scholarly journals Differential Incorporation of Cholesterol by Sindbis Virus Grown in Mammalian or Insect Cells

2009 ◽  
Vol 83 (18) ◽  
pp. 9113-9121 ◽  
Author(s):  
Amanda Hafer ◽  
Rebecca Whittlesey ◽  
Dennis T. Brown ◽  
Raquel Hernandez
Keyword(s):  
Cell Membrane ◽  
Cell Lines ◽  
Insect Cell ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Insect Cells ◽  
Virus Assembly ◽  
Critical Role ◽  
Insect Cell Line

ABSTRACT Cholesterol has been shown to be essential for the fusion of alphaviruses with artificial membranes (liposomes). Cholesterol has also been implicated as playing an essential and critical role in the processes of entry and egress of alphaviruses in living cells. Paradoxically, insects, the alternate host for alphaviruses, are cholesterol auxotrophs and contain very low levels of this sterol. To further evaluate the role of cholesterol in the life cycle of alphaviruses, the cholesterol levels of the alphavirus Sindbis produced from three different mosquito (Aedes albopictus) cell lines; one other insect cell line, Sf21 from Spodoptera frugiperda; and BHK (mammalian) cells were measured. Sindbis virus was grown in insect cells under normal culture conditions and in cells depleted of cholesterol by growth in serum delipidated by using Cab-O-sil, medium treated with methyl-β-cyclodextrin, or serum-free medium. The levels of cholesterol incorporated into the membranes of the cells and into the virus produced from these cells were determined. Virus produced from these treated and untreated cells was compared to virus grown in BHK cells under standard conditions. The ability of insect cells to produce Sindbis virus after delipidation was found to be highly cell specific and not dependent on the level of cholesterol in the cell membrane. A very low level of cholesterol was required for the generation of wild-type levels of infectious Sindbis virus from delipidated cells. The data show that one role of the virus membrane is structural, providing the stability required for infectivity that may not be provided by the delipidated membranes in some cells. These data show that the amount of cholesterol in the host cell membrane in and of itself has no effect on the process of virus assembly or on the ability of virus to infect cells. Rather, these data suggest that the cholesterol dependence reported for infectivity and assembly of Sindbis virus is a reflection of differences in the insect cell lines used and the methods of delipidation.

Recovery of infective juveniles of the entomopathogenic nematode Steinernema carpocapsae via factors produced by insect cells and symbiotic bacteria

Nematology ◽  
2009 ◽  
Vol 11 (4) ◽  
pp. 611-618 ◽  
Author(s):  
Shingo Kikuta ◽  
Takashi Kiuchi ◽  
Fugaku Aoki ◽  
Masao Nagata
Keyword(s):  
Cell Lines ◽  
Insect Cell ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Entomopathogenic Nematode ◽  
Insect Cell Line ◽  
Fruit Fly ◽  
Steinernema Carpocapsae ◽  
Symbiotic Bacteria ◽  
Insect Cell Lines

Abstract Entomopathogenic nematodes, Steinernema carpocapsae, show 'recovery' from the dauer form as infective juveniles (IJ) up to fourth-stage juveniles when host invasion occurs. This recovery also occurs within an insect cell line culturing system. Here we addressed the factor(s) that induce recovery. When IJ were exposed to cell medium obtained from the cultivation of Sf9 cell lines derived from armyworms (Spodoptera frugiperda), approximately 50% of IJ recovered after 4 h. By 16 h, 90% of the IJ had undergone recovery. Other insect cell lines such as silkworm (Bombyx mori)-derived BmN cells and fruit fly (Drosophila melanogaster)-derived S2 cells also secreted the recovery inducing factor(s). By contrast, mammalian cells (NIH/3T3 and HeLa) had no effect on nematode recovery. Our data also suggest that symbiotic bacteria are involved in IJ recovery; axenic IJ did not recover in the cell-cultured medium. When symbiotic bacteria isolated from IJ were propagated within the cell-cultured medium, the supernatant gained recovery-inducing activity against axenic IJ. From these results, we conclude that IJ recovery in S. carpocapsae is induced by multiple factor(s) secreted from insect cells and symbiont bacteria.

scholarly journals Role of a conserved tripeptide in the endodomain of Sindbis virus glycoprotein E2 in virus assembly and function

2006 ◽  
Vol 87 (3) ◽  
pp. 657-664 ◽  
Author(s):  
John West ◽  
Dennis T. Brown
Keyword(s):  
Sindbis Virus ◽  
Structural Changes ◽  
Virus Assembly ◽  
Critical Role ◽  
E2 Glycoprotein ◽  
Conserved Domain ◽  
Specific Association ◽  
And Function ◽  
Hydrophobic Cleft

Envelopment of Sindbis virus (SV) at the plasma membrane begins with the interaction of the E2 glycoprotein endodomain with a hydrophobic cleft in the surface of the pre-assembled nucleocapsid. The driving force for this budding event is thought to reside in this virus type-specific association at the surface of the cell. The specific amino acids involved in this interaction have not been identified; however, it has been proposed that a conserved motif (TPY) at aa 398–400 in the E2 tail plays a critical role in this interaction. This interaction has been examined with virus containing mutations at two positions in this conserved domain, T398A and Y400N. The viruses produced have very low infectivity (as determined by particle : p.f.u. ratios); however, there appears to be no defect in assembly, as the virus has wild-type density and electron microscopy shows assembled particles with no obvious aberrant structural changes. The loss of infectivity in the double mutant is accompanied by the loss of the ability to fuse cells after brief exposure to acid pH. These data support the idea that these residues are vital for production of infectious/functional virus; however, they are dispensable for assembly. These results, combined with other published observations, expand our understanding of the interaction of the E2 endodomain with the capsid protein.

scholarly journals Mutations in the Endodomain of Sindbis Virus Glycoprotein E2 Define Sequences Critical for Virus Assembly

2006 ◽  
Vol 80 (9) ◽  
pp. 4458-4468 ◽  
Author(s):  
John West ◽  
Raquel Hernandez ◽  
Davis Ferreira ◽  
Dennis T. Brown
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Insect Cells ◽  
Virus Assembly ◽  
Virus Production ◽  
Initial Step ◽  
Membrane Composition ◽  
Blood Sucking

ABSTRACT Envelopment of Sindbis virus at the plasma membrane is a multistep process in which an initial step is the association of the E2 protein via a cytoplasmic endodomain with the preassembled nucleocapsid. Sindbis virus is vectored in nature by blood-sucking insects and grows efficiently in a number of avian and mammalian vertebrate hosts. The assembly of Sindbis virus, therefore, must occur in two very different host cell environments. Mammalian cells contain cholesterol which insect membranes lack. This difference in membrane composition may be critical in determining what requirements are placed on the E2 tail for virus assembly. To examine the interaction between the E2 tail and the nucleocapsid in Sindbis virus, we have produced substitutions and deletions in a region of the E2 tail (E2 amino acids 408 to 415) that is initially integrated into the endoplasmic reticulum. This sequence was identified as being critical for nucleocapsid binding in an in vitro peptide protection assay. The effects of these mutations on virus assembly and function were determined in both vertebrate and invertebrate cells. Amino acid substitutions (at positions E2: 408, 410, 411, and 413) reduced infectious virus production in a position-dependent fashion but were not efficient in disrupting assembly in mammalian cells. Deletions in the E2 endodomain (Δ406-407, Δ409-411, and Δ414-417) resulted in the failure to assemble virions in mammalian cells. Electron microscopy of BHK cells transfected with these mutants revealed assembly of nucleocapsids that failed to attach to membranes. However, introduction of these deletion mutants into insect cells resulted in the assembly of virus-like particles but no assayable infectivity. These data help define protein interactions critical for virus assembly and suggest a fundamental difference between Sindbis virus assembly in mammalian and insect cells.

scholarly journals Single Amino Acid Insertions at the Junction of the Sindbis Virus E2 Transmembrane Domain and Endodomain Disrupt Virus Envelopment and Alter Infectivity

2005 ◽  
Vol 79 (12) ◽  
pp. 7682-7697 ◽  
Author(s):  
Raquel Hernandez ◽  
Davis Ferreira ◽  
Christine Sinodis ◽  
Katherine Litton ◽  
Dennis T. Brown
Keyword(s):  
Amino Acid ◽  
Sindbis Virus ◽  
Insect Cells ◽  
Virus Assembly ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Insertion Mutant ◽  
Bhk Cells ◽  
Virus Particles

ABSTRACT The final steps in the envelopment of Sindbis virus involve specific interactions of the E2 endodomain with the virus nucleocapsid. Deleting E2 K at position 391 (E2 ΔK391) resulted in the disruption of virus assembly in mammalian cells but not insect cells (host range mutant). This suggested unique interactions of the E2 ΔK391 endodomain with the different biochemical environments of the mammalian and insect cell lipid bilayers. To further investigate the role of the amino acid residues located at or around position E2 391 and constraints on the length of the endodomain on virus assembly, amino acid insertions/substitutions at the transmembrane/endodomain junction were constructed. An additional K was inserted at amino acid position 392 (KK391/392), a K→F substitution at position 391 was constructed (F391), and an additional F was inserted at 392 (FF391/392). These changes should lengthen the endodomain in the KK391/392 insertion mutant or shorten the endodomain in the FF391/392 mutant. The mutant FF391/392 grown in BHK cells formed virus particles containing extruded material not found on wild-type virus. This characteristic was not seen in FF391/392 virus grown in insect cells. The mutant KK391/392 grown in BHK cells was defective in the final membrane fission reaction, producing multicored or conjoined virus particles. The production of these aberrant particles was ameliorated when the KK391/392 mutant was grown in insect cells. These data indicate that there is a critical minimal spanning distance from the E2 membrane proximal amino acid at position 391 and the conserved E2 Y400 residue. The observed phenotypes of these mutants also invoke an important role of the specific host membrane lipid composition on virus architecture and infectivity.

Autophagy Modulators and Neuroinflammation

2020 ◽  
Vol 27 (6) ◽  
pp. 955-982 ◽  
Author(s):  
Kyoung Sang Cho ◽  
Jang Ho Lee ◽  
Jeiwon Cho ◽  
Guang-Ho Cha ◽  
Gyun Jee Song
Keyword(s):  
Neurodegenerative Disorders ◽  
Neurological Disorders ◽  
Mammalian Cells ◽  
Critical Role ◽  
Therapeutic Agents ◽  
Mechanisms Of Action ◽  
Scientific Interest ◽  
Therapeutic Tools ◽  
Underlying Mechanisms

Background: Neuroinflammation plays a critical role in the development and progression of various neurological disorders. Therefore, various studies have focused on the development of neuroinflammation inhibitors as potential therapeutic tools. Recently, the involvement of autophagy in the regulation of neuroinflammation has drawn substantial scientific interest, and a growing number of studies support the role of impaired autophagy in the pathogenesis of common neurodegenerative disorders. Objective: The purpose of this article is to review recent research on the role of autophagy in controlling neuroinflammation. We focus on studies employing both mammalian cells and animal models to evaluate the ability of different autophagic modulators to regulate neuroinflammation. Methods: We have mostly reviewed recent studies reporting anti-neuroinflammatory properties of autophagy. We also briefly discussed a few studies showing that autophagy modulators activate neuroinflammation in certain conditions. Results: Recent studies report neuroprotective as well as anti-neuroinflammatory effects of autophagic modulators. We discuss the possible underlying mechanisms of action of these drugs and their potential limitations as therapeutic agents against neurological disorders. Conclusion: Autophagy activators are promising compounds for the treatment of neurological disorders involving neuroinflammation.

scholarly journals Mammals Preferred: Reassortment of Batai and Bunyamwera orthobunyavirus Occurs in Mammalian but Not Insect Cells

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1702
Author(s):  
Anna Heitmann ◽  
Frederic Gusmag ◽  
Martin G. Rathjens ◽  
Maurice Maurer ◽  
Kati Frankze ◽  
...  
Keyword(s):  
Cell Lines ◽  
Insect Cell ◽  
Viral Genome ◽  
Insect Cells ◽  
Central Africa ◽  
Genome Segment ◽  
Reassortant Virus ◽  
Mammalian Cell Lines ◽  
Insect Cell Lines ◽  
Human Infections

Reassortment is a viral genome-segment recomposition known for many viruses, including the orthobunyaviruses. The co-infection of a host cell with two viruses of the same serogroup, such as the Bunyamwera orthobunyavirus and the Batai orthobunyavirus, can give rise to novel viruses. One example is the Ngari virus, which has caused major outbreaks of human infections in Central Africa. This study aimed to investigate the potential for reassortment of Bunyamwera orthobunyavirus and the Batai orthobunyavirus during co-infection studies and the replication properties of the reassortants in different mammalian and insect cell lines. In the co-infection studies, a Ngari-like virus reassortant and a novel reassortant virus, the Batunya virus, arose in BHK-21 cells (Mesocricetus auratus). In contrast, no reassortment was observed in the examined insect cells from Aedes aegypti (Aag2) and Aedes albopictus (U4.4 and C6/36). The growth kinetic experiments show that both reassortants are replicated to higher titers in some mammalian cell lines than the parental viruses but show impaired growth in insect cell lines.

scholarly journals Direct lysosome-based autophagy of lipid droplets in hepatocytes

2020 ◽  
Vol 117 (51) ◽  
pp. 32443-32452
Author(s):  
Ryan J. Schulze ◽  
Eugene W. Krueger ◽  
Shaun G. Weller ◽  
Katherine M. Johnson ◽  
Carol A. Casey ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Lipid Droplets ◽  
Critical Role ◽  
Specific Protein ◽  
Lipid Homeostasis ◽  
Dynamic Interactions ◽  
Double Membrane ◽  
Live Cell Microscopy ◽  
Cell Microscopy

Hepatocytes metabolize energy-rich cytoplasmic lipid droplets (LDs) in the lysosome-directed process of autophagy. An organelle-selective form of this process (macrolipophagy) results in the engulfment of LDs within double-membrane delimited structures (autophagosomes) before lysosomal fusion. Whether this is an exclusive autophagic mechanism used by hepatocytes to catabolize LDs is unclear. It is also unknown whether lysosomes alone might be sufficient to mediate LD turnover in the absence of an autophagosomal intermediate. We performed live-cell microscopy of hepatocytes to monitor the dynamic interactions between lysosomes and LDs in real-time. We additionally used a fluorescent variant of the LD-specific protein (PLIN2) that exhibits altered fluorescence in response to LD interactions with the lysosome. We find that mammalian lysosomes and LDs undergo interactions during which proteins and lipids can be transferred from LDs directly into lysosomes. Electron microscopy (EM) of primary hepatocytes or hepatocyte-derived cell lines supports the existence of these interactions. It reveals a dramatic process whereby the lipid contents of the LD can be “extruded” directly into the lysosomal lumen under nutrient-limited conditions. Significantly, these interactions are not affected by perturbations to crucial components of the canonical macroautophagy machinery and can occur in the absence of double-membrane lipoautophagosomes. These findings implicate the existence of an autophagic mechanism used by mammalian cells for the direct transfer of LD components into the lysosome for breakdown. This process further emphasizes the critical role of lysosomes in hepatic LD catabolism and provides insights into the mechanisms underlying lipid homeostasis in the liver.

scholarly journals The Structure of Sindbis Virus Produced from Vertebrate and Invertebrate Hosts as Determined by Small-Angle Neutron Scattering

2010 ◽  
Vol 84 (10) ◽  
pp. 5270-5276 ◽  
Author(s):  
Lilin He ◽  
Amanda Piper ◽  
Flora Meilleur ◽  
Dean A. A. Myles ◽  
Raquel Hernandez ◽  
...  
Keyword(s):  
Neutron Scattering ◽  
Small Angle ◽  
Host Species ◽  
Mammalian Cells ◽  
Small Angle Neutron Scattering ◽  
Sindbis Virus ◽  
Insect Cells ◽  
Lipid Membrane ◽  
Scattering Data ◽  
The Impact

ABSTRACT The complex natural cycle of vectored viruses that transition between host species, such as between insects and mammals, makes understanding the full life cycle of the virus an incredibly complex problem. Sindbis virus, an arbovirus and prototypic alphavirus having an inner protein shell and an outer glycoprotein coat separated by a lipid membrane, is one example of a vectored virus that transitions between vertebrate and insect hosts. While evidence of host-specific differences in Sindbis virus has been observed, no work has been performed to characterize the impact of the host species on the structure of the virus. Here, we report the first study of the structural differences between Sindbis viruses grown in mammalian and insect cells, which were determined by small-angle neutron scattering (SANS), a nondestructive technique that did not decrease the infectivity of the Sindbis virus particles studied. The scattering data and modeling showed that, while the radial position of the lipid bilayer did not change significantly, it was possible to conclude that it did have significantly more cholesterol when the virus was grown in mammalian cells. Additionally, the outer protein coat was found to be more extended in the mammalian Sindbis virus. The SANS data also demonstrated that the RNA and nucleocapsid protein share a closer interaction in the mammalian-cell-grown virus than in the virus from insect cells.

scholarly journals Dual Role for Transforming Growth Factor β-Dependent Signaling in Trypanosoma cruzi Infection of Mammalian Cells

2000 ◽  
Vol 68 (4) ◽  
pp. 2077-2081 ◽  
Author(s):  
Belinda S. Hall ◽  
Miercio A. Pereira
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Transforming Growth Factor ◽  
Growth Arrest ◽  
Transforming Growth Factor Β

ABSTRACT Expression of functional transforming growth factor β (TGF-β) receptors (TβR) is required for the invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi. However, the precise role of this host cell signaling complex in T. cruzi infection is unknown. To investigate the role of the TGF-β signaling pathway, infection levels were studied in the mink lung epithelial cell lines JD1, JM2, and JM3. These cells express inducible mutant TβR1 proteins that cannot induce growth arrest in response to TGF-β but still transmit the signal for TGF-β-dependent gene expression. In the absence of mutant receptor expression, trypomastigotes invaded the cells at a low level. Induction of the mutant receptors caused an increase in infection in all three cell lines, showing that the requirement for TGF-β signaling at invasion can be divorced from TGF-β-induced growth arrest. TGF-β pretreatment of mink lung cells expressing wild-type TβR1 caused a marked enhancement of infection, but no enhancement was seen in JD1, JM2, and JM3 cells, showing that the ability of TGF-β to stimulate infection is associated with growth arrest. Likewise, expression of SMAD7 or SMAD2SA, inhibitors of TGF-β signaling, did not block infection by T. cruzi but did block the enhancement of infection by TGF-β. Taken together, these results show that there is a dual role for TGF-β signaling in T. cruzi infection. The initial invasion of the host cell is independent of both TGF-β-dependent gene expression and growth arrest, but TGF-β stimulation of infection requires a fully functional TGF-β signaling pathway.

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