Role of N-Linked Glycans on Bunyamwera Virus Glycoproteins in Intracellular Trafficking, Protein Folding, and Virus Infectivity

). 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.Sindbis virus (SINV), the prototype alphavirus in the family Togaviridae, is an arthropod-borne virus that cycles between mosquitoes and wild birds and causes summertime outbreaks of rashes and arthritis in humans (17, 34). In mice, SINV causes encephalomyelitis and serves as a useful model for the study of alphavirus infections of the central nervous system (CNS). The virion is enveloped and has icosahedral symmetry, with a single linear strand of positive-sense RNA surrounded by a capsid and two transmembrane glycoproteins, E1 and E2, that heterodimerize and then form trimeric spikes on the surface. Each of the viral glycoproteins has two N-linked glycosylation sites at E1 positions 139 and 245 (E1-139 and E1-245) and at E2 positions 196 and 318 (E2-196 and E2-318) that are used (4, 42). Some glycosylation is required for proper folding and transport of the glycoproteins, as total ablation of glycosylation by treatment with tunicamycin results in failure of E1 and E2 to be transported through the Golgi apparatus to the cell surface (31, 32).The type of carbohydrate, high-mannose carbohydrate or complex oligosaccharide, at a glycosylation site varies with the type and growth state of the cell infected (3,18,23,28,29,35,51). In insect cells, all sites have high-mannose sugars (22) due to the absence of N-acetylglucosaminyl-, galactosyl-, and sialyltransferases (5). In vertebrate cells, the distribution of complex and high-mannose carbohydrates is largely determined by the accessibility of the site to processing enzymes in the Golgi apparatus (23).E1 has an extracellular domain that contains the internal fusion peptide. The carbohydrate at E1-139 consists of complex oligosaccharides in vertebrate cell...

Section: Discussionmentioning

confidence: 99%

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

Knight

Schultz

Kent

et al. 2009

“…Vero E6 (ATCC C1008), BHK-21, and BSR-T7/5 cells (4) were maintained as described previously (47). Working stocks of wild-type (wt) and mutant BUNV were grown in BHK-21 cells, and titers were determined by plaque assay as detailed previously (57).…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Both Gn and Gc are type I integral transmembrane proteins that are modified by N-linked glycosylation. The signal for Golgi retention and targeting of the BUNV glycoproteins was mapped to the transmembrane domain of the Gn protein, and heterodimerization between Gn and Gc is crucial for the Golgi transportation and maturation of the larger Gc protein (27,47,50). The BUNV Gn protein consists of 302 residues (M r , 32,000) with a predicted cytoplasmic tail (CT) domain of 78 amino acids (residues 225 to 302).…”

mentioning

confidence: 99%

Role of the Cytoplasmic Tail Domains of Bunyamwera Orthobunyavirus Glycoproteins Gn and Gc in Virus Assembly and Morphogenesis

Shi

Kohl

et al. 2007

Self Cite

The M RNA genome segment of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, encodes a precursor polyprotein that is proteolytically cleaved to yield two structural proteins, Gn and Gc, and a nonstructural protein called NSm. Gn and Gc are type I integral transmembrane glycoproteins. The Gn protein contains a predicted cytoplasmic tail (CT) of 78 residues, and Gc has a shorter CT of 25 residues. Little is known about the role of the Gn and Gc CT domains in the virus replication cycle. We generated a series of mutant glycoprotein precursor constructs containing either deletions or alanine substitutions in the CT domains of Gn and Gc. We examined the effects of these mutations on glycoprotein maturation, cell surface expression, and low pH-induced syncytium formation. In addition, the effects of these mutations were also assessed using a reverse genetics-based virus assembly assay and a virus rescue system. Our results show that the CT domains of both Gn and Gc play crucial roles in BUNV-mediated membrane fusion, virus assembly, and morphogenesis.

“…Even though glycosylation of E rns , E1, or E2 protein may play a significant role in the CSFV viral replication cycle, the function of added oligosaccharides is not known. In general, glycosylation of enveloped virus structural proteins has been shown to be important for receptor binding, membrane fusion, penetration, virus budding, and infectivity as analyzed in cultured cells (1,5,9,34,35). However, the significance of viral envelope protein glycosylation in virus replication, pathogenesis, and virulence in the natural host is unknown.…”

mentioning

confidence: 99%

N-Linked Glycosylation Status of Classical Swine Fever Virus Strain Brescia E2 Glycoprotein Influences Virulence in Swine

Risatti

Holinka

Sainz

et al. 2007

E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.