scholarly journals Vaccinia Virus Envelope D8L Protein Binds to Cell Surface Chondroitin Sulfate and Mediates the Adsorption of Intracellular Mature Virions to Cells

1999 ◽  
Vol 73 (10) ◽  
pp. 8750-8761 ◽  
Author(s):  
Jye-Chian Hsiao ◽  
Che-Sheng Chung ◽  
Wen Chang
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Chondroitin Sulfate ◽  
Virus Entry ◽  
Viral Envelope ◽  
Binding Assays ◽  
Viral Envelope Protein ◽  
Virus Envelope ◽  
Virus Growth ◽  
Control Virus

ABSTRACT We previously showed that an envelope A27L protein of intracellular mature virions (IMV) of vaccinia virus binds to cell surface heparan sulfate during virus infection. In the present study we identified another viral envelope protein, D8L, that binds to chondroitin sulfate on cells. Soluble D8L protein interferes with the adsorption of wild-type vaccinia virions to cells, indicating a role in virus entry. To explore the interaction of cell surface glycosaminoglycans and vaccinia virus, we generated mutant viruses from a control virus, WR32-7/Ind14K (A27L+ D8L+) to be defective in expression of either the A27L or the D8L gene (A27L+D8L− or A27L− D8L+) or both (A27L− D8L−). The A27L+D8L+ and A27L− D8L+ mutants grew well in BSC40 cells, consistent with previous observations. However, the IMV titers of A27L+ D8L− and A27L− D8L− viruses in BSC40 cells were reduced, reaching only 10% of the level for the control virus. The data suggested an important role for D8L protein in WR32-7/Ind14K virus growth in cell cultures. A27L protein, on the other hand, could not complement the functions of D8L protein. The low titers of the A27L+ D8L− and A27L−D8L− mutant viruses were not due to defects in the morphogenesis of IMV, and the mutant virions demonstrated a brick shape similar to that of the control virions. Furthermore, the infectivities of the A27L+ D8L− and A27L−D8L− mutant virions were 6 to 10% of that of the A27L+ D8L+ control virus. Virion binding assays revealed that A27L+ D8L− and A27L− D8L− mutant virions bound less well to BSC40 cells, indicating that binding of viral D8L protein to cell surface chondroitin sulfate could be important for vaccinia virus entry.

scholarly journals Vaccinia Virus Interactions with the Cell Membrane Studied by New Chromatic Vesicle and Cell Sensor Assays

2006 ◽  
Vol 81 (3) ◽  
pp. 1140-1147 ◽  
Author(s):  
Z. Orynbayeva ◽  
S. Kolusheva ◽  
N. Groysman ◽  
N. Gavrielov ◽  
L. Lobel ◽  
...  
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Lipid Bilayers ◽  
Growth Factor Receptor ◽  
Viral Envelope ◽  
Virus Infectivity ◽  
Membrane Interactions ◽  
Viral Interactions ◽  
Epidermal Growth ◽  
Virus Interactions

ABSTRACT The potential danger of cross-species viral infection points to the significance of understanding the contributions of nonspecific membrane interactions with the viral envelope compared to receptor-mediated uptake as a factor in virus internalization and infection. We present a detailed investigation of the interactions of vaccinia virus particles with lipid bilayers and with epithelial cell membranes using newly developed chromatic biomimetic membrane assays. This analytical platform comprises vesicular particles containing lipids interspersed within reporter polymer units that emit intense fluorescence following viral interactions with the lipid domains. The chromatic vesicles were employed as membrane models in cell-free solutions and were also incorporated into the membranes of epithelial cells, thereby functioning as localized membrane sensors on the cell surface. These experiments provide important insight into membrane interactions with and fusion of virions and the kinetic profiles of these processes. In particular, the data emphasize the significance of cholesterol/sphingomyelin domains (lipid rafts) as a crucial factor promoting bilayer insertion of the viral particles. Our analysis of virus interactions with polymer-labeled living cells exposed the significant role of the epidermal growth factor receptor in vaccinia virus infectivity; however, the data also demonstrated the existence of additional non-receptor-mediated mechanisms contributing to attachment of the virus to the cell surface and its internalization.

scholarly journals Isolation and Metagenomic Identification of Avian Leukosis Virus Associated with Mortality in Broiler Chicken

2016 ◽  
Vol 2016 ◽  
pp. 1-4
Author(s):  
Faruku Bande ◽  
Siti Suri Arshad ◽  
Abdul Rahman Omar
Keyword(s):  
Sequence Homology ◽  
Mdck Cells ◽  
Avian Leukosis Virus ◽  
Viral Envelope ◽  
Homology Search ◽  
Economic Losses ◽  
Viral Envelope Protein ◽  
Isolation And Identification ◽  
Virus Growth ◽  
Transmission Electron

Avian leukosis virus (ALV) belongs to the family Retroviridae and causes considerable economic losses to the poultry industry. Following an outbreak associated with high mortality in a broiler flock in northern part of Malaysia, kidney tissues from affected chickens were submitted for virus isolation and identification in chicken embryonated egg and MDCK cells. Evidence of virus growth was indicated by haemorrhage and embryo mortality in egg culture. While viral growth in cell culture was evidenced by the development of cytopathic effects. The isolated virus was purified by sucrose gradient and identified using negative staining transmission electron microscopy. Further confirmation was achieved through next-generation sequencing and nucleotide sequence homology search. Analysis of the viral sequences using the NCBI BLAST tool revealed 99-100% sequence homology with exogenous ALV viral envelope protein. Phylogenetic analysis based on partial envelope sequences showed the Malaysian isolate clustered with Taiwanese and Japanese ALV strains, which were closer to ALV subgroup J, ALV subgroup E, and recombinant A/E isolates. Based on these findings, ALV was concluded to be associated with the present outbreak. It was recommended that further studies should be conducted on the molecular epidemiology and pathogenicity of the identified virus isolate.

scholarly journals Role of Receptor-Mediated Endocytosis in the Formation of Vaccinia Virus Extracellular Enveloped Particles

2005 ◽  
Vol 79 (7) ◽  
pp. 4080-4089 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Adaptor Protein ◽  
Viral Proteins ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Viral Envelope Proteins

ABSTRACT Infectious intracellular mature vaccinia virus particles are wrapped by cisternae, which may arise from trans-Golgi or early endosomal membranes, and are transported along microtubules to the plasma membrane where exocytosis occurs. We used EH21, a dominant-negative form of Eps15 that is an essential component of clathrin-coated pits, to investigate the extent and importance of endocytosis of viral envelope proteins from the cell surface. Several recombinant vaccinia viruses that inducibly or constitutively express an enhanced green fluorescent protein (GFP)-EH21 fusion protein were constructed. Expression of GFP-EH21 blocked uptake of transferrin, a marker for clathrin-mediated endocytosis, as well as association of adaptor protein-2 with clathrin-coated pits. When GFP-EH21 was expressed, there were increased amounts of viral envelope proteins, including A33, A36, B5, and F13, in the plasma membrane, and their internalization was inhibited. Wrapping of virions appeared to be qualitatively unaffected as judged by electron microscopy, a finding consistent with a primary trans-Golgi origin of the cisternae. However, GFP-EH21 expression caused a 50% reduction in released enveloped virions, decreased formation of satellite plaques, and delayed virus spread, indicating an important role for receptor-mediated endocytosis. Due to dynamic interconnection between endocytic and exocytic pathways, viral proteins recovered from the plasma membrane could be used by trans-Golgi or endosomal cisternae to form new viral envelopes. Adherence of enveloped virions to unrecycled viral proteins on the cell surface may also contribute to decreased virus release in the presence of GFP-EH21. In addition to a salvage function, the retrieval of viral proteins from the cell surface may reduce immune recognition.

Is CD4 sufficient for HIV entry? Cell surface molecules involved in HIV infection

1993 ◽  
Vol 342 (1299) ◽  
pp. 67-73 ◽  
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Cell Adhesion Molecules ◽  
Virus Entry ◽  
Neural Cells ◽  
Cell Surface Molecules ◽  
Virus Envelope ◽  
Surface Molecules ◽  
Hiv Entry ◽  
Hiv 1

HIV-1, HIV-2 and SIV each bind to CD4 as the first step in virus entry. However, alternative receptors may also be used. HIV-1 binds to glycolipids with terminal galactosylceramide residues on neural cells; opsonized virus binds to Fc receptors; HIV-2 can infect certain CD4-negative cells. Further receptors may also play a role in CD4-mediated infection, including cell adhesion molecules and possibly cell surface proteinases. After binding to CD4, immunodeficiency viruses require secondary molecules to effect fusion between the virus envelope and the cell membrane; these accessory requirements differ between HIV-1, HIV-2 and SIV.

scholarly journals Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2

2013 ◽  
Vol 87 (23) ◽  
pp. 12552-12561 ◽  
Author(s):  
Kazuya Shirato ◽  
Miyuki Kawase ◽  
Shutoku Matsuyama
Keyword(s):  
Middle East ◽  
Cell Surface ◽  
Serine Protease ◽  
Virus Entry ◽  
Cathepsin L ◽  
Vero Cells ◽  
Middle East Respiratory Syndrome ◽  
Simultaneous Treatment ◽  
Virus Growth ◽  
Transmembrane Serine Protease

The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than after infection with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-l-leucylamindo-3-methylbutane ethyl ester, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells, which were derived from lung, but these characters differed from those of mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line.

scholarly journals A Multiplexed Cell-Based Assay for the Identification of Modulators of Pre-Membrane Processing as a Target against Dengue Virus

2015 ◽  
Vol 20 (5) ◽  
pp. 616-626 ◽  
Author(s):  
Zachary D. Stolp ◽  
Cameron A. Smurthwaite ◽  
Connor Reed ◽  
Wesley Williams ◽  
Andre Dharmawan ◽  
...  
Keyword(s):  
Cell Surface ◽  
Large Scale ◽  
Secretory Pathway ◽  
Viral Envelope ◽  
Viral Envelope Protein ◽  
Membrane Processing ◽  
A Cell ◽  
Novel Drug ◽  
First Time ◽  
Large Scale Screening

The DenV pre-membrane protein (prM) is a crucial chaperone for the viral envelope protein, preventing premature fusion with vesicles during viral export. prM molecules in immature particles are cleaved by host proteases, leading to mature fusogenic virions. Blockade of prM cleavage would restrict fusion and represents a novel druggable opportunity against DenV. We have thus established a cell-based platform to monitor prM processing that relies on an engineered two-tag scaffold that travels to the cell surface through the secretory pathway. The assay discriminates between a single cell-surface tag when prM is cleaved and two tags when it is not, as detected through fluorescent-coupled antibodies by flow cytometry. The assay, miniaturized into a 96-well plate format, was multiplexed with the HIV-1 envelope boundary, also cleaved in the same pathway. A pilot screen against 1280 compounds was executed, leading to the identification of a potential active and corroborating the robustness of our assay for large-scale screening. We describe for the first time a cell-based assay that monitors DenV prM processing within the classical secretory pathway, which was exploited to identify a potential novel drug against DenV.

scholarly journals Antiviral Peptides Targeting the West Nile Virus Envelope Protein

2006 ◽  
Vol 81 (4) ◽  
pp. 2047-2055 ◽  
Author(s):  
Fengwei Bai ◽  
Terrence Town ◽  
Deepti Pradhan ◽  
Jonathan Cox ◽  
Ashish ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Envelope Protein ◽  
Phage Display Library ◽  
Viral Envelope ◽  
Host Cells ◽  
Viral Envelope Protein ◽  
Virus Envelope ◽  
West Nile ◽  
Inhibition Concentration

ABSTRACT West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 μM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 μM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.

scholarly journals Vaccinia Virus A25 and A26 Proteins Are Fusion Suppressors for Mature Virions and Determine Strain-Specific Virus Entry Pathways into HeLa, CHO-K1, and L Cells

2010 ◽  
Vol 84 (17) ◽  
pp. 8422-8432 ◽  
Author(s):  
Shu-Jung Chang ◽  
Yu-Xun Chang ◽  
Roza Izmailyan ◽  
Yin-Liang Tang ◽  
Wen Chang
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Membrane Fusion ◽  
Virus Entry ◽  
Low Ph ◽  
Viral Envelope ◽  
Viral Fusion ◽  
Entry Pathway ◽  
L Cells ◽  
Wide Range

ABSTRACT Mature vaccinia virus enters cells through either fluid-phase endocytosis/macropinocytosis or plasma membrane fusion. This may explain the wide range of host cell susceptibilities to vaccinia virus entry; however, it is not known how vaccinia virus chooses between these two pathways and which viral envelope proteins determine such processes. By screening several recombinant viruses and different strains, we found that mature virions containing the vaccinia virus A25 and A26 proteins entered HeLa cells preferentially through a bafilomycin-sensitive entry pathway, whereas virions lacking these two proteins entered through a bafilomycin-resistant pathway. To investigate whether the A25 and A26 proteins contribute to entry pathway specificity, two mutant vaccinia viruses, WRΔA25L and WRΔA26L, were subsequently generated from the wild-type WR strain. In contrast to the WR strain, both the WRΔA25L and WRΔA26L viruses became resistant to bafilomycin, suggesting that the removal of the A25 and A26 proteins bypassed the low-pH endosomal requirement for mature virion entry. Indeed, WRΔA25L and WRΔA26L virus infections of HeLa, CHO-K1, and L cells immediately triggered cell-to-cell fusion at a neutral pH at 1 to 2 h postinfection (p.i.), providing direct evidence that viral fusion machinery is readily activated after the removal of the A25 and A26 proteins to allow virus entry through the plasma membrane. In summary, our data support a model that on vaccinia mature virions, the viral A25 and A26 proteins are low-pH-sensitive fusion suppressors whose inactivation during the endocytic route results in viral and cell membrane fusion. Our results also suggest that during virion morphogenesis, the incorporation of the A25 and A26 proteins into mature virions may help restrain viral fusion activity until the time of infections.

scholarly journals Vaccinia Virus 4c (A26L) Protein on Intracellular Mature Virus Binds to the Extracellular Cellular Matrix Laminin

2006 ◽  
Vol 81 (5) ◽  
pp. 2149-2157 ◽  
Author(s):  
Wen-Ling Chiu ◽  
Chi-Long Lin ◽  
Min-Hsiang Yang ◽  
Der-Lii M. Tzou ◽  
Wen Chang
Keyword(s):  
Vaccinia Virus ◽  
Envelope Protein ◽  
Binding Activity ◽  
Viral Envelope ◽  
Wild Type ◽  
Viral Envelope Protein ◽  
Reading Frame ◽  
Mature Virus ◽  
Laminin Binding

ABSTRACT Vaccinia virus intracellular mature virus (IMV) binds to glycosaminoglycans (GAGs) on cells via three virion proteins, H3L, A27L, and D8L. In this study, we demonstrated that binding of IMV to BSC40 cells was competitively inhibited by soluble laminin but not by fibronectin or collagen V, suggesting that this cell surface extracellular matrix (ECM) protein may play a role in vaccinia virus entry. Moreover, IMV infection of GAG− sog9 cells was also inhibited by laminin, demonstrating that virion binding to laminin does not involve a prior interaction with GAGs. Furthermore, comparative envelope protein analyses of wild-type vaccinia virus strain Western Reserve, which binds to laminin, and of a mutant virus, IA27L, which does not, showed that the A26L open reading frame (ORF), encoding an envelope protein, was mutated in IA27L, resulting in A26L being absent from the IMV. Expression of the wild-type A26L ORF in IA27L resulted in laminin binding activity. Moreover, recombinant A26L protein bound to laminin in vitro with a high affinity, providing direct evidence that A26L is the laminin binding protein on IMV. In summary, these results reveal a novel role for the vaccinia viral envelope protein A26L in binding to the ECM protein laminin, an association that is proposed to facilitate IMV entry.

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