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.

scholarly journals Single-Site Glycoprotein Mutants Inhibit a Late Event in Sindbis Virus Assembly

2016 ◽  
Vol 90 (18) ◽  
pp. 8372-8380 ◽  
Author(s):  
Joseph Magliocca ◽  
Ricardo Vancini ◽  
Raquel Hernandez ◽  
Dennis T. Brown
Keyword(s):  
Amino Acid ◽  
Sindbis Virus ◽  
Deletion Mutant ◽  
Virus Assembly ◽  
Single Site ◽  
Progeny Virus ◽  
Virus Protein ◽  
Baby Hamster Kidney ◽  
Bhk Cells ◽  
Virus Particles

ABSTRACTA panel of Sindbis virus mutants that were suspected to have deficiencies in one or more aspects of their replication cycles was examined in baby hamster kidney (BHK) cells. These included an amino acid deletion (ΔH230) and substitution (H230A) in the Sindbis glycoprotein E1_H230 and similar mutants in E2_G209 (G209A, G209D, and ΔG209). Neither H230 mutation produced a measurable titer, but repeated passaging of the H230A mutant in BHK cells produced a second-site compensatory mutant (V231I) that partially rescued both H230 mutants. Electron micrograph (EM) images of these mutants showed assembled viral nucleocapsids but no completed, mature virions. EM of the compensatory mutant strains showed complete virus particles, but these now formed paracrystalline arrays. None of the E2_G209 substitution mutants had any effect on virus production; however, the deletion mutant (ΔG209) showed a very low titer when grown at 37°C and no titer when grown at 28°C. When the deletion mutant grown at 28°C was examined by EM, partially budded virions were observed at the cell surface.35S labeling of this mutant confirmed the presence of mutant virus protein in the transfected BHK cell lysate. We conclude that H230 is essential for the assembly of complete infectious Sindbis virus virions and that the presence of an amino acid at E2 position 209 is required for complete budding of Sindbis virus particles although several different amino acids can be at this location without affecting the titer.IMPORTANCEOur data show the importance of single-site mutations at E1_H230 and E2_G209 in Sindbis virus glycoproteins. These sites have been shown to affect assembly and antibody binding in previous studies. Our data indicate that mutation of one histidine residue in E1 is detrimental to the assembly of Sindbis virus particles in baby hamster kidney cells. Repeated passaging leads to a second-site substitution that partially restores the titer although EM still shows an altered phenotype. Substitutions at position G209 in E2 have no effect on titer, but deletion of this residue greatly reduces titer and again prevents assembly. When this mutant is grown at a lower temperature, virus particles bud from the host cell, but budding arrests before the progeny virus escapes. These results allow us to conclude that these sites have essential roles in assembly, and E2_G209 shows us a new viral egress phenotype.

scholarly journals Identification of Adult Mouse Neurovirulence Determinants of the Sindbis Virus Strain AR86

2005 ◽  
Vol 79 (7) ◽  
pp. 4219-4228 ◽  
Author(s):  
Mehul S. Suthar ◽  
Reed Shabman ◽  
Kenya Madric ◽  
Cassandra Lambeth ◽  
Mark T. Heise
Keyword(s):  
Spinal Cord ◽  
Amino Acid ◽  
Virus Strain ◽  
Sindbis Virus ◽  
Adult Mouse ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Valuable Insight ◽  
Wild Type ◽  
Genetic Elements

ABSTRACT Sindbis virus infection of mice has provided valuable insight into viral and host factors that contribute to virus-induced neurologic disease. In an effort to further define the viral genetic elements that contribute to adult mouse neurovirulence, the neurovirulent Sindbis virus strain AR86 was compared to the closely related (22 single amino acid coding changes and the presence or absence of an 18-amino-acid sequence in nsP3 [positions 386 to 403]) but avirulent Girdwood strain. Initial studies using chimeric viruses demonstrated that genetic elements within the nonstructural and structural coding regions contributed to AR86 neurovirulence. Detailed mapping studies identified three major determinants in the nonstructural region, at nsP1 538 (Ile to Thr; avirulent to virulent), an 18-amino-acid deletion in nsP3 (positions 386 to 403), and nsP3 537 (opal to Cys; avirulent to virulent), as well as a single determinant in the structural genes at E2 243 (Leu to Ser; avirulent to virulent), which were essential for AR86 adult mouse neurovirulence. Replacing these codons in AR86 with those found in Girdwood resulted in the attenuation of AR86, while the four corresponding AR86 changes in the Girdwood genetic background increased virulence to the level of wild-type AR86. The attenuating mutations did not adversely affect viral replication in vitro, and the attenuated viruses established infection in the brain and spinal cord as efficiently as the virulent viruses. However, the virus containing the four virulence determinants grew to higher levels in the spinal cord at late times postinfection, suggesting that the virus containing the four attenuating determinants either failed to spread or was cleared more efficiently than the wild-type virus.

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 Role of Conserved Cysteines in the Alphavirus E3 Protein

2008 ◽  
Vol 83 (6) ◽  
pp. 2584-2591 ◽  
Author(s):  
Megan M. Parrott ◽  
Sarah A. Sitarski ◽  
Randy J. Arnold ◽  
Lora K. Picton ◽  
R. Blake Hill ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Viral Entry ◽  
Disulfide Bonds ◽  
Functional Role ◽  
Sindbis Virus ◽  
Virus Assembly ◽  
Wild Type ◽  
Virus Particles ◽  
Extracellular Glycoprotein

ABSTRACT Alphavirus particles are covered by 80 glycoprotein spikes that are essential for viral entry. Spikes consist of the E2 receptor binding protein and the E1 fusion protein. Spike assembly occurs in the endoplasmic reticulum, where E1 associates with pE2, a precursor containing E3 and E2 proteins. E3 is a small, cysteine-rich, extracellular glycoprotein that mediates proper folding of pE2 and its subsequent association with E1. In addition, cleavage of E3 from the assembled spike is required to make the virus particles efficiently fusion competent. We have found that the E3 protein in Sindbis virus contains one disulfide bond between residues Cys19 and Cys25. Replacing either of these two critical cysteines resulted in mutants with attenuated titers. Replacing both cysteines with either alanine or serine resulted in double mutants that were lethal. Insertion of additional cysteines based on E3 proteins from other alphaviruses resulted in either sequential or nested disulfide bond patterns. E3 sequences that formed sequential disulfides yielded virus with near-wild-type titers, while those that contained nested disulfide bonds had attenuated activity. Our data indicate that the role of the cysteine residues in E3 is not primarily structural. We hypothesize that E3 has an enzymatic or functional role in virus assembly, and these possibilities are further discussed.

scholarly journals Modification of the 5′ Terminus of Sindbis Virus Genomic RNA Allows nsP4 RNA Polymerases with Nonaromatic Amino Acids at the N Terminus To Function in RNA Replication

2003 ◽  
Vol 77 (4) ◽  
pp. 2301-2309 ◽  
Author(s):  
Yukio Shirako ◽  
Ellen G. Strauss ◽  
James H. Strauss
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Minus Strand ◽  
Genomic Rna ◽  
Wild Type ◽  
N Terminus

ABSTRACT We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40°C upon addition of AU, AUA, or AUU to the 5′ terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3′-UA-5′ is required at the 3′ terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5′ end of the genomic RNA provides unpaired 3′-UA-5′ at the 3′ end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3′-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3′ terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.

scholarly journals Optimal Replication of Human Cytomegalovirus Correlates with Endocytosis of Glycoprotein gpUL132

2010 ◽  
Vol 84 (14) ◽  
pp. 7039-7052 ◽  
Author(s):  
Barbara Kropff ◽  
Yvonne Koedel ◽  
William Britt ◽  
Michael Mach
Keyword(s):  
Amino Acid ◽  
Human Cytomegalovirus ◽  
Intracellular Localization ◽  
Surface Expression ◽  
Wild Type Virus ◽  
Wild Type ◽  
Recombinant Viruses ◽  
Carboxy Terminal ◽  
Golgi Network

ABSTRACT Envelopment of a herpesvirus particle is a complex process of which much is still to be learned. We previously identified the glycoprotein gpUL132 of human cytomegalovirus (HCMV) as an envelope component of the virion. In its carboxy-terminal portion, gpUL132 contains at least four motifs for sorting of transmembrane proteins to endosomes; among them are one dileucine-based signal and three tyrosine-based signals of the YXXØ and NPXY (where X stands for any amino acid, and Ø stands for any bulky hydrophobic amino acid) types. To investigate the role of each of these trafficking signals in intracellular localization and viral replication, we constructed a panel of expression plasmids and recombinant viruses in which the signals were rendered nonfunctional by mutagenesis. In transfected cells wild-type gpUL132 was mainly associated with the trans-Golgi network. Consecutive mutation of the trafficking signals resulted in increasing fractions of the protein localized at the cell surface, with gpUL132 mutated in all four trafficking motifs predominantly associated with the plasma membrane. Concomitant with increased surface expression, endocytosis of mutant gpUL132 was reduced, with a gpUL132 expressing all four motifs in mutated form being almost completely impaired in endocytosis. The replication of recombinant viruses harboring mutations in single trafficking motifs was comparable to replication of wild-type virus. In contrast, viruses containing mutations in three or four of the trafficking signals showed pronounced deficits in replication with a reduction of approximately 100-fold. Moreover, recombinant viruses expressing gpUL132 with three or four trafficking motifs mutated failed to incorporate the mutant protein into the virus particle. These results demonstrate a role of endocytosis of an HCMV envelope glycoprotein for incorporation into the virion and optimal virus replication.

scholarly journals SC-23 * THE ROLE OF SINGLE AMINO ACID POLYMORPHISMS IN GLIOMA STEM CELL PHENOTYPES

2014 ◽  
Vol 16 (suppl 5) ◽  
pp. v202-v202
Author(s):  
C. L. Nilsson ◽  
A. Vegvari ◽  
E. Mostovenko ◽  
C. F. Lichti ◽  
D. Fenyo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Amino Acid ◽  
Single Amino Acid ◽  
Glioma Stem Cell ◽  
Single Amino Acid Polymorphisms ◽  
Cell Phenotypes

scholarly journals Infection of Human Dendritic Cells by a Sindbis Virus Replicon Vector Is Determined by a Single Amino Acid Substitution in the E2 Glycoprotein

2000 ◽  
Vol 74 (24) ◽  
pp. 11849-11857 ◽  
Author(s):  
Jason P. Gardner ◽  
Ilya Frolov ◽  
Silvia Perri ◽  
Yaying Ji ◽  
Mary Lee MacKichan ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Sindbis Virus ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
E2 Glycoprotein ◽  
Human Dendritic Cells ◽  
Antigen Presenting

ABSTRACT The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55Gag elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.

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.

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