scholarly journals Lyn kinase regulates egress of flaviviruses in autophagosome-derived organelles

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ming Yuan Li ◽  
Trupti Shivaprasad Naik ◽  
Lewis Yu Lam Siu ◽  
Oreste Acuto ◽  
Eric Spooner ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Rab Gtpases ◽  
Wild Type ◽  
Virus Particles ◽  
Cellular Processes ◽  
Lyn Kinase ◽  
Exit Route ◽  
Viral Egress ◽  
Infected Cells ◽  
Circulating Antibodies

Abstract Among the various host cellular processes that are hijacked by flaviviruses, few mechanisms have been described with regard to viral egress. Here we investigate how flaviviruses exploit Src family kinases (SFKs) for exit from infected cells. We identify Lyn as a critical component for secretion of Dengue and Zika infectious particles and their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs, Lyn in particular, block virus secretion. Lyn−/− cells are impaired in virus release and are rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We establish that virus particles are secreted in two distinct populations – one as free virions and the other enclosed within membranes. Lyn is critical for the latter, which consists of proteolytically processed, infectious virus progenies within autophagosome-derived vesicles. This process depends on Ulk1, Rab GTPases and SNARE complexes implicated in secretory but not degradative autophagy and occur with significantly faster kinetics than the conventional secretory pathway. Our study reveals a previously undiscovered Lyn-dependent exit route of flaviviruses in LC3+ secretory organelles that enables them to evade circulating antibodies and might affect tissue tropism.

scholarly journals Activation of Src-family kinases orchestrate secretion of flaviviruses by targeting mature progeny virions to secretory autophagosomes

2020 ◽  
Author(s):  
Ming Yuan Li ◽  
Trupti Shivaprasad Naik ◽  
Lewis Yu Lam Siu ◽  
Oreste Acuto ◽  
Eric Spooner ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Src Family Kinases ◽  
Tissue Tropism ◽  
Membrane Association ◽  
Wild Type ◽  
Virus Transport ◽  
Virus Particles ◽  
Cellular Processes ◽  
Infected Cells ◽  
Circulating Antibodies

SummaryAmong the various host cellular processes that are hijacked by flaviviruses, very few mechanisms have been described with regard to viral secretion. Here we investigated how flaviviruses exploit the Src family kinases (SFKs) for exit from infected cells. We isolated three members of the SFK family – Src, Fyn and Lyn – that were specifically activated during secretion of Dengue and Zika or their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs blocked virus secretion, most significantly upon Lyn-deficiency. Lyn-/- cells were severely impaired in virus release, and were rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We further established that Lyn, via its palmitoylation-dependent membrane association, triggered post-Golgi virus transport in specialised Rab11 and Transferrin receptor positive organelles resembling secretory autophagosomes, and distinct from conventional exocytic vesicles. In the absence of Lyn activity or its aberrant membrane association, virions were sorted into the lysosomal pathway for degradation. This mode of export was specifically triggered by processed, and mature, but not by furin-resistant virus particles, and occurred with significantly faster kinetics than the conventional secretory pathway. Our study therefore charts a previously undiscovered Lyn-dependent exit strategy, triggered by flaviviruses in secretory autophagosomes that might enable them to evade circulating antibodies and dictate tissue tropism.

scholarly journals Investigation of orf virus structure and morphogenesis using recombinants expressing FLAG-tagged envelope structural proteins: evidence for wrapped virus particles and egress from infected cells

2009 ◽  
Vol 90 (3) ◽  
pp. 614-625 ◽  
Author(s):  
Joanne L. Tan ◽  
Norihito Ueda ◽  
Andrew A. Mercer ◽  
Stephen B. Fleming
Keyword(s):  
Fluorescent Microscopy ◽  
Cellular Membrane ◽  
Immunogold Labelling ◽  
Orf Virus ◽  
Structural Proteins ◽  
Post Translational Modification ◽  
Virus Particles ◽  
Viral Egress ◽  
Infected Cells ◽  
Almost All

Orf virus (ORFV) is the type species of the genus Parapoxvirus, but little is known about the structure or morphogenesis of the virus. In contrast, the structure and morphogenesis of vaccinia virus (VACV) has been extensively studied. VACV has two main infectious forms, mature virion (MV) and extracellular virion (EV). The MV is wrapped by two additional membranes derived from the trans-Golgi to produce a wrapped virion (WV), the outermost of which is lost by cellular membrane fusion during viral egress to form the EV. Genome sequencing of ORFV has revealed that it has homologues of almost all of the VACV structural genes. Notable exceptions are A36R, K2L, A56R and B5R, which are associated with WV and EV envelopes. This study investigated the morphogenesis and structure of ORFV by fusing FLAG peptide to the structural proteins 10 kDa, F1L and ORF-110 to form recombinant viruses. 10 kDa and F1L are homologues of VACV A27L and H3L MV membrane proteins, whilst ORF-110 is homologous to VACV A34R, an EV membrane protein. Immunogold labelling of FLAG proteins on virus particles isolated from lysed cells showed that FLAG–F1L and FLAG–10 kDa were displayed on the surface of infectious particles, whereas ORF-110–FLAG could not be detected. Western blot analysis of solubilized recombinant ORF-110–FLAG particles revealed that ORF-110–FLAG was abundant and undergoes post-translational modification indicative of endoplasmic reticulum trafficking. Fluorescent microscopy confirmed the prediction that ORF-110–FLAG localized to the Golgi in virus-infected cells. Finally, immunogold labelling of EVs showed that ORF-110–FLAG became exposed on the surface of EV-like particles as a result of egress from the cell.

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 The Src Family Kinase c-Yes Is Required for Maturation of West Nile Virus Particles

2005 ◽  
Vol 79 (18) ◽  
pp. 11943-11951 ◽  
Author(s):  
Alec J. Hirsch ◽  
Guruprasad R. Medigeshi ◽  
Heather L. Meyers ◽  
Victor DeFilippis ◽  
Klaus Früh ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Secretory Pathway ◽  
Virus Assembly ◽  
Cellular Protein ◽  
Viral Envelope ◽  
Src Family Kinase ◽  
West Nile ◽  
Virus Particles ◽  
Cellular Genes ◽  
Infected Cells

ABSTRACT The role of cellular genes in West Nile virus (WNV) replication is not well understood. Examination of cellular transcripts upregulated during WNV infection revealed an increase in the expression of the src family kinase (SFK) c-Yes. WNV-infected cell lines treated with the SFK inhibitor PP2 demonstrated a 2- to 4-log decrease in viral titers, suggesting that SFK activity is required for completion of the viral replication cycle. RNA interference mediated knock-down of c-Yes, but not c-Src, and similarly reduced virus yield, specifically implicating c-Yes in WNV production. Interestingly, PP2 treatment did not reduce intracellular levels of either viral RNA or protein, suggesting that the drug does not act on the early stages of replication. However, endoglycosidase H (endoH) digestion of the viral envelope (E) glycoprotein revealed that the acquisition of endoH-resistant glycans by E, but not endogenous major histocompatibility complex class I, was reduced in PP2-treated cells, demonstrating that E specifically does not traffic beyond the endoplasmic reticulum in the absence of SFK activity. Electron microscopy further revealed that PP2-treated WNV-infected cells accumulated an increased number of virions in the ER compared to untreated cells. Therefore, we conclude that inhibition of SFK activity did not interfere with virus assembly but prevented transit of virions through the secretory pathway. These results identify c-Yes as a cellular protein that is involved in WNV assembly and egress.

scholarly journals Poliovirus 3A Protein Limits Interleukin-6 (IL-6), IL-8, and Beta Interferon Secretion during Viral Infection

2001 ◽  
Vol 75 (17) ◽  
pp. 8158-8165 ◽  
Author(s):  
Dana A. Dodd ◽  
Thomas H. Giddings ◽  
Karla Kirkegaard
Keyword(s):  
Protein Secretion ◽  
Interleukin 6 ◽  
Secretory Pathway ◽  
Mutant Virus ◽  
Cytokine Secretion ◽  
Wild Type Virus ◽  
General Mechanism ◽  
Wild Type ◽  
Beta Interferon ◽  
Infected Cells

ABSTRACT During viral infections, the host secretory pathway is crucial for both innate and acquired immune responses. For example, the export of most proinflammatory and antiviral cytokines, which recruit lymphocytes and initiate antiviral defenses, requires traffic through the host secretory pathway. To investigate potential effects of the known inhibition of cellular protein secretion during poliovirus infection on pathogenesis, cytokine secretion from cells infected with wild-type virus and with 3A-2, a mutant virus carrying an insertion in viral protein 3A which renders the virus defective in the inhibition of protein secretion, was tested. We show here that cells infected with 3A-2 mutant virus secrete greater amounts of cytokines interleukin-6 (IL-6), IL-8, and beta interferon than cells infected with wild-type poliovirus. Increased cytokine secretion from the mutant-infected cells can be attributed to the reduced inhibition of host protein secretion, because no significant differences between 3A-2- and wild-type-infected cells were observed in the inhibition of viral growth, host cell translation, or the ability of wild-type- or 3A-2-infected cells to support the transcriptional induction of beta interferon mRNA. We surmise that the wild-type function of 3A in inhibiting ER-to-Golgi traffic is not required for viral replication in tissue culture but, by altering the amount of secreted cytokines, could have substantial effects on pathogenesis within an infected host. The global inhibition of protein secretion by poliovirus may reflect a general mechanism by which pathogens that do not require a functional protein secretory apparatus can reduce the native immune response and inflammation associated with infection.

scholarly journals Septins suppress the release of vaccinia virus from infected cells

2018 ◽  
Vol 217 (8) ◽  
pp. 2911-2929 ◽  
Author(s):  
Julia Pfanzelter ◽  
Serge Mostowy ◽  
Michael Way
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Membrane Trafficking ◽  
Live Cell Imaging ◽  
Actin Polymerization ◽  
Antiviral Effect ◽  
Cellular Processes ◽  
Viral Egress ◽  
Infected Cells ◽  
Cell Spread

Septins are conserved components of the cytoskeleton that play important roles in many fundamental cellular processes including division, migration, and membrane trafficking. Septins can also inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella. We found that septins are recruited to vaccinia virus immediately after its fusion with the plasma membrane during viral egress. RNA interference–mediated depletion of septins increases virus release and cell-to-cell spread, as well as actin tail formation. Live cell imaging reveals that septins are displaced from the virus when it induces actin polymerization. Septin loss, however, depends on the recruitment of the SH2/SH3 adaptor Nck, but not the activity of the Arp2/3 complex. Moreover, it is the recruitment of dynamin by the third Nck SH3 domain that displaces septins from the virus in a formin-dependent fashion. Our study demonstrates that septins suppress vaccinia release by “entrapping” the virus at the plasma membrane. This antiviral effect is overcome by dynamin together with formin-mediated actin polymerization.

scholarly journals The Morphology and Composition of Influenza A Virus Particles Are Not Affected by Low Levels of M1 and M2 Proteins in Infected Cells

2005 ◽  
Vol 79 (12) ◽  
pp. 7926-7932 ◽  
Author(s):  
Svetlana V. Bourmakina ◽  
Adolfo García-Sastre
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles ◽  
M1 Protein ◽  
Infected Cells ◽  
Low Levels ◽  
Viral Components

ABSTRACT We generated a recombinant influenza A virus (Mmut) that produced low levels of matrix (M1) and M2 proteins in infected cells. Mmut virus propagated to significantly lower titers than did wild-type virus in cells infected at low multiplicity. By contrast, virion morphology and incorporation of viral proteins and vRNAs into virus particles were similar to those of wild-type virus. We propose that a threshold amount of M1 protein is needed for the assembly of viral components into an infectious particle and that budding is delayed in Mmut virus-infected cells until sufficient levels of M1 protein accumulate at the plasma membrane.

scholarly journals Endocytic Trafficking Routes of Wild Type and ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator

2004 ◽  
Vol 15 (6) ◽  
pp. 2684-2696 ◽  
Author(s):  
Martina Gentzsch ◽  
Xiu-Bao Chang ◽  
Liying Cui ◽  
Yufeng Wu ◽  
Victor V. Ozols ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Secretory Pathway ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Early Endosomes ◽  
Δf508 Cftr ◽  
Cystic Fibrosis Transmembrane

Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. ΔF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and ΔF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16°C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations ΔF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis.

scholarly journals Assembly and Maturation of the Flavivirus Kunjin Virus Appear To Occur in the Rough Endoplasmic Reticulum and along the Secretory Pathway, Respectively

2001 ◽  
Vol 75 (22) ◽  
pp. 10787-10799 ◽  
Author(s):  
Jason M. Mackenzie ◽  
Edwin G. Westaway
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rough Endoplasmic Reticulum ◽  
Envelope Protein ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Immunogold Labeling ◽  
Virus Particles ◽  
Infected Cells ◽  
Assembly Site

ABSTRACT The intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER). Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.

scholarly journals Rescue of Recombinant Marburg Virus from cDNA Is Dependent on Nucleocapsid Protein VP30

2006 ◽  
Vol 80 (2) ◽  
pp. 1038-1043 ◽  
Author(s):  
Sven Enterlein ◽  
Viktor Volchkov ◽  
Michael Weik ◽  
Larissa Kolesnikova ◽  
Valentina Volchkova ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Ebola Virus ◽  
Marburg Virus ◽  
Wild Type Virus ◽  
Binding Motif ◽  
Wild Type ◽  
Virus Particles ◽  
Virus Recombinant ◽  
Infected Cells ◽  
Virus Recovery

ABSTRACT Here we report recovery of infectious Marburg virus (MARV) from a full-length cDNA clone. Compared to the wild-type virus, recombinant MARV showed no difference in terms of morphology of virus particles, intracellular distribution in infected cells, and growth kinetics. The nucleocapsid protein VP30 of MARV and Ebola virus (EBOV) contains a Zn-binding motif which is important for the function of VP30 as a transcriptional activator in EBOV, whereas its role for MARV is unclear. It has been reported previously that MARV VP30 is able to support transcription in an EBOV-specific minigenome system. When the Zn-binding motif was destroyed, MARV VP30 was shown to be inactive in the EBOV system. While it was not possible to rescue recombinant MARV when the VP30 plasmid was omitted from transfection, MARV VP30 with a destroyed Zn-binding motif and EBOV VP30 were able to mediate virus recovery. In contrast, rescue of recombinant EBOV was not supported by EBOV VP30 containing a mutated Zn-binding domain.

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