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Infectivities of pseudotype viruses for adherent cell lines (A) and cell lines in suspension (B). Each cell line was infected with the four pseudotype viruses (VSVG*-G, VSVG*-EdHF, VSVG*-KAHF, and VSVG*), and infectivity titers were measured by counting the number of GFP-expressing cells.
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The Edmonston strain of measles virus (MV) that utilizes the human CD46 as the cellular receptor produced cytopathic effects (CPE) in all of the primate cell lines examined. In contrast, the wild-type MV strains isolated in a marmoset B-cell line B95a (the KA and Ichinose strains) replicated and produced CPE in some but not all of the primate lymph...
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... was prepared without supplying envelope proteins, so that the cell's susceptibility to VSVG* will reflect virus entry independent of VSV or MV envelope proteins. Figure 4A shows the susceptibility of adherent cell lines to pseudotype viruses. VSVG*-G infected all of the cell lines tested, with titers ranging from 10 6.0 to 10 8.7 infectious units/ml (as measured by counting the number of GFP-expressing cells). ...
Context 2
... susceptibility of cell lines in suspension to pseudotype viruses was also examined (Fig. 4B). All human cell lines tested were susceptible to VSVG*-EdHF, while neither of the mouse cell lines was. VSVG*-KAHF titers were 2 to 3 logs higher than VSVG* titers on B95-8, Raji, Ramos, BJAB- B95-8, and MT-2. VSVG* showed a high infectivity titer on C91/PL, making it impossible to evaluate virus entry into this cell line dependent on ...
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... The presence of this machinery at least in part determines the cellular tropism of the virus, generally defined as the cell type that is permissive to infection. These host factors include proteins and pathways required for each step in the viral life cycle including viral entry, viral processing, viral replication, and viral particle assembly and release Puelles et al., 2020;Schneider-Schaulies, 2000;Tatsuo et al., 2000). However, even within a given cell type that is known to be infectable by a particular virus, only a subset of cells are infected (Heldt et al., 2015;Melms et al., 2021;Ravindra et al., 2021;Russell et al., 2018;Snijder et al., 2009;Snijder & Pelkmans, 2011). ...
The ability of a virus to infect a cell type is at least in part determined by the presence of host factors required for the viral life cycle. However, even within cell types that express known factors needed for infection, not every cell is equally susceptible, suggesting that our knowledge of the full spectrum of factors that promote infection is incomplete. Profiling the most susceptible subsets of cells within a population may reveal additional factors that promote infection. However, because viral infection dramatically alters the state of the cell, new approaches are needed to reveal the state of these cells prior to infection with virus. Here, we used single-cell clone tracing to retrospectively identify and characterize lung epithelial cells that are highly susceptible to infection with SARS-CoV-2. The transcriptional state of these highly susceptible cells includes markers of retinoic acid signaling and epithelial differentiation. Loss of candidate factors identified by our approach revealed that many of these factors play roles in viral entry. Moreover, a subset of these factors exert control over the infectable cell state itself, regulating the expression of key factors associated with viral infection and entry. Analysis of patient samples revealed the heterogeneous expression of these factors across both cells and patients in vivo. Further, the expression of these factors is upregulated in particular inflammatory pathologies. Altogether, our results show that the variable expression of intrinsic cell states is a major determinant of whether a cell can be infected by SARS-CoV-2.
... LRP6 is widely expressed in embryonic and adult tissues (61), and it is highly conserved between different species (62). Such expression profiles and high sequence identities fit nicely with the known broad cell tropism that is mediated by CDV-OP, which is known to infect various canine cells (22,48) as well as SLAM-and N4-negative human Jurkat and African green monkey Vero cells (63,64). LRP6 consists of a large ectodomain (EC), a transmembrane domain (TM), and a short cytosolic tail (65). ...
Wild-type canine distemper virus (CDV) is an important pathogen of dogs as well as wildlife that can infect immune and epithelial cells through two known receptors: the signaling lymphocytic activation molecule (SLAM) and nectin-4, respectively. Conversely, the ferret and egg-adapted CDV-Onderstepoort strain (CDV-OP) is employed as an effective vaccine for dogs. CDV-OP also exhibits promising oncolytic properties, such as its abilities to infect and kill multiple cancer cells in vitro. Interestingly, several cancer cells do not express SLAM or nectin-4, suggesting the presence of a yet unknown entry factor for CDV-OP. By conducting a genome-wide CRISPR/Cas9 knockout (KO) screen in CDV-OP-susceptible canine mammary carcinoma P114 cells, which neither express SLAM nor nectin-4, we identified low-density lipoprotein receptor-related protein 6 (LRP6) as a host factor that promotes CDV-OP infectivity. Whereas the genetic ablation of LRP6 rendered cells resistant to infection, ectopic expression in resistant LRP6KO cells restored susceptibility. Furthermore, multiple functional studies revealed that (i) the overexpression of LRP6 leads to increased cell-cell fusion, (ii) a soluble construct of the viral receptor-binding protein (solHOP) interacts with a soluble form of LRP6 (solLRP6), (iii) an H-OP point mutant that prevents interaction with solLRP6 abrogates cell entry in multiple cell lines once transferred into recombinant viral particles, and (iv) vesicular stomatitis virus (VSV) pseudotyped with CDV-OP envelope glycoproteins loses its infectivity in LRP6KO cells. Collectively, our study identified LRP6 as the long sought-after cell entry receptor of CDV-OP in multiple cell lines, which set the molecular bases to refine our understanding of viral-cell adaptation and to further investigate its oncolytic properties. IMPORTANCE Oncolytic viruses (OV) have gathered increasing interest in recent years as an alternative option to treat cancers. The Onderstepoort strain of canine distemper virus (CDV-OP), an enveloped RNA virus belonging to the genus Morbillivirus, is employed as a safe and efficient vaccine for dogs against distemper disease. Importantly, although CDV-OP can infect and kill multiple cancer cell lines, the basic mechanisms of entry remain to be elucidated, as most of those transformed cells do not express natural receptors (i.e., SLAM and nectin-4). In this study, using a genome-wide CRISPR/Cas9 knockout screen, we describe the discovery of LRP6 as a novel functional entry receptor for CDV-OP in various cancer cell lines and thereby uncover a basic mechanism of cell culture adaptation. Since LRP6 is upregulated in various cancer types, our data provide important insights in order to further investigate the oncolytic properties of CDV-OP.
... The VSV machinery produces structural proteins that bud through the cellular plasma membrane and will incorporate foreign glycoproteins that are present. These newly produced ppVSV particles mimic the entry pathways of the viruses from which their glycoproteins are derived, but are not replication-competent since they lack genes encoding a glycoprotein and cannot produce infectious progeny [23][24][25]. Recombinant VSV (rVSV) particles are replication-competent viruses generated from altered VSV genomes in which the foreign glycoproteins have been cloned into the genome in place of VSV-G. ...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the most recent global pandemic that has caused more than a million deaths around the world. The spike glycoprotein (S) drives the entry and fusion of this virus and is the main determinant of cell tropism. To explore S requirements for entry under BSL2 conditions, S has been pseudotyped onto vesicular stomatitis virus (VSV) or retroviral particles with varied success. Several alterations to S were demonstrated to improve pseudoparticle titers, but they have not been systematically compared. In this study, we produced pseudotyped VSV particles with multiple modifications to S, including truncation, mutation, and tagging strategies. The main objective of this study was to determine which modifications of the S protein optimize cell surface expression, incorporation into pseudotyped particles, and pseudoparticle entry. Removal of the last 19 residues of the cytoplasmic tail produced a hyper-fusogenic S, while removal of 21 residues increased S surface production and VSV incorporation. Additionally, we engineered a replication-competent VSV (rVSV) virus to produce the S-D614G variant with a truncated cytoplasmic tail. While the particles can be used to assess S entry requirements, the rVSV∆G/SMet1D614G∆21 virus has a poor specific infectivity (particle to infectious titer ratio).
... To show the important roles of hSLAM-Met29 and MVH-Phe549 in the context of virus infection, cell lines constitutively expressing mutated hSLAM (M29S and M29F) or non-mutated hSLAM were generated. For these experiments, we used Jurkat cells, a human T cell-derived cell line that does not express SLAM (Tatsuo et al., 2000a). The cell lines were named Jurkat/hSLAM-M29S, Jurkat/hSLAM-M29F, and Jurkat/ hSLAM, respectively. ...
Measles virus (MV) is a human pathogen that is classified in the genus Morbillivirus in the family Paramyxoviridae together with several non-human animal morbilliviruses. They cause severe systemic infections by using signaling lymphocytic activation molecule (SLAM) and poliovirus receptor-like 4 expressed on immune and epithelial cells, respectively, as receptors. The viral hemagglutinin (H) protein is responsible for the receptor-binding. Previously determined structures of MV-H and SLAM complexes revealed a major binding interface between the SLAM V domain and MV-H with four binding components (sites 1–4) in the interface. We studied the MV-H and human SLAM (hSLAM) complex structure in further detail by in silico analyses and determined missing regions or residues in the previously determined complex structures. These analyses showed that, in addition to sites 1–4, MV-H establishes a unique interaction with the extreme N-terminal region (ExNTR) of hSLAM. The first principles calculation-based fragment molecular orbital computation method revealed that methionine at position 29 (hSLAM-Met29) is the key residue for the interaction. hSLAM-Met29 was predicted to establish a CH-π interaction with phenylalanine at position 549 of MV-H (MVH-Phe549). A cell-cell fusion assay showed that the hSLAM-Met29 and MVH-Phe549 interaction is important for hSLAM-dependent MV membrane fusion. Furthermore, Jurkat cell lines expressing hSLAM with or without Met29 and recombinant MV possessing the H protein with or without Phe549 showed that the hSLAM-Met29 and MVH-Phe549 interaction enhanced hSLAM-dependent MV infection by ~10-fold. We speculate that in the evolutionary history of morbilliviruses, this interaction may have contributed to MV adaptation to humans because this interaction is unique for MV and only MV uses hSLAM efficiently among morbilliviruses.
... The cells were transfected (when~75-80% confluent) with plasmids encoding, as indicated, VSV-G (plasmid backbone: pCAGGS), EBOV-GPΔ (plasmid backbone: VRC6002), or Measles F and H (plasmid backbone: PCXN2), with polyethylenimine (PEI; Polysciences, Inc Cat# 23966). Measles F and H (Edmonston strain) plasmids were generously provided by Dr. Yusuke Yanagi of Kyushu University This strain of measles virus was reported to use CD46 as its receptor on non-lymphoid cells [49]. 24 hours later, cells were infected with pre-titered VSV-ΔG helper virus encoding GFP (from a plaque eluate) at 37˚C for 1 hr, washed extensively with PBS, and then incubated overnight at 37˚C in growth medium. ...
Influenza virus is an enveloped virus wrapped in a lipid bilayer derived from the host cell plasma membrane. Infection by influenza virus is dependent on these host cell lipids, which include sphingolipids. Here we examined the role of the sphingolipid, glucosylceramide, in influenza virus infection by knocking out the enzyme responsible for its synthesis, glucosylceramide synthase (UGCG). We observed diminished influenza virus infection in HEK 293 and A549 UGCG knockout cells and demonstrated that this is attributed to impaired viral entry. We also observed that entry mediated by the glycoproteins of other enveloped viruses that enter cells by endocytosis is also impaired in UGCG knockout cells, suggesting a broader role for UGCG in viral entry by endocytosis.
... 23966). Measles F plasmid was generously provided by Yusuke Yanagi of Kyushu University (65). The next day, cells were infected with VSV-ΔG helper virus (from plaque eluate, with previously measured titer) encoding GFP for 1 h at 37°C, washed extensively with phosphate-buffered saline (PBS), and then cultured in growth medium overnight at 37°C. ...
Influenza virus is the pathogen responsible for the second largest pandemic in human history. A better understanding of how influenza virus enters host cells may lead to the development of more-efficacious therapies against emerging strains of the virus. Here we show that the glycosphingolipid metabolizing enzyme glucosylceramidase is required for optimal influenza virus trafficking to late endosomes and for consequent fusion, entry, and infection. We also provide evidence that promotion of influenza virus entry by glucosylceramidase extends to other endosome-entering viruses and is due to a general requirement for this enzyme, and hence for optimal levels of glucosylceramide, for efficient trafficking of endogenous cargos, such as the epidermal growth factor (EGF) receptor, along the endocytic pathway. This work therefore has implications for the basic process of endocytosis as well as for pathogenic processes, including virus entry and Gaucher disease.
... The recombinant MVs were prepared as described previously (58) and titrated on Vero/hSLAM cells by plaque assay. VSVΔG*-G was prepared and titrated on 293T cells as previously described (47,59). (60,61). ...
Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV.
IMPORTANCE
Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. This neurological complication is almost always fatal, and there is currently no effective treatment for it. Mechanisms by which MV invades the CNS and causes the disease remain to be elucidated. We have previously shown that fusion-enhancing substitutions in the fusion protein of MVs isolated from SSPE patients contribute to MV spread in neurons. In this study, we demonstrate that MV bearing the hyperfusogenic mutant fusion protein spreads between human neurons in a cell-to-cell manner. Spread of the virus was inhibited by a fusion inhibitor peptide and antibodies against the MV hemagglutinin, indicating that both the hemagglutinin and hyperfusogenic fusion protein play important roles in MV spread between human neurons. The findings help us better understand the disease process of SSPE.
... The pseudotype viruses were thus expected to show similar behaviors to RV during the process of virus entry, and the subsequent processes of gene expression are dependent on the VSV replication machinery. In the initial experiment, only RV envelope E2 and E1 proteins were provided in trans to generate the GFP gene-and FLuc gene-encoding pseudotype viruses, VSV GFP -RV/E2E1 and VSV FLuc -RV/E2E1, respectively, like other VSV pseudotype viruses 13,[19][20][21][22][23][24][25][26][27][28][29][30] . The infectivity titers for VSV GFP -RV/E2E1 and VSV FLuc -RV/E2E1 were 10-fold higher than those of the counterpart control viruses, VSV GFP -∆G and VSV FLuc -∆G, respectively, which lack envelope glycoproteins, in Vero cells ( Fig. 2A, B). ...
... expression plasmid using branched polyethylenimine (Sigma-Aldrich). At 32 h posttransfection, the cells were infected with VSV GFP -G at a multiplicity of infection (MOI) of 3.0 13,16,22,30,63 . The VSV GFP -G genome lacks the G gene, which is replaced with the GFP gene 13 . ...
... VSV GFP -MV/FH and VSV FLuc -MV/ FH were also generated similarly to VSV GFP -RV/CE2E1 and VSV FLuc -RV/CE2E1, respectively, using pCXN-Ed-F and pCA7PS-Ed-H 30, 59, 60 instead of pcDNA3.1-CE2E1. To generate VSV GFP -MV/FH and VSV FLuc -MV/FH, a fusion-blocking peptide (Z-D-Phe-Phe-Gly) (The Peptide Institute, Osaka, Japan) was used to inhibit syncytium formation during the production of these pseudotype viruses, as reported previously 30,59 . VSVΔG/GFP-*G 63 Electron microscopy image of viral particles. ...
Rubella virus (RV) generally causes a systemic infection in humans. Viral cell tropism is a key determinant of viral pathogenesis, but the tropism of RV is currently poorly understood. We analyzed various human cell lines and determined that RV only establishes an infection efficiently in particular non-immune cell lines. To establish an infection the host cells must be susceptible and permissible. To assess the susceptibility of individual cell lines, we generated a pseudotype vesicular stomatitis virus bearing RV envelope proteins (VSV-RV/CE2E1). VSV-RV/CE2E1 entered cells in an RV envelope protein-dependent manner, and thus the infection was neutralized completely by an RV-specific antibody. The infection was Ca²⁺-dependent and inhibited by endosomal acidification inhibitors, further confirming the dependency on RV envelope proteins for the VSV-RV/CE2E1 infection. Human non-immune cell lines were mostly susceptible to VSV-RV/CE2E1, while immune cell lines were much less susceptible than non-immune cell lines. However, susceptibility of immune cells to VSV-RV/CE2E1 was increased upon stimulation of these cells. Our data therefore suggest that immune cells are generally less susceptible to RV infection than non-immune cells, but the susceptibility of immune cells is enhanced upon stimulation.
... For PPRV isolation from clinical specimens, monkey cells expressing goat SLAM are more sensitive than those expressing cattle SLAM [Adombi et al., 2011]. B95a cells express a high level of SLAM on the cell surface [Tatsuo et al., 2000a] and hence serve as a common cell line for the isolation of MV, CDV, RPV and PPRV. ...
Systems biology refers to system-wide changes in biological components such as RNA/DNA (genomics), protein (proteomics) and lipids (lipidomics). In this review, we provide comprehensive information about morbillivirus replication. Besides discussing the role of individual viral/host proteins in virus replication, we also discuss how systems-level analyses could improve our understanding of morbillivirus replication, host-pathogen interaction, immune response and disease resistance. Finally, we discuss how viroinformatics is likely to provide important insights for understanding genome-genome, genome-protein and protein-protein interactions.
... influenza HA, luciferase) could be replicated by providing the G protein in a helper cell line, which is used for growing stocks of resultant VLPs, e.g. [48][49][50][51][52][53][54][55][56][57][58]. This type of VSV construct is generally classified as BSL1 e.g. ...
Peste des petits ruminants (PPR) is a viral disease of sheep and goats that is spreading through many countries in the developing world. Work on the virus is often restricted to studies of attenuated vaccine strains or to work in laboratories that have high containment facilities. We have created a helper cell dependent form of PPR virus by removing the entire RNA polymerase gene and complementing it with polymerase made constitutively in a cell line. The resultant L-deleted virus grows efficiently in the L-expressing cell line but not in other cells. Virus made with this system is indistinguishable from normal virus when used in diagnostic assays, and can be grown in normal facilities without the need for high level biocontainment. The L-deleted virus will thus make a positive contribution to the control and study of this important disease.
