Heparan Sulfate Binding Can Contribute to the Neurovirulence of Neuroadapted and Nonneuroadapted Sindbis Viruses

). 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: 53%

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

Knight

Schultz

Kent

et al. 2009

“…Possible mechanisms of attenuation for 3= DP vectors include competition between the authentic subgenomic promoter and the artificial second promoter for limiting viral factors and/or an unidentified effect upon virus packaging or host responses to infection associated with this particular change in the structure of the alphavirus genome. Overall, our results should substantially temper conclusions drawn from experiments utilizing DP viruses expressing fLuc or fluorescent proteins that have been used to track virus replication in animals (3,6,27,28), especially later in infection.…”

Section: Discussionmentioning

confidence: 60%

“…While a number of studies have examined expression of fLuc from DP viruses (3,6,24,28) using in vivo imaging, nLuc has yet to be fully tested and may compromise some sensitivity in comparison to fLuc due to a blue shift of light emitted resulting in increased potential for absorption by animal tissues (16). However, this may be compensated by the increased luminescence per molecule of nLuc as well as resistance of nLuc to pH and temperature variations and lack of ATP requirement compared with fLuc (16).…”

Section: Discussionmentioning

confidence: 99%

Stable, High-Level Expression of Reporter Proteins from Improved Alphavirus Expression Vectors To Track Replication and Dissemination during Encephalitic and Arthritogenic Disease

Sun

Gardner

Watson

et al. 2014

Self Cite

108

127

Engineered alphavirus vectors expressing reporters of infection have been used for a number of years due to their relatively low costs for analysis of virus replication and the capacity to utilize imaging systems for longitudinal measurements of growth within single animals. In general, these vectors have been derived from Old World alphaviruses using a second viral subgenomic promoter to express the transgenes, placed either immediately after the nonstructural proteins or at the 3= end of the viral coding sequences. However, the relevance of these vectors to natural infections is questionable, as they have not been rigorously tested for virulence in vivo in comparison with parental viruses or for the retention of the reporter during replication. Here, we report construction of new expression vectors for two Old World arthritogenic alphaviruses (Sindbis and Chikungunya viruses) and two New World encephalitic alphaviruses (eastern and Venezuelan equine encephalitis viruses) based upon either fusion of the reporter protein in frame within nonstructural protein 3 (nsP3) or insertion of the reporter as a cleavable element between the capsid and PE2 structural proteins. We have compared these with a traditional 3= double subgenomic promoter virus expressing either a large, firefly luciferase (fLuc; 1,650 nucleotides), or small, NanoLuc (nLuc; 513 nucleotides), luminescent reporter protein. Results indicate that the nLuc is substantially more stable than fLuc during repeated rounds of infection regardless of the transgene location. However, the capsid-PE2 insertion and nsP3 fusion viruses exhibit the most authentic mimicking of parental virus infection regardless of expressed protein. IMPORTANCEAs more antiviral therapeutics and vaccines are developed, rapid and accurate in vivo modeling of their efficacy will be required. However, current alphavirus vectors expressing reporters of infection have not been extensively tested for accurate mimicking of the infection characteristics of unmodified parental viruses. Additionally, use of in vivo imaging systems detecting light emitted from luciferase reporters can significantly decrease costs associated with efficacy studies by minimizing numbers of animals. Herein we report development and testing of new expression vectors for Sindbis, Chikungunya, and eastern and Venezuelan equine encephalitis viruses and demonstrate that a small (ϳ500-nucleotide) reporter gene (NanoLuc; Promega) is very stable and causes a disease severity similar to that caused by unmodified parental viruses. In contrast, expression of larger reporters is very rapidly lost with virus replication and can be significantly attenuating. The utility of NanoLuc for in vivo imaging is also demonstrated.

“…To eliminate the possibility that the expression pattern variation was related to the differences in infectivity, we expressed DENV and WNV NS1 ectopically using a double subgenomic Sindbis virus (SINV) (53,55). The complete DENV (amino acids 1 to 352) and WNV (amino acids 1 to 352) NS1 genes were cloned downstream of their respective 24-or 25-amino-acid leader sequences (C terminus of the E gene) and a second 26S subgenomic promoter of SINV (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant wild-type or chimeric NS1 was expressed transgenically using a double subgenomic Sindbis virus (SINV) expression system. Recombinant SINVs were constructed using the infectious clone p39-E2H55K70 (p39HK; gift of W. Klimstra, Pittsburgh, PA) as described previously (55). The wild-type or chimeric NS1 gene was digested with NotI from the pSTBlue vector.…”

mentioning

confidence: 99%

A Short N-Terminal Peptide Motif on Flavivirus Nonstructural Protein NS1 Modulates Cellular Targeting and Immune Recognition

Youn¹,

Cho

Fremont

et al. 2010

Flavivirus NS1 is a versatile nonstructural glycoprotein, with intracellular NS1 functioning as an essential cofactor for viral replication and cell surface and secreted NS1 antagonizing complement activation. Even though NS1 has multiple functions that contribute to virulence, the genetic determinants that regulate the spatial distribution of NS1 in cells among different flaviviruses remain uncharacterized. Here, by creating a panel of West Nile virus-dengue virus (WNV-DENV) NS1 chimeras and site-specific mutants, we identified a novel, short peptide motif immediately C-terminal to the signal sequence cleavage position that regulates its transit time through the endoplasmic reticulum and differentially directs NS1 for secretion or plasma membrane expression. Exchange of two amino acids within this motif reciprocally changed the cellular targeting pattern of DENV or WNV NS1. For WNV, this substitution also modulated infectivity and antibody-induced phagocytosis of infected cells. Analysis of a mutant lacking all three conserved N-linked glycosylation sites revealed an independent requirement of N-linked glycans for secretion but not for plasma membrane expression of WNV NS1. Collectively, our experiments define the requirements for cellular targeting of NS1, with implications for the protective host responses, immune antagonism, and association with the host cell sorting machinery. These studies also suggest a link between the effects of NS1 on viral replication and the levels of secreted or cell surface NS1.