The role of host immunocompetence in neuroinvasion of Sindbis virus

1999 ◽  
Vol 144 (6) ◽  
pp. 1159-1171 ◽  
Author(s):  
S. Lustig ◽  
M. Halevy ◽  
D. Ben-Nathan ◽  
C. M. Rice ◽  
D. Kobiler
Keyword(s):  
Sindbis Virus ◽  
Host Immunocompetence

scholarly journals Role of Sindbis Virus Capsid Protein Region II in Nucleocapsid Core Assembly and Encapsidation of Genomic RNA

2008 ◽  
Vol 82 (9) ◽  
pp. 4461-4470 ◽  
Author(s):  
Ranjit Warrier ◽  
Benjamin R. Linger ◽  
Barbara L. Golden ◽  
Richard J. Kuhn
Keyword(s):  
Amino Acids ◽  
Capsid Protein ◽  
Sindbis Virus ◽  
Rna Virus ◽  
Genomic Rna ◽  
Viral Glycoproteins ◽  
Infected Cells ◽  
Viral Genomic Rna ◽  
Region Ii

ABSTRACT Sindbis virus is an enveloped positive-sense RNA virus in the alphavirus genus. The nucleocapsid core contains the genomic RNA surrounded by 240 copies of a single capsid protein. The capsid protein is multifunctional, and its roles include acting as a protease, controlling the specificity of RNA that is encapsidated into nucleocapsid cores, and interacting with viral glycoproteins to promote the budding of mature virus and the release of the genomic RNA into the newly infected cell. The region comprising amino acids 81 to 113 was previously implicated in two processes, the encapsidation of the viral genomic RNA and the stable accumulation of nucleocapsid cores in the cytoplasm of infected cells. In the present study, specific amino acids within this region responsible for the encapsidation of the genomic RNA have been identified. The region that is responsible for nucleocapsid core accumulation has considerable overlap with the region that controls encapsidation specificity.

scholarly journals ISG15 Arg151 and the ISG15-Conjugating Enzyme UbE1L Are Important for Innate Immune Control of Sindbis Virus

2008 ◽  
Vol 83 (4) ◽  
pp. 1602-1610 ◽  
Author(s):  
Nadia V. Giannakopoulos ◽  
Elena Arutyunova ◽  
Caroline Lai ◽  
Deborah J. Lenschow ◽  
Arthur L. Haas ◽  
...  
Keyword(s):  
Virus Infection ◽  
Survival Benefit ◽  
Sindbis Virus ◽  
Innate Immune ◽  
Protein Conjugates ◽  
Antiviral Effects ◽  
Antiviral Function ◽  
Alpha Beta

ABSTRACT Interferon (IFN)-stimulated gene 15 (ISG15) is a ubiquitin-like molecule that conjugates to target proteins via a C-terminal LRLRGG motif and has antiviral function in vivo. We used structural modeling to predict human ISG15 (hISG15) residues important for interacting with its E1 enzyme, UbE1L. Kinetic analysis revealed that mutation of arginine 153 to alanine (R153A) ablated hISG15-hUbE1L binding and transthiolation of UbcH8. Mutation of other predicted UbE1L-interacting residues had minimal effects on the transfer of ISG15 from UbE1L to UbcH8. The capacity of hISG15 R153A to form protein conjugates in 293T cells was markedly diminished. Mutation of the homologous residue in mouse ISG15 (mISG15), arginine 151, to alanine (R151A) also attenuated protein ISGylation following transfection into 293T cells. We assessed the role of ISG15-UbE1L interactions in control of virus infection by constructing double subgenomic Sindbis viruses that expressed the mISG15 R151A mutant. While expression of mISG15 protected alpha/beta-IFN-receptor-deficient (IFN-αβR−/−) mice from lethality following Sindbis virus infection, expression of mISG15 R151A conferred no survival benefit. The R151A mutation also attenuated ISG15's ability to decrease Sindbis virus replication in IFN-αβR−/− mice or prolong survival of ISG15−/− mice. The importance of UbE1L was confirmed by demonstrating that mice lacking this ISG15 E1 enzyme were highly susceptible to Sindbis virus infection. Together, these data support a role for protein conjugation in the antiviral effects of ISG15.

scholarly journals Rubella virus-like replicon particles: analysis of encapsidation determinants and non-structural roles of capsid protein in early post-entry replication

2012 ◽  
Vol 93 (3) ◽  
pp. 516-525 ◽  
Author(s):  
Claudia Claus ◽  
Wen-Pin Tzeng ◽  
U. G Liebert ◽  
Teryl K. Frey
Keyword(s):  
Capsid Protein ◽  
Rubella Virus ◽  
Sindbis Virus ◽  
Packaging Signal ◽  
Initiation Of Replication ◽  
Replicase Proteins ◽  
Infected Cells ◽  
In Trans ◽  
Post Entry

Rubella virus (RUBV) contains a plus-strand RNA genome with two ORFs, one encoding the non-structural replicase proteins (NS-ORF) and the second encoding the virion structural proteins (SP-ORF). This study describes development and use of a trans-encapsidation system for the assembly of infectious RUBV-like replicon particles (VRPs) containing RUBV replicons (self replicating genomes with the SP-ORF replaced with a reporter gene). First, this system was used to map signals within the RUBV genome that mediate packaging of viral RNA. Mutations within a proposed packaging signal did not significantly affect relative packaging efficiency. The insertion of various fragments derived from the RUBV genome into Sindbis virus replicons revealed that there are several regions within the RUBV genome capable of enhancing encapsidation of heterologous replicon RNAs. Secondly, the trans-encapsidation system was used to analyse the effect of alterations within the capsid protein (CP) on release of VRPs and subsequent initiation of replication in newly infected cells. Deletion of the N-terminal eight amino acids of the CP reduced VRP titre significantly, which could be partially complemented by native CP provided in trans, indicating that this mutation affected an entry or post-entry event in the replication cycle. To test this hypothesis, the trans-encapsidation system was used to demonstrate the rescue of a lethal deletion within P150, one of the virus replicase proteins, by CP contained within the virus particle. This novel finding substantiated the functional role of CP in early post-entry replication.

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 The Cholesterol Requirement for Sindbis Virus Entry and Exit and Characterization of a Spike Protein Region Involved in Cholesterol Dependence

1999 ◽  
Vol 73 (5) ◽  
pp. 4272-4278 ◽  
Author(s):  
Yanping E. Lu ◽  
Todd Cassese ◽  
Margaret Kielian
Keyword(s):  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Virus Entry ◽  
Single Point ◽  
Endocytic Pathway ◽  
Progeny Virus ◽  
Single Point Mutation ◽  
Entry And Exit ◽  
Wild Type

ABSTRACT Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV. Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV,srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had thesrf-3 amino acid sequence from E1 position 224 to 235 showed increases of ∼100-fold in infection and ∼250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGMexit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region.

On the infectivity of the Sindbis virus nucleocapsid

1970 ◽  
Vol 16 (12) ◽  
pp. 1273-1283 ◽  
Author(s):  
P. Dobos ◽  
P. Faulkner
Keyword(s):  
Infected Cell ◽  
Viral Replication ◽  
Cell Cultures ◽  
Sindbis Virus ◽  
Envelope Proteins ◽  
Intact Virus

The nucleocapsid fraction derived either from Sindbis virions or from infected cell cultures was studied with respect to its ability to adsorb to susceptible cells as well as to initiate stages in viral replication. Neither preparation was adsorbed to BHK 21 cells under conditions where virion adsorption could be demonstrated. In addition parental RNA from virions was shown to be converted to an RNAase-resistant 16–20 S form, whereas no evidence for the penetration and conversion of RNA present in viral cores was observed. These studies emphasize the role of envelope proteins in facilitating entry of intact virus into susceptible cells.

scholarly journals Location and Role of Free Cysteinyl Residues in the Sindbis Virus E1 and E2 Glycoproteins

2007 ◽  
Vol 81 (12) ◽  
pp. 6231-6240 ◽  
Author(s):  
Christopher B. Whitehurst ◽  
Erik J. Soderblom ◽  
Michelle L. West ◽  
Raquel Hernandez ◽  
Michael B. Goshe ◽  
...  
Keyword(s):  
Virus Particle ◽  
Disulfide Bonds ◽  
Sindbis Virus ◽  
Rna Virus ◽  
Virus Maturation ◽  
Virus Particles ◽  
Native Virus ◽  
Mature Virus Particle ◽  
Liquid Chromatography Tandem Mass

ABSTRACT Sindbis virus is a single-stranded positive-sense RNA virus. It is composed of 240 copies of three structural proteins: E1, E2, and capsid. These proteins form a mature virus particle composed of two nested T=4 icosahedral shells. A complex network of disulfide bonds in the E1 and E2 glycoproteins is developed through a series of structural intermediates as virus maturation occurs (M. Mulvey and D. T. Brown, J. Virol. 68:805-812, 1994; M. Carleton et al., J. Virol. 71:1558-1566, 1997). To better understand the nature of this disulfide network, E1 and E2 cysteinyl residues were labeled with iodoacetamide in the native virus particle and analyzed by liquid chromatography-tandem mass spectrometry. This analysis identified cysteinyl residues of E1 and E2, which were found to be label accessible in the native virus particle, as well as those that were either label inaccessible or blocked by their involvement in disulfide bonds. Native virus particles alkylated with iodoacetamide demonstrated a 4-log decrease in viral infectivity. This suggests that the modification of free cysteinyl residues results in the loss of infectivity by destabilizing the virus particle or that a rearrangement of disulfide bonds, which is required for infectivity, is blocked by the modification. Although modification of these residues prevented infectivity, it did not alter the ability of virus to fuse cells after exposure to acidic pH; thus, modification of free cysteinyl residues biochemically separated the process of infection from the process of membrane fusion.

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