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.

Trypanosoma cruzi heparin-binding proteins mediate the adherence of epimastigotes to the midgut epithelial cells of Rhodnius prolixus

Parasitology ◽  
2012 ◽  
Vol 139 (6) ◽  
pp. 735-743 ◽  
Author(s):  
F. O. R. OLIVEIRA ◽  
C. R. ALVES ◽  
F. SOUZA-SILVA ◽  
C. M. CALVET ◽  
L. M. C. CÔRTES ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Rhodnius Prolixus ◽  
Insect Vector ◽  
Heparin Binding ◽  
Molecular Masses ◽  
Pre Treatment

SUMMARYHeparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.

scholarly journals Differential Incorporation of Cholesterol by Sindbis Virus Grown in Mammalian or Insect Cells

2009 ◽  
Vol 83 (18) ◽  
pp. 9113-9121 ◽  
Author(s):  
Amanda Hafer ◽  
Rebecca Whittlesey ◽  
Dennis T. Brown ◽  
Raquel Hernandez
Keyword(s):  
Cell Membrane ◽  
Cell Lines ◽  
Insect Cell ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Insect Cells ◽  
Virus Assembly ◽  
Critical Role ◽  
Insect Cell Line

ABSTRACT Cholesterol has been shown to be essential for the fusion of alphaviruses with artificial membranes (liposomes). Cholesterol has also been implicated as playing an essential and critical role in the processes of entry and egress of alphaviruses in living cells. Paradoxically, insects, the alternate host for alphaviruses, are cholesterol auxotrophs and contain very low levels of this sterol. To further evaluate the role of cholesterol in the life cycle of alphaviruses, the cholesterol levels of the alphavirus Sindbis produced from three different mosquito (Aedes albopictus) cell lines; one other insect cell line, Sf21 from Spodoptera frugiperda; and BHK (mammalian) cells were measured. Sindbis virus was grown in insect cells under normal culture conditions and in cells depleted of cholesterol by growth in serum delipidated by using Cab-O-sil, medium treated with methyl-β-cyclodextrin, or serum-free medium. The levels of cholesterol incorporated into the membranes of the cells and into the virus produced from these cells were determined. Virus produced from these treated and untreated cells was compared to virus grown in BHK cells under standard conditions. The ability of insect cells to produce Sindbis virus after delipidation was found to be highly cell specific and not dependent on the level of cholesterol in the cell membrane. A very low level of cholesterol was required for the generation of wild-type levels of infectious Sindbis virus from delipidated cells. The data show that one role of the virus membrane is structural, providing the stability required for infectivity that may not be provided by the delipidated membranes in some cells. These data show that the amount of cholesterol in the host cell membrane in and of itself has no effect on the process of virus assembly or on the ability of virus to infect cells. Rather, these data suggest that the cholesterol dependence reported for infectivity and assembly of Sindbis virus is a reflection of differences in the insect cell lines used and the methods of delipidation.

scholarly journals DC-SIGN and L-SIGN Can Act as Attachment Receptors for Alphaviruses and Distinguish between Mosquito Cell- and Mammalian Cell-Derived Viruses

2003 ◽  
Vol 77 (22) ◽  
pp. 12022-12032 ◽  
Author(s):  
William B. Klimstra ◽  
Elizabeth M. Nangle ◽  
M. Shane Smith ◽  
Andrew D. Yurochko ◽  
Kate D. Ryman
Keyword(s):  
Mammalian Cells ◽  
Reticuloendothelial System ◽  
Host Cells ◽  
Specific Monoclonal Antibody ◽  
Complex Carbohydrate ◽  
Virus Binding ◽  
Virus Particles ◽  
Mosquito Cells ◽  
A Cell ◽  
Arbovirus Infection

ABSTRACT C-type lectins such as DC-SIGN and L-SIGN, which bind mannose-enriched carbohydrate modifications of host and pathogen proteins, have been shown to bind glycoproteins of several viruses and facilitate either cis or trans infection. DC-SIGN and L-SIGN are expressed in several early targets of arbovirus infection, including dendritic cells (DCs) and cells of the reticuloendothelial system. In the present study, we show that DC-SIGN and L-SIGN can function as attachment receptors for Sindbis (SB) virus, an arbovirus of the Alphavirus genus. Human monocytic THP-1 cells stably transfected with DC-SIGN or L-SIGN were permissive for SB virus replication, while untransfected controls were essentially nonpermissive. The majority of control THP-1 cells were permissive when attachment and entry steps were eliminated through electroporation of virus transcripts. Infectivity for the DC-SIGN/L-SIGN-expressing cells was largely blocked by yeast mannan, EDTA, or a DC-SIGN/L-SIGN-specific monoclonal antibody. Infection of primary human DCs by SB virus was also dependent upon SIGN expression by similar criteria. Furthermore, production of virus particles in either C6/36 mosquito cells or CHO mammalian cells under conditions that limited complex carbohydrate content greatly increased SB virus binding to and infection of THP-1 cells expressing these lectins. C6/36-derived virus also was much more infectious for primary human DCs than CHO-derived virus. These results suggest that (i) lectin molecules such as DC-SIGN and L-SIGN may represent common attachment receptor molecules for arthropod-borne viruses, (ii) arbovirus particles produced in and delivered by arthropod vectors may preferentially target vertebrate host cells bearing these or similar lectin molecules, and (iii) a cell line has been identified that can productively replicate alphaviruses but is deficient in attachment receptors.

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 A glycan shield on chimpanzee CD4 protects against infection by primate lentiviruses (HIV/SIV)

2019 ◽  
Vol 116 (23) ◽  
pp. 11460-11469 ◽  
Author(s):  
Cody J. Warren ◽  
Nicholas R. Meyerson ◽  
Alex C. Stabell ◽  
Will T. Fattor ◽  
Gregory K. Wilkerson ◽  
...  
Keyword(s):  
Cell Receptor ◽  
Glycosylation Site ◽  
Surface Glycoprotein ◽  
Target Cells ◽  
Virus Binding ◽  
Binding Interface ◽  
Binding Surface ◽  
Primate Lentiviruses ◽  
And Function ◽  
Hiv 1

Pandemic HIV-1 (group M) emerged following the cross-species transmission of a simian immunodeficiency virus from chimpanzees (SIVcpz) to humans. Primate lentiviruses (HIV/SIV) require the T cell receptor CD4 to enter into target cells. By surveying the sequence and function of CD4 in 50 chimpanzee individuals, we find that all chimpanzee CD4 alleles encode a fixed, chimpanzee-specific substitution (34T) that creates a glycosylation site on the virus binding surface of the CD4 receptor. Additionally, a single nucleotide polymorphism (SNP) has arisen in chimpanzee CD4 (68T) that creates a second glycosylation site on the same virus-binding interface. This substitution is not yet fixed, but instead alleles containing this SNP are still circulating within chimpanzee populations. Thus, all allelic versions of chimpanzee CD4 are singly glycosylated at the virus binding surface, and some allelic versions are doubly glycosylated. Doubly glycosylated forms of chimpanzee CD4 reduce HIV-1 and SIVcpz infection by as much as two orders of magnitude. Full restoration of virus infection in cells bearing chimpanzee CD4 requires reversion of both threonines at sites 34 and 68, destroying both of the glycosylation sites, suggesting that the effects of the glycans are additive. Differentially glycosylated CD4 receptors were biochemically purified and used in neutralization assays and microscale thermophoresis to show that the glycans on chimpanzee CD4 reduce binding affinity with the lentiviral surface glycoprotein, Env. These glycans create a shield that protects CD4 from being engaged by viruses, demonstrating a powerful form of host resistance against deadly primate lentiviruses.

scholarly journals A Single Mutation in the E2 Glycoprotein Important for Neurovirulence Influences Binding of Sindbis Virus to Neuroblastoma Cells

2002 ◽  
Vol 76 (12) ◽  
pp. 6302-6310 ◽  
Author(s):  
Peiyu Lee ◽  
Ronald Knight ◽  
Jolanda M. Smit ◽  
Jan Wilschut ◽  
Diane E. Griffin
Keyword(s):  
Virus Strain ◽  
Sindbis Virus ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Neural Cells ◽  
Single Mutation ◽  
Virus Binding ◽  
Critical Determinant ◽  
Bhk Cells ◽  
E2 Glycoprotein

ABSTRACT The amino acid at position 55 of the E2 glycoprotein (E255) of Sindbis virus (SV) is a critical determinant of SV neurovirulence in mice. Recombinant virus strain TE (E255 = histidine) differs only at this position from virus strain 633 (E255= glutamine), yet TE is considerably more neurovirulent than 633. TE replicates better than 633 in a neuroblastoma cell line (N18), but similarly in BHK cells. Immunofluorescence staining showed that most N18 cells were infected by TE at a multiplicity of infection (MOI) of 50 to 500 and by 633 only at an MOI of 5,000, while both viruses infected essentially 100% of BHK cells at an MOI of 5. When exposed to pH 5, TE and 633 viruses fused to similar extents with liposomes derived from BHK or N18 cell lipids, but fusion with N18-derived liposomes was less extensive (15 to 20%) than fusion with BHK-derived liposomes (∼50%). Binding of TE and 633 to N18, but not BHK, cells was dependent on the medium used for virus binding. Differences between TE and 633 binding to N18 cells were evident in Dulbecco's modified Eagle medium (DMEM), but not in RPMI. In DMEM, the binding efficiency of 633 decreased significantly as the pH was raised from 6.5 to 8.0, while that of TE did not change. The same pattern was observed with RPMI when the ionic strength of RPMI was increased to that of DMEM. TE bound better to heparin-Sepharose than 633, but this difference was not pH dependent. Growth of N18 and BHK cells in sodium chlorate to eliminate all sulfation decreased virus-cell binding, suggesting the involvement of sulfated molecules on the cell surface. Taken together, the presence of glutamine at E255 impairs SV binding to neural cells under conditions characteristic of interstitial fluid. We conclude that mutation to histidine participates in or stabilizes the interaction between the virus and the surface of neural cells, contributing to greater neurovirulence.

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 Single Amino Acid Insertions at the Junction of the Sindbis Virus E2 Transmembrane Domain and Endodomain Disrupt Virus Envelopment and Alter Infectivity

2005 ◽  
Vol 79 (12) ◽  
pp. 7682-7697 ◽  
Author(s):  
Raquel Hernandez ◽  
Davis Ferreira ◽  
Christine Sinodis ◽  
Katherine Litton ◽  
Dennis T. Brown
Keyword(s):  
Amino Acid ◽  
Sindbis Virus ◽  
Insect Cells ◽  
Virus Assembly ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Insertion Mutant ◽  
Bhk Cells ◽  
Virus Particles

ABSTRACT The final steps in the envelopment of Sindbis virus involve specific interactions of the E2 endodomain with the virus nucleocapsid. Deleting E2 K at position 391 (E2 ΔK391) resulted in the disruption of virus assembly in mammalian cells but not insect cells (host range mutant). This suggested unique interactions of the E2 ΔK391 endodomain with the different biochemical environments of the mammalian and insect cell lipid bilayers. To further investigate the role of the amino acid residues located at or around position E2 391 and constraints on the length of the endodomain on virus assembly, amino acid insertions/substitutions at the transmembrane/endodomain junction were constructed. An additional K was inserted at amino acid position 392 (KK391/392), a K→F substitution at position 391 was constructed (F391), and an additional F was inserted at 392 (FF391/392). These changes should lengthen the endodomain in the KK391/392 insertion mutant or shorten the endodomain in the FF391/392 mutant. The mutant FF391/392 grown in BHK cells formed virus particles containing extruded material not found on wild-type virus. This characteristic was not seen in FF391/392 virus grown in insect cells. The mutant KK391/392 grown in BHK cells was defective in the final membrane fission reaction, producing multicored or conjoined virus particles. The production of these aberrant particles was ameliorated when the KK391/392 mutant was grown in insect cells. These data indicate that there is a critical minimal spanning distance from the E2 membrane proximal amino acid at position 391 and the conserved E2 Y400 residue. The observed phenotypes of these mutants also invoke an important role of the specific host membrane lipid composition on virus architecture and infectivity.

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