Polarized glycoprotein targeting affects the spread of measles virus in vitro and in vivo

We have shown previously that basolateral targeting of plasmid-encoded measles virus (MV) F and H protein is dependent on single tyrosine residues in the cytoplasmic tails of the glycoproteins and is essential for fusion activity in polarized epithelial cells. Here, we present data on the functional importance of polarized glycoprotein expression for the cytopathic properties of infectious MV in culture and for pathogenesis in vivo. By the introduction of single point mutations, we generated recombinant viruses in which the basolateral targeting signal of either one or both glycoproteins was destroyed (tyrosine mutants). As a consequence, the mutated glycoproteins were predominantly expressed on the apical membrane of polarized Madin–Darby canine kidney cells. In contrast to parental MV, none of these virus mutants was able to spread by syncytia formation in polarized cells showing that the presence of both MV glycoproteins at the basolateral cell surface is required for cell-to-cell fusion in vitro. Using cotton rats as an animal model that allows MV replication in the respiratory tract, we showed that basolateral glycoprotein targeting is also of importance for the spread of infection in vivo. Whereas parental MV was able to spread laterally within the respiratory epithelium and from there to cells in the underlying tissue, tyrosine mutants infected only single epithelial and very few subepithelial cells. These data strongly suggest that basolateral targeting of MV glycoproteins helps to overcome the epithelial barrier and thereby facilitates the systemic spread of MV infection in vivo.

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Live-Attenuated Measles Virus Vaccine Targets Dendritic Cells and Macrophages in Muscle of Nonhuman Primates

Journal of Virology ◽

10.1128/jvi.02924-14 ◽

2014 ◽

Vol 89 (4) ◽

pp. 2192-2200 ◽

Cited By ~ 35

Author(s):

Linda J. Rennick ◽

Rory D. de Vries ◽

Thomas J. Carsillo ◽

Ken Lemon ◽

Geert van Amerongen ◽

...

Keyword(s):

Measles Virus ◽

Nonhuman Primates ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Vaccine Virus ◽

Target Cells ◽

Recombinant Viruses ◽

Green Fluorescent

ABSTRACTAlthough live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replicationin vivoare still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MVEZ. Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMVEZEGFP(1), rMVEZEGFP(3), and rMVEZEGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were comparedin vitroby growth curves, which indicated that rMVEZEGFP(1) was overattenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMVEZEGFP(1) and rMVEZEGFP(6) did not induce satisfactory serum antibody responses, whereas bothin vitroandin vivorMVEZEGFP(3) was functionally equivalent to the commercial MVEZ-containing vaccine. Intramuscular vaccination of macaques with rMVEZEGFP(3) resulted in the identification of EGFP+cells in the muscle at days 3, 5, and 7 postvaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV.IMPORTANCEEven though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicatesin vivo. This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious.

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mSphere of Influence: Understanding Virus-Host Interactions Requires a Multifaceted Approach

mSphere ◽

10.1128/msphere.00105-20 ◽

2020 ◽

Vol 5 (2) ◽

Author(s):

Rory D. de Vries

Keyword(s):

Measles Virus ◽

Ex Vivo ◽

Viral Pathogenesis ◽

Model Systems ◽

Host Immune System ◽

Recombinant Viruses ◽

Host Interactions ◽

Link Type

ABSTRACT Rory de Vries works in the field of viral pathogenesis and focuses on interactions between respiratory viruses (or corresponding vaccines) and the host immune system. In this mSphere of Influence article, he reflects on how the articles “Predominant infection of CD150+ lymphocytes and dendritic cells during measles virus infection of macaques” by R. L. de Swart et al. (R. L. de Swart, M. Ludlow, L. de Witte, Y. Yanagi, et al., PLoS Pathog 3:e178, 2007, https://doi.org/10.1371/journal.ppat.0030178) and “Long-term measles-induced immunomodulation increases overall childhood infectious disease mortality” by M. J. Mina et al. (M. J. Mina, C. J. Metcalf, R. L. de Swart, A. D. M. E. Osterhaus, and B. T. Grenfell, Science 348:694–699, 2015, https://doi.org/10.1126/science.aaa3662) made an impact on him. These articles studied interactions between measles virus and the host and influenced him by making two important points. (i) It is crucial to use nonadapted (recombinant) viruses in disease-relevant model systems when studying virus-host interactions. (ii) Studying viral pathogenesis requires a combination of in vitro, ex vivo, and in vivo studies, and a group of researchers with multiple expertises. He learned that only when all these aspects are combined, can one truly answer the question: “How does a virus cause disease?”

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Activation of the proto-oncogene p60c-src by point mutations in the SH2 domain

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2855-2862.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2855-2862

Author(s):

M C O'Brien ◽

Y Fukui ◽

H Hanafusa

Keyword(s):

Kinase Activity ◽

Single Point ◽

Point Mutations ◽

Soft Agar ◽

Specific Protein ◽

Sh2 Domain ◽

Single Point Mutation ◽

Src Gene

To investigate the importance of a conserved region spanning residues 137 to 241 in the noncatalytic domain of p60c-src (SH2 region), we used oligonucleotide-directed mutagenesis to change residues that are highly conserved in this region. Chicken embryo fibroblasts infected with a p60c-src variant containing arginine instead of tryptophan at residue 148 (W148R) appeared more rounded than cells overexpressing a normal c-src gene, and they formed colonies in soft agar. p60c-src variants containing serine instead of arginine at residue 155 (R155S) or isoleucine instead of glycine at residue 170 (G170I) also appeared transformed and were anchorage independent, but to a lesser extent than W148R. Mutation of residue 201 from histidine to leucine (H201L) had no observable effect. The in vitro kinase activity of cells infected with W148R or G170I was elevated twofold. Expression of p60W148R (or, to a lesser extent, of p60G170I) increased the number of proteins phosphorylated on tyrosine in infected cells. All of the mutants were phosphorylated in vivo on Tyr-527, instead of Tyr-416 as observed for p60v-src. Immunoprecipitated p60W148R and p60G170I were found to be associated with a phosphatidylinositol kinase activity, a factor which appears to be necessary for transformation by tyrosine-specific protein kinases. These results show that a single point mutation in the SH2 region of the cellular src gene can activate its transforming potential. This type of activation is in a new category of alterations at the amino terminus that activate but do not cause a shift in phosphorylation at the carboxy terminus.

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BST2/Tetherin Overexpression Modulates Morbillivirus Glycoprotein Production to Inhibit Cell–Cell Fusion

Viruses ◽

10.3390/v11080692 ◽

2019 ◽

Vol 11 (8) ◽

pp. 692 ◽

Cited By ~ 5

Author(s):

James T. Kelly ◽

Stacey Human ◽

Joseph Alderman ◽

Fatoumatta Jobe ◽

Leanne Logan ◽

...

Keyword(s):

Cell Fusion ◽

Measles Virus ◽

Viral Fusion ◽

Large Reduction ◽

Glycosyl Phosphatidylinositol ◽

Species Specific ◽

Syncytia Formation ◽

Cell Cell

The measles virus (MeV), a member of the genus Morbillivirus, is an established pathogen of humans. A key feature of morbilliviruses is their ability to spread by virus–cell and cell–cell fusion. The latter process, which leads to syncytia formation in vitro and in vivo, is driven by the viral fusion (F) and haemagglutinin (H) glycoproteins. In this study, we demonstrate that MeV glycoproteins are sensitive to inhibition by bone marrow stromal antigen 2 (BST2/Tetherin/CD317) proteins. BST2 overexpression causes a large reduction in MeV syncytia expansion. Using quantitative cell–cell fusion assays, immunolabeling, and biochemistry we further demonstrate that ectopically expressed BST2 directly inhibits MeV cell–cell fusion. This restriction is mediated by the targeting of the MeV H glycoprotein, but not other MeV proteins. Using truncation mutants, we further establish that the C-terminal glycosyl-phosphatidylinositol (GPI) anchor of BST2 is required for the restriction of MeV replication in vitro and cell–cell fusion. By extending our study to the ruminant morbillivirus peste des petits ruminants virus (PPRV) and its natural host, sheep, we also confirm this is a broad and cross-species specific phenotype.

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Activation of the proto-oncogene p60c-src by point mutations in the SH2 domain.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2855 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2855-2862 ◽

Cited By ~ 35

Author(s):

M C O'Brien ◽

Y Fukui ◽

H Hanafusa

Keyword(s):

Kinase Activity ◽

Single Point ◽

Point Mutations ◽

Soft Agar ◽

Specific Protein ◽

Sh2 Domain ◽

Single Point Mutation ◽

Src Gene

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Compensatory mechanisms in resistant Anopheles gambiae AcerKis and KdrKis neurons modulate insecticide-based mosquito control

Communications Biology ◽

10.1038/s42003-021-02192-0 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Stéphane Perrier ◽

Eléonore Moreau ◽

Caroline Deshayes ◽

Marine El-Adouzi ◽

Delphine Goven ◽

...

Keyword(s):

Sodium Channel ◽

Anopheles Gambiae ◽

Calcium Concentration ◽

Mosquito Control ◽

Point Mutations ◽

Resting Membrane Potential ◽

Compensatory Mechanisms ◽

Pyrethroid Insecticides

AbstractIn the malaria vector Anopheles gambiae, two point mutations in the acetylcholinesterase (ace-1R) and the sodium channel (kdrR) genes confer resistance to organophosphate/carbamate and pyrethroid insecticides, respectively. The mechanisms of compensation that recover the functional alterations associated with these mutations and their role in the modulation of insecticide efficacy are unknown. Using multidisciplinary approaches adapted to neurons isolated from resistant Anopheles gambiae AcerKis and KdrKis strains together with larval bioassays, we demonstrate that nAChRs, and the intracellular calcium concentration represent the key components of an adaptation strategy ensuring neuronal functions maintenance. In AcerKis neurons, the increased effect of acetylcholine related to the reduced acetylcholinesterase activity is compensated by expressing higher density of nAChRs permeable to calcium. In KdrKis neurons, changes in the biophysical properties of the L1014F mutant sodium channel, leading to enhance overlap between activation and inactivation relationships, diminish the resting membrane potential and reduce the fraction of calcium channels available involved in acetylcholine release. Together with the lower intracellular basal calcium concentration observed, these factors increase nAChRs sensitivity to maintain the effect of low concentration of acetylcholine. These results explain the opposite effects of the insecticide clothianidin observed in AcerKis and KdrKis neurons in vitro and in vivo.

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Characterization of LDD-2633 as a Novel RET Kinase Inhibitor with Anti-Tumor Effects in Thyroid Cancer

Pharmaceuticals ◽

10.3390/ph14010038 ◽

2021 ◽

Vol 14 (1) ◽

pp. 38

Author(s):

Hyo Jeong Lee ◽

Pyeonghwa Jeong ◽

Yeongyu Moon ◽

Jungil Choi ◽

Jeong Doo Heo ◽

...

Keyword(s):

Thyroid Cancer ◽

Thyroid Carcinoma ◽

Kinase Inhibitor ◽

Point Mutations ◽

Carcinoma Cells ◽

Medullary Thyroid ◽

Potent Inhibition ◽

Kinase Inhibitory Activity

Rearranged during transfection (RET), a receptor tyrosine kinase, is activated by glial cell line-derived neurotrophic factor family ligands. Chromosomal rearrangement or point mutations in RET are observed in patients with papillary thyroid and medullary thyroid carcinomas. Oncogenic alteration of RET results in constitutive activation of RET activity. Therefore, inhibiting RET activity has become a target in thyroid cancer therapy. Here, the anti-tumor activity of a novel RET inhibitor was characterized in medullary thyroid carcinoma cells. The indirubin derivative LDD-2633 was tested for RET kinase inhibitory activity. In vitro, LDD-2633 showed potent inhibition of RET kinase activity, with an IC50 of 4.42 nM. The growth of TT thyroid carcinoma cells harboring an RET mutation was suppressed by LDD-2633 treatment via the proliferation suppression and the induction of apoptosis. The effects of LDD-2633 on the RET signaling pathway were examined; LDD-2633 inhibited the phosphorylation of the RET protein and the downstream molecules Shc and ERK1/2. Oral administration of 20 or 40 mg/kg of LDD-2633 induced dose-dependent suppression of TT cell xenograft tumor growth. The in vivo and in vitro experimental results supported the potential use of LDD-2633 as an anticancer drug for thyroid cancers.

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Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL

International Journal of Molecular Sciences ◽

10.3390/ijms22010434 ◽

2021 ◽

Vol 22 (1) ◽

pp. 434

Author(s):

Yuria Jang ◽

Hong Moon Sohn ◽

Young Jong Ko ◽

Hoon Hyun ◽

Wonbong Lim

Keyword(s):

Nuclear Translocation ◽

Critical Role ◽

Osteoclast Differentiation ◽

Point Mutations ◽

Tartrate Resistant Acid Phosphatase ◽

Mice Model ◽

Gsk 3Β ◽

Rank Signaling

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

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Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.7.1811 ◽

1998 ◽

Vol 42 (7) ◽

pp. 1811-1814 ◽

Cited By ~ 45

Author(s):

Leonardo K. Basco ◽

Rachida Tahar ◽

Pascal Ringwald

Keyword(s):

Dihydrofolate Reductase ◽

Point Mutations ◽

Gene Mutations ◽

Adult Patients ◽

Wild Type ◽

Dihydropteroate Synthase ◽

Reductase Gene ◽

Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

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Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6866 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6866-6875 ◽

Cited By ~ 20

Author(s):

D C Hagen ◽

L Bruhn ◽

C A Westby ◽

G F Sprague

Keyword(s):

Saccharomyces Cerevisiae ◽

Binding Site ◽

Dna Sequences ◽

Point Mutations ◽

Transcription Activation ◽

Specific Gene ◽

Binding Assays ◽

Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

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