in the later phase of PPV infection. Therefore, our data suggest that autophagy can enhance PPV replication and promote the occurrence of lysosomal-damage-associated non-apoptotic cell death in PPV-infected porcine placental trophoblasts.

scholarly journals Antiviral Role of IFITM Proteins in Classical Swine Fever Virus Infection

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 126 ◽  
Author(s):  
Cheng Li ◽  
Hongqing Zheng ◽  
Yifan Wang ◽  
Wang Dong ◽  
Yaru Liu ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Mammalian Cells ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Infected Cells ◽  
The Relationship ◽  
Antiviral Role

The proteins IFITM1, IFITM2, and IFITM3 are host effectors against a broad range of RNA viruses whose roles in classical swine fever virus (CSFV) infection had not yet been reported. We investigated the effect of these proteins on CSFV replication in mammalian cells. The proteins were overexpressed and silenced using lentiviruses. Confocal microscopy was used to determine the distribution of these proteins in the cells, and immunofluorescence colocalization analysis was used to evaluate the relationship between IFITMs and the CSFV endosomal pathway, including early endosomes, late endosomes, and lysosomes. IFITM1, IFITM2, or IFITM3 overexpression significantly inhibited CSFV replication, whereas protein knockdown enhanced CSFV replication. In porcine alveolar macrophages (PAMs), IFITM1 was mainly located at the cell surface, whereas IFITM2 and IFITM3 were mainly located in the cytoplasm. Following CSFV infection, the distribution of IFITM1 changed. IFITM1, IFITM2, and IFITM3 colocalization with Lamp1, IFITM2 with Rab5 and Rab7, and IFITM3 with Rab7 were observed in CSFV-infected cells. Collectively, these results provide insights into the possible mechanisms associated with the anti-CSFV action of the IFITM family.

scholarly journals Herpesvirus Capsid Association with the Nuclear Pore Complex and Viral DNA Release Involve the Nucleoporin CAN/Nup214 and the Capsid Protein pUL25

2009 ◽  
Vol 83 (13) ◽  
pp. 6610-6623 ◽  
Author(s):  
David Pasdeloup ◽  
Danielle Blondel ◽  
Anabela L. Isidro ◽  
Frazer J. Rixon
Keyword(s):  
Capsid Protein ◽  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
Cellular Interactions ◽  
Viral Dna ◽  
Microtubule Network ◽  
Infected Cells ◽  
Dna Release ◽  
Simplex Virus

ABSTRACT After penetrating the host cell, the herpesvirus capsid is transported to the nucleus along the microtubule network and docks to the nuclear pore complex before releasing the viral DNA into the nucleus. The viral and cellular interactions involved in the docking process are poorly characterized. However, the minor capsid protein pUL25 has recently been reported to be involved in viral DNA uncoating. Here we show that herpes simplex virus type 1 (HSV-1) capsids interact with the nucleoporin CAN/Nup214 in infected cells and that RNA silencing of CAN/Nup214 delays the onset of viral DNA replication in the nucleus. We also show that pUL25 interacts with CAN/Nup214 and another nucleoporin, hCG1, and binds to the pUL36 and pUL6 proteins, two other components of the herpesvirus particle that are known to be important for the initiation of infection and viral DNA release. These results identify CAN/Nup214 as being a nuclear receptor for the herpesvirus capsid and pUL25 as being an interface between incoming capsids and the nuclear pore complex and as being a triggering element for viral DNA release into the nucleus.

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