scholarly journals Mutations at the Alphavirus E2/E1 inter-dimer interface have host-specific phenotypes

2021 ◽  
Author(s):  
Sophia C. Ren ◽  
Shefah A. Qazi ◽  
Brian Towell ◽  
Joseph CY Wang ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Viral Entry ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Particle Surface ◽  
Antiviral Drug ◽  
Particle Morphology ◽  
Mutant Virus ◽  
New Host ◽  
Particle Assembly ◽  
Mosquito Cells

ABSTRACTAlphaviruses are enveloped viruses that are transmitted by an arthropod vector to vertebrate hosts. Alphaviruses have glycoprotein spikes on their particle surface which are essential for viral entry. Each of the 80 spikes on the surface of an alphavirus particle consists of a trimer of E2-E1 heterodimers. Two types of interactions make up the spikes: (1) interactions between E2 and E1 of the same heterodimer called intra-dimer contacts, and (2) inter-dimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (called E1’). We hypothesized that the inter-dimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus where we replaced six inter-dimeric residues in Sindbis virus (WT SINV) with those from Chikungunya virus (CHIKV); the mutant is called CPB. CPB grew slower and to lower levels than WT SINV in mammalian cells, but not mosquito cells. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to capsid protein, and defects in particle morphology compared to mosquito cells. CPB transported glycoproteins to the plasma membrane in similar amounts to WT SINV in mammalian cells. Two revertants, E2-H333N and E1-S247L, restored particle assembly to different degrees. The viruses were visualized by cryo-EM. We determined that the spikes of CPB had a different conformation than WT SINV or the revertants. We conclude that the inter-dimer mutant, CPB, has host-dependent defects in spike trimerization and/or particle budding in mammalian cells.IMPORTANCEAlphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as an emerging pathogen with a global distribution. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This is important because it identifies a region in the spike that could be a target for antiviral drug design.

scholarly journals Role of N-Linked Glycosylation for Sindbis Virus Infection and Replication in Vertebrate and Invertebrate Systems

2009 ◽  
Vol 83 (11) ◽  
pp. 5640-5647 ◽  
Author(s):  
Ronald L. Knight ◽  
Kimberly L. W. Schultz ◽  
Rebekah J. Kent ◽  
Meera Venkatesan ◽  
Diane E. Griffin
Keyword(s):  
Mammalian Cells ◽  
Sindbis Virus ◽  
Glycosylation Site ◽  
Surface Glycoprotein ◽  
Heparin Binding ◽  
Virus Binding ◽  
Bhk Cells ◽  
Mosquito Cells ◽  
Intrathoracic Inoculation

ABSTRACT Each Sindbis virus (SINV) surface glycoprotein has two sites for N-linked glycosylation (E1 positions 139 and 245 [E1-139 and E1-245] and E2 positions 196 and 318 [E2-196 and E2-318]). Studies of SINV strain TE12 mutants with each site eliminated identified the locations of carbohydrates by cryo-electron microscopy (S. V. Pletnev et al., Cell 105:127-136, 2001). In the current study, the effects of altered glycosylation on virion infectivity, growth in cells of vertebrates and invertebrates, heparin binding, virulence in mice, and replication in mosquitoes were assessed. Particle-to-PFU ratios for E1-139 and E2-196 mutant strains were similar to that for TE12, but this ratio for the E1-245 mutant was 100-fold lower than that for TE12. Elimination of either E2 glycosylation site increased virus binding to heparin and increased replication in BHK cells. Elimination of either E1 glycosylation site had no effect on heparin binding but resulted in an approximately 10-fold decrease in virus yield from BHK cells compared to the TE12 amount. No differences in pE2 processing were detected. E2-196 and E2-318 mutants were more virulent in mice after intracerebral inoculation, while E1-139 and E1-245 mutants were less virulent. The E1-245 mutant showed impaired replication in C7/10 mosquito cells and in Culex quinquefasciatus after intrathoracic inoculation. We conclude that the increased replication and virulence of E2-196 and E2-318 mutants are primarily due to increased efficiency of binding to heparan sulfate on mammalian cells. Lack of glycosylation at E1-139 or E1-245 impairs replication in vertebrate cells, while E1-245 also severely affects replication in invertebrate cells.

scholarly journals Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells

2008 ◽  
Vol 82 (20) ◽  
pp. 10088-10101 ◽  
Author(s):  
Rodion Gorchakov ◽  
Natalia Garmashova ◽  
Elena Frolova ◽  
Ilya Frolov
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Protein Complexes ◽  
Nonstructural Proteins ◽  
Cell Nuclei ◽  
High Concentration ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Rna Complexes

ABSTRACT Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

scholarly journals The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

2021 ◽  
Author(s):  
Monica L Husby ◽  
Souad Amiar ◽  
Laura I Prugar ◽  
Emily A David ◽  
Caroline B Plescia ◽  
...  
Keyword(s):  
Viral Entry ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Particle Morphology ◽  
Matrix Proteins ◽  
In Vitro Assays ◽  
Virus Particles ◽  
Viral Budding

Phosphatidylserine (PS) has been shown to be a critical lipid factor in the assembly and spread of numerous lipid enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA approved drug, fendiline, to reduce PS clustering subsequently reducing virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were employed including imaging, viral budding and viral entry assays. Fendiline reduced the PS content in mammalian cells and PS in the plasma membrane, reducing the ability of VP40 to form new virus particles. Further, particles that do form from fendiline treated cells have altered particle morphology and decreased infectivity capacity. These complementary studies reveal the mechanism by which filovirus matrix proteins cluster PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.

scholarly journals Antiviral Compounds for Blocking Arboviral Transmission in Mosquitoes

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 108
Author(s):  
Shengzhang Dong ◽  
George Dimopoulos
Keyword(s):  
High Throughput Screening ◽  
Mammalian Cells ◽  
Symbiotic Bacterium ◽  
Screening Methods ◽  
Human Pathogens ◽  
Inhibitory Effects ◽  
Antiviral Compounds ◽  
Mosquito Cells ◽  
Recent Emergence ◽  
Arboviral Transmission

Mosquito-borne arthropod-borne viruses (arboviruses) such as the dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) are important human pathogens that are responsible for significant global morbidity and mortality. The recent emergence and re-emergence of mosquito-borne viral diseases (MBVDs) highlight the urgent need for safe and effective vaccines, therapeutics, and vector-control approaches to prevent MBVD outbreaks. In nature, arboviruses circulate between vertebrate hosts and arthropod vectors; therefore, disrupting the virus lifecycle in mosquitoes is a major approach for combating MBVDs. Several strategies were proposed to render mosquitoes that are refractory to arboviral infection, for example, those involving the generation of genetically modified mosquitoes or infection with the symbiotic bacterium Wolbachia. Due to the recent development of high-throughput screening methods, an increasing number of drugs with inhibitory effects on mosquito-borne arboviruses in mammalian cells were identified. These antivirals are useful resources that can impede the circulation of arboviruses between arthropods and humans by either rendering viruses more vulnerable in humans or suppressing viral infection by reducing the expression of host factors in mosquitoes. In this review, we summarize recent advances in small-molecule antiarboviral drugs in mammalian and mosquito cells, and discuss how to use these antivirals to block the transmission of MBVDs.

Sindbis virus maturation in cultured mosquito cells is sensitive to actinomycin D

Virology ◽  
1981 ◽  
Vol 110 (2) ◽  
pp. 292-301 ◽  
Author(s):  
Ursula Scheefers-Borchel ◽  
Hans Scheefers ◽  
Judy Edwards ◽  
Dennis T. Brown
Keyword(s):  
Sindbis Virus ◽  
Actinomycin D ◽  
Virus Maturation ◽  
Mosquito Cells

scholarly journals Effects of inducing or inhibiting apoptosis on Sindbis virus replication in mosquito cells

2008 ◽  
Vol 89 (11) ◽  
pp. 2651-2661 ◽  
Author(s):  
Hua Wang ◽  
Carol D. Blair ◽  
Ken E. Olson ◽  
Rollie J. Clem
Keyword(s):  
Virus Replication ◽  
Persistent Infection ◽  
Sindbis Virus ◽  
Actinomycin D ◽  
Virus Production ◽  
Cell Types ◽  
Lytic Infection ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Apoptotic Genes

Sindbis virus (SINV) is a mosquito-borne virus in the genus Alphavirus, family Togaviridae. Like most alphaviruses, SINVs exhibit lytic infection (apoptosis) in many mammalian cell types, but are generally thought to cause persistent infection with only moderate cytopathic effects in mosquito cells. However, there have been several reports of apoptotic-like cell death in mosquitoes infected with alphaviruses or flaviviruses. Given that apoptosis has been shown to be an antiviral response in other systems, we have constructed recombinant SINVs that express either pro-apoptotic or anti-apoptotic genes in order to test the effects of inducing or inhibiting apoptosis on SINV replication in mosquito cells. Recombinant SINVs expressing the pro-apoptotic genes reaper (rpr) from Drosophila or michelob_x (mx) from Aedes aegypti caused extensive apoptosis in cells from the mosquito cell line C6/36, thus changing the normal persistent infection observed with SINV to a lytic infection. Although the infected cells underwent apoptosis, high levels of virus replication were still observed during the initial infection. However, virus production subsequently decreased compared with persistently infected cells, which continued to produce high levels of virus over the next several days. Infection of C6/36 cells with SINV expressing the baculovirus caspase inhibitor P35 inhibited actinomycin D-induced caspase activity and protected infected cells from actinomycin D-induced apoptosis, but had no observable effect on virus replication. This study is the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells.

scholarly journals The Host Range Phenotype Displayed by a Sindbis Virus Glycoprotein Variant Results from Virion Aggregation and Retention on the Surface of Mosquito Cells

2000 ◽  
Vol 74 (23) ◽  
pp. 11398-11406 ◽  
Author(s):  
Karl W. Boehme ◽  
Vsevolod L. Popov ◽  
Hans W. Heidner
Keyword(s):  
Cell Surface ◽  
Host Range ◽  
Sindbis Virus ◽  
Cell Type ◽  
Virus Variant ◽  
Cytoplasmic Vacuoles ◽  
Mosquito Cells ◽  
Transmission Electron ◽  
Maturation Defect ◽  
Processing Properties

ABSTRACT The Sindbis virus variant NE2G216 is a PE2-containing host range mutant that is growth restricted in cultured mosquito cells (C6/36) due to inefficient release of virions from this cell type. The maturation defect of NE2G216 has been linked to the structures of N-linked oligosaccharides synthesized by arthropod cells. Analysis of C6/36 cells infected with NE2G216 by transmission electron microscopy revealed the presence of dense virus aggregates within cytoplasmic vacuoles and virus aggregates adhered to the cell surface. The virus aggregation phenotype of NE2G216 was reproduced in vertebrate cells (Pro-5) by the addition of 1-deoxymannojirimycin, an inhibitor of carbohydrate processing which limits the processing of N-linked oligosaccharides to structures that are structurally similar, albeit not identical, to those synthesized in C6/36 cells. We conclude that defective maturation of NE2G216 in mosquito cells is due to virion aggregation and retention on the cell surface and that this phenotype is directly linked to the carbohydrate-processing properties of these cells.

Vapor phase synthesis of Al-doped titania powders

1994 ◽  
Vol 9 (5) ◽  
pp. 1241-1249 ◽  
Author(s):  
Kamal M. Akhtar ◽  
Sotiris E. Pratsinis ◽  
Sebastian V.R. Mastrangelo
Keyword(s):  
Vapor Phase ◽  
Flow Reactor ◽  
Particle Surface ◽  
Particle Morphology ◽  
High Concentration ◽  
Phase Synthesis ◽  
Transmission Electron ◽  
Significant Enrichment ◽  
Vapor Phase Synthesis ◽  
Doped Titania

The role of aluminum as dopant in gas phase synthesis of titania powders was experimentally investigated in an aerosol flow reactor between 1300 and 1700 K. Titania was produced by vapor phase oxidation of titanium tetrachloride in the presence of dopant aluminum trichloride vapor. The presence of aluminum altered the particle morphology from polyhedral to irregular crystals. Energy dispersive analysis and transmission electron microscopy indicated that the powders were mixtures of crystalline titania and amorphous alumina. Analysis by XPS indicated significant enrichment of aluminum on the particle surface. Some aluminum titanate (up to 17% by volume) was formed at 1700 K when a high concentration of AlCl3 was used (AlCl3/TiCl4 ≥ 0.07). Measurements of lattice parameters by x-ray diffraction indicated that aluminum formed a solid solution in titania. While titania synthesized in the absence of aluminum was about 90% anatase, the introduction of aluminum resulted in pure rutile at AlCl3/TiCl4 = 0.07. The effects of aluminum on titania phase composition and morphology are explained by the creation of oxygen vacancies in the titania crystallites and by the enhancement of the sintring rate of titania grains.

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