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Many (+)-strand RNA viruses use subgenomic (SG) RNAs as messengers for protein expression, or to regulate their viral life cycle. Three different mechanisms have been described for the synthesis of SG RNAs. The first mechanism involves internal initiation on a (−)-strand RNA template and requires an internal SGP promoter. The second mechanism makes a prematurely terminated (−)-strand RNA which is...
Human glioblastoma cells (SF268) develop apoptosis, as characterized by DNA fragmentation and caspase activation, upon infection with Enterovirus 71 (EV71). To determine the step in virus replication that triggers apoptosis, the authors used ultraviolet (UV)-inactivated virus, inhibitors of protein and viral RNA synthesis, and chloroquine to block virus uncoating. Activation of caspase-3 was detected...
We describe here an in vitro system for template-dependent initiation and synthesis of a Sindbis virus (SV) subgenomic (SG) RNA transcript. The critical components of this system were (1) a minus-strand promoter-template corresponding to the region of the SV genome from nt 7441 to nt 7772 (−157 to +175 relative to the SG RNA transcription initiation site at nt 7598), and (2) a p15 fraction from cells...
Enterovirus 71 (EV71) infection may be asymptomatic or may cause diarrhea, rashes, and hand, foot, and mouth disease (HFMD). However, EV71 also has the potential to cause severe neurological disease. To date, little is known about the molecular mechanisms of host response to EV71 infection. In this report, we utilized cDNA microarray to profile the kinetics and patterns of host gene expression in...
The human glioblastoma SF268 cell line was used to investigate the induction of apoptosis by the 3C protease of enterovirus 71 (EV71). Transient expression in these cells of the wild-type 3C protein encoded by EV71 induced morphological alterations typical of apoptosis, including generation of apoptotic bodies. Degradation of cellular DNA in nucleosomes was also observed. When two of the amino acids...
SV PZF is a mutant of Sindbis virus (SV) which we selected on the basis of its ability to replicate in mosquito cells treated with pyrazofurin (PZF), a drug which inhibits pyrimidine nucleotide biosynthesis (Lin et al., 2000, Virology 272, 61–71). Three mutations, A6627U, A7543U, and C7593A, were identified in the nsP4 (the viral RNA polymerase) coding region, which were required for the PZF-resistant...
Pyrazofurin (PZF), a cytidine analog and an inhibitor of orotate monophosphate decarboxylase, has been shown to decrease the levels of UTP and CTP in treated cells. When Sindbis virus (SV)-infected Aedes albopictus cells were treated with PZF, the yield of virus was reduced 100- to 1000-fold. By serial passage of our standard SV STD in Ae. albopictus cells in the presence of increasing concentrations...
In order to obtain a mutant of Sindbis virus (SV) with a low methionine-resistant (LMR) phenotype, i.e., able to replicate in methionine-deprived Aedes albopictus mosquito cells, standard SV (SV STD ) was passaged 17 times in mosquito cells maintained in a low methionine medium and then plaque-purified, also in mosquito cells. Although the virus obtained by this procedure, SV LM17 ...
SV MPA is a mutant of Sindbis virus, selected for its ability to replicate in mycophenolic acid (MPA)-treated mosquito cells. SV MPA has another phenotype: although able to replicate normally in primary cultures of chick embryo fibroblasts (CEF), its replication is restricted in secondary cultures prepared from aged primary CEF cultures. The mutations responsible for these phenotypes...
JKT-7400 virus is an orbivirus originally isolated fromCulexmosquitoes. In earlier work we had described the viral structural proteins and presented evidence suggesting that a minor protein, VP6, located in the viral core was the viral guanylyltransferase. We now show that gradient-purified JKT-7400 virions possess a methyltransferase (MTase) activity which can use GTP or GDP as the methyl acceptor...
JKT-7400 virus, an orbivirus originally isolated fromCulexmosquitos, was plaque purified and adapted toAedes albopictusmosquito cells. Conditions which enhance viral cytopathic effect and optimize plaque formation are described. In contrast to bluetongue virus, the prototype orbivirus, no replication of JKT-7400 virus in vertebrate cells was observed. The core particle of JKT-7400 virus contains 10...
Stable transfectants of Hela cells were isolated which expressed either the full-length 540-amino-acid Sindbis virus (SV) nonstructural protein, nsP1 (the form encoded by the mutant, SV LM21 ), or one of four forms with a carboxyl-terminal deletion. SV LM21 , in contrast to standard SV (SV STD ), can replicate in Hela cells maintained in low-methionine (LM) medium. Expression...
It has been suggested that four amino acids which are absolutely conserved in the nsP1 nonstructural proteins encoded by togaviruses and in the homologous proteins encoded by plant viruses in the Sindbis virus (SV) superfamily may constitute a “methyltransferase motif.” In the Sindbis virus nsP1 protein (540 amino acids) these four amino acids are represented by His 39 , Arg 91 , Asp...
Experiments many years ago showed that reducing the ionic strength of the medium in which Sindbis virus-infected chick embryo fibroblasts were maintained prevented the release of progeny virus into the medium. It was proposed at that time that the susceptibility of virus release to inhibition by low ionic strength (LIS) medium might be determined by the properties of the viral envelope proteins. We...
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