scholarly journals Peptides from cytomegalovirus UL130 and UL131 proteins induce high titer antibodies that block viral entry into mucosal epithelial cells

Vaccine ◽  
2011 ◽  
Vol 29 (15) ◽  
pp. 2705-2711 ◽  
Author(s):  
Frances M. Saccoccio ◽  
Anne L. Sauer ◽  
Xiaohong Cui ◽  
Amy E. Armstrong ◽  
EL-Sayed E. Habib ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Viral Entry ◽  
High Titer

scholarly journals Genome sequences of human cytomegalovirus strain TB40/E variants propagated in fibroblasts and epithelial cells

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ahmed Al Qaffas ◽  
Salvatore Camiolo ◽  
Mai Vo ◽  
Alexis Aguiar ◽  
Amine Ourahmane ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Sequence Data ◽  
Laboratory Strain ◽  
Serial Passage ◽  
Wild Type Virus ◽  
Protein Coding ◽  
Genetic Changes ◽  
Long Read

AbstractThe advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.

scholarly journals Disruption of Adherens Junctions Liberates Nectin-1 To Serve as Receptor for Herpes Simplex Virus and Pseudorabies Virus Entry

2002 ◽  
Vol 76 (14) ◽  
pp. 7203-7208 ◽  
Author(s):  
Miri Yoon ◽  
Patricia G. Spear
Keyword(s):  
Herpes Simplex ◽  
Epithelial Cells ◽  
Viral Entry ◽  
Pseudorabies Virus ◽  
Adherens Junctions ◽  
Cell Junctions ◽  
Immunoglobulin Superfamily ◽  
Calcium Depletion ◽  
Normal Medium ◽  
Herpes Simplex Viruses

ABSTRACT Nectin-1, a cell adhesion molecule belonging to the immunoglobulin superfamily, can bind to virion glycoprotein D (gD) to mediate entry of herpes simplex viruses (HSV) and pseudorabies virus (PRV). Nectin-1 colocalizes with E-cadherin at adherens junctions in epithelial cells. The disruption of cell junctions can result in the redistribution of nectin-1. To determine whether disruption of junctions by calcium depletion influenced the susceptibility of epithelial cells to viral entry, Madin-Darby canine kidney cells expressing endogenous nectin-1 or transfected human nectin-1 were tested for the ability to bind soluble forms of viral gD and to be infected by HSV and PRV, before and after calcium depletion. Confocal microscopy revealed that binding of HSV and PRV gD was localized to adherens junctions in cells maintained in normal medium but was distributed, along with nectin-1, over the entire cell surface after calcium depletion. Both the binding of gD and the fraction of cells that could be infected by HSV-1 and PRV were enhanced by calcium depletion. Taken together, these results provide evidence that nectin-1 confined to adherens junctions in epithelial cells is not very accessible to virus, whereas dissociation of cell junctions releases nectin-1 to serve more efficiently as an entry receptor.

Immunogenicity Evaluation of Chimeric Subunit Vaccine Comprising Adhesion Coli Surface Antigens from Enterotoxigenic Escherichia coli

2019 ◽  
Vol 29 (1-6) ◽  
pp. 91-100
Author(s):  
Dorna Khoobbakht ◽  
Shohreh Zare Karizi ◽  
Mohammad Javad  Motamedi ◽  
Rouhollah Kazemi ◽  
Pooneh Roghanian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Subunit Vaccine ◽  
Fusion Gene ◽  
Intestinal Epithelial Cells ◽  
High Titer ◽  
Chimeric Protein ◽  
Amino Acid Sequences ◽  
Intestinal Epithelial ◽  
E Coli

Enterotoxigenic <i>Escherichia coli</i> (ETEC) is the most common agent of diarrhea morbidity in developing countries. ETEC adheres to host intestinal epithelial cells via various colonization factors. The CooD and CotD proteins play a significant role in bacteria binding to the intestinal epithelial cells as adhesin tip subunits of CS1 and CS2 pili. The purpose here was to design a new construction containing <i>cooD</i> and <i>cotD</i> genes and use several types of bioinformatics software to predict the structural and immunological properties of the designed antigen. The fusion gene was synthesized with codon bias of <i>E. coli</i> in order to increase the expression level of the protein. The amino acid sequences, protein structure, and immunogenicity properties of potential antigens were analyzed in silico. The chimeric protein was expressed in <i>E. coli</i>BL21 (DE3). The antigenicity of the recombinant proteins was verified by Western blotting and ELISA. In order to assess the induced immunity, the immunized mice were challenged with wild-type ETEC by an intraperitoneal route. Immunological analyses showed the production of a high titer of IgG serum with no sign of serum-mucosal IgA antibody response. The result of the challenge assay showed that 30% of immunized mice survived. The results of this study showed that CooD-CotD recombinant protein can stimulate immunity against ETEC. The designed chimera could be a prototype for the subunit vaccine, which is worthy of further consideration.

scholarly journals Preventing SARS-CoV-2 infection by blocking a tissue serine protease

2020 ◽  
Vol 7 ◽  
pp. 204993612093307
Author(s):  
Katherine C. Jankousky ◽  
Jonathan Schultz ◽  
Samuel Windham ◽  
Andrés F. Henao-Martínez ◽  
Carlos Franco-Paredes ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Severe Acute Respiratory Syndrome ◽  
Serine Protease ◽  
Viral Entry ◽  
Clinical Impact ◽  
Spike Protein ◽  
Viral Pathogens ◽  
Pharmacological Targets ◽  
Alternative Approach ◽  
Viral Surface

Currently, there are no proven pharmacologic interventions to reduce the clinical impact and prevent complications of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, the cause of the ongoing Coronavirus Disease of 2019 (COVID-19) pandemic. Selecting specific pharmacological targets for the treatment of viral pathogens has traditionally relied in blockage of specific steps in their replicative lifecycle in human cells. However, an alternative approach is reducing the molecular cleavage of the viral surface spike protein of SARS-CoV-2 to prevent viral entry into epithelial cells.

scholarly journals Cross-Dressing the Virion: the Transcapsidation of Adeno-Associated Virus Serotypes Functionally Defines Subgroups

2004 ◽  
Vol 78 (9) ◽  
pp. 4421-4432 ◽  
Author(s):  
Joseph E. Rabinowitz ◽  
Dawn E. Bowles ◽  
Susan M. Faust ◽  
Julie G. Ledford ◽  
Scott E. Cunningham ◽  
...  
Keyword(s):  
Viral Entry ◽  
Fluorescent Protein ◽  
High Titer ◽  
Threshold Effect ◽  
Structural Proteins ◽  
Adeno Associated Virus ◽  
Serotype 1 ◽  
Cross Dressing ◽  
Helper Plasmids ◽  
Green Fluorescent

ABSTRACT For all adeno-associated virus (AAV) serotypes, 60 monomers of the Vp1, Vp2, and Vp3 structural proteins assemble via an unknown mechanism to form an intact capsid. In an effort to better understand the properties of the capsid monomers and their role in viral entry and infection, we evaluated whether monomers from distinct serotypes can be mixed to form infectious particles with unique phenotypes. This transcapsidation approach consisted of the transfection of pairwise combinations of AAV serotype 1 to 5 helper plasmids to produce mosaic capsid recombinant AAV (rAAV). All ratios (19:1, 3:1, 1:1, 1:3, and 1:19) of these mixtures were able to replicate the green fluorescent protein transgene and to produce capsid proteins. A high-titer rAAV was obtained with mixtures that included either serotype 1, 2, or 3, whereas an rAAV of intermediate titer was obtained from serotype 5 mixtures. Only mixtures containing the AAV4 capsid exhibited reduced packaging capacity. The binding profiles of the mixed-virus preparations to either heparin sulfate (HS) or mucin agarose revealed that only AAV3-AAV5 mixtures at the 3:1 ratio exhibited duality in binding. All other mixtures displayed either an abrupt shift or a gradual alteration in the binding profile to the respective ligand upon increase of a capsid component that conferred either HS or mucin binding. The transduction of cell lines was used to further evaluate the phenotypes of these transcapsidated virions. Three transduction profiles were observed: (i) small to no change regardless of ratio, (ii) a gradual increase in transduction consistent with titration of a second capsid component, or (iii) an abrupt increase in transduction (threshold effect) dependent on the specific ratios used. Interestingly, an unexpected synergistic effect in transduction was observed when AAV1 helper constructs were combined with type 2 or type 3 recipient helpers. Further studies determined that at least two components contributed to this observed synergy: (i) heparin-mediated binding from AAV2 and (ii) an unidentified enhancement activity from AAV1 structural proteins. Using this procedure of mixing different AAV helper plasmids to generate “cross-dressed” AAV virions, we propose an additional means of classifying new AAV serotypes into subgroups based on functional approaches to analyze AAV capsid assembly, receptor-mediated binding, and virus trafficking. Exploitation of this approach in generating custom-designed AAV vectors should be of significant value to the field of gene therapy.

scholarly journals Cytoplasmic tail truncation of SARS-CoV-2 Spike protein enhances titer of pseudotyped vectors but masks the effect of the D614G mutation

2021 ◽  
Author(s):  
Hsu-Yu Chen ◽  
Chun Huang ◽  
Lu Tian ◽  
Xiaoli Huang ◽  
Chennan Zhang ◽  
...  
Keyword(s):  
Viral Entry ◽  
High Titer ◽  
Cytoplasmic Tail ◽  
Cell Types ◽  
Spike Protein ◽  
Tangential Flow Filtration ◽  
Tangential Flow ◽  
Protein Functions ◽  
Flow Filtration ◽  
The Impact

The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV) and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein’s function. Here, we optimized concentration methods for SARS-CoV-2 Spike pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers, but had no impact on sensitivity to convalescent serum inhibition. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but that this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions.

scholarly journals A Dual-Fluorescence Labeling Pseudovirus for Real-Time Imaging of Single SARS-CoV-2 Entry in Respiratory Epithelial Cells

2020 ◽  
Author(s):  
Yingxin Ma ◽  
Guobin Mao ◽  
Guoqiang Wu ◽  
Minghai Chen ◽  
Fujun Qin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Host Cell ◽  
Viral Entry ◽  
Alveolar Epithelial Cells ◽  
Fluorescence Labeling ◽  
Viral Core ◽  
Alveolar Epithelial ◽  
Dual Color ◽  
Particle Level ◽  
High Level

Abstract The pseudovirus strategy makes studies of highly pathogenic viruses feasible without the restriction of high-level biosafety facility, thus greatly contributing to virology and being used in research of SARS-CoV-2. Here, we generated a dual-color pseudo-SARS-CoV-2 virus using an HIV-1 pseudovirus production system and the SARS-CoV-2 spike (S) glycoprotein, of which the membrane was labeled with lipophilic dye (DiO) and the genomic RNA-related viral protein R (Vpr) of the viral core were fused with mCherry. With this dual-color labeling strategy, not only the movement of the whole virus but also the fate of the labeled components can be traced. The pseudovirions were applied to track viral entry at a single particle level in four types of the human respiratory cells: nasal epithelial cells (HNEpC), pulmonary alveolar epithelial cells (HPAEpiC), bronchial epithelial cells (BEP-2D), and oral epithelial cells (HOEC). Pseudo-SARS-CoV-2 entered into the host cell and released viral core into the cytoplasm，which clearly indicates that the host entry mainly occurred through endocytosis. The infection efficiency was found to be correlated with the expression of the known receptor of SARS-CoV-2, angiotensin-converting 2 (ACE2) on the host cell surface. We believe that the dual-color fluorescence labeled pseudovirus system created in this study can be a useful tool in SARS-CoV-2/COVID-19 for many purposes.

scholarly journals Conjunctival epithelial cells resist productive SARS-CoV-2 infection

2021 ◽  
Author(s):  
Majlinda Lako ◽  
Robert M Jackson ◽  
Catherine F Hatton ◽  
Jarmila S Spegarova ◽  
Maria Georgiou ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Viral Entry ◽  
Ocular Surface ◽  
Cell Types ◽  
Productive Infection ◽  
Conjunctival Epithelium ◽  
Genome Expression ◽  
Open Question ◽  
Air Liquid Interface ◽  
Conjunctival Epithelial Cells

Although tropism of SARS-CoV-2 for respiratory tract epithelial cells is well established, an open question is whether the conjunctival epithelium is also a target for SARS-CoV-2. Conjunctival epithelial cells, which express viral entry receptors ACE2 and TMPRSS2, constitute the largest exposed epithelium of the ocular surface tissue, and may represent a relevant viral entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of progenitor, basal and superficial epithelial cells and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA Seq, with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to SARS-CoV-2 genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-Kβ activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplants.

The novel antiviral properties of brassicasterol against human adenovirus

2021 ◽  
Author(s):  
Peifeng Yu ◽  
Dan Lou ◽  
Lifeng Qi ◽  
Zewei Chen
Keyword(s):  
Dna Replication ◽  
Epithelial Cells ◽  
Viral Entry ◽  
Human Adenovirus ◽  
Airway Epithelial Cells ◽  
Dependent Manner ◽  
The Novel ◽  
Airway Epithelial ◽  
Infected Cells ◽  
Dose Dependent

Aim: To investigate whether brassicasterol has inhibitory effects against adenovirus (AdV). Materials and methods: The antiviral effects of brassicasterol against AdV 3 and 7 were tested in human airway epithelial cells. Brassicasterol cytotoxicity was assessed by WST-1 assay. AdV DNA was quantified by qPCR. Results: Brassicasterol inhibited AdV 3 and 7 infection of airway epithelial cells in a dose-dependent manner. Similarly, brassicasterol also inhibited AdV 3 and 7 production in infected cells. No apparent cytotoxicity of brassicasterol was detected. Further study showed that brassicasterol inhibited AdV DNA replication, but had no impact on viral entry of cells and viral genome import to nucleus. Conclusion: Brassicasterol exerts anti-AdV effects probably through the inhibition of viral DNA replication.

scholarly journals A cytomegalovirus DNA vaccine induces antibodies that block viral entry into fibroblasts and epithelial cells

Vaccine ◽  
2015 ◽  
Vol 33 (51) ◽  
pp. 7328-7336 ◽  
Author(s):  
Michael A. McVoy ◽  
Ronzo Lee ◽  
Frances M. Saccoccio ◽  
Jukka Hartikka ◽  
Larry R. Smith ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Dna Vaccine ◽  
Viral Entry
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