Evolution of Sindbis virus with a low-methionine-resistant phenotype is dependent both on a pre-existing mutation and on the methionine concentration in the medium.

SVlm21 is a mutant of Sindbis virus which was isolated by serial passage of virus in mosquito cells maintained in low-methionine medium; it therefore has a low-methionine-resistant (LMR) phenotype. This phenotype requires mutations at nts 319 and 321; these mutations result in Arg to Leu and Ser to Cys changes at positions 87 and 88 respectively in the viral methyl transferase, nsP1. To better understand the genesis of SVlm21, we carried out serial passages of viruses having only one of these amino acid changes, but in mosquito cells maintained in normal methionine-medium.

Host factors in enterovirus 71 replication.

Enterovirus 71 (EV71) infections continue to remain an important public health problem around the world, especially in the Asia-Pacific region. There is a significant mortality rate following such infections, and there is neither any proven therapy nor a vaccine for EV71. This has spurred much fundamental research into the replication of the virus.

Subgenomic messenger RNAs: mastering regulation of (+)-strand RNA virus life cycle.

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.

hnRNP A1 interacts with the genomic and subgenomic RNA promoters of Sindbis virus and is required for the synthesis of G and SG RNA.

Background: Sindbis virus (SV) is the prototype of alphaviruses which are a group of widely distributed human and animal pathogens. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an RNA-binding protein that shuttles between the nucleus and the cytoplasm. Our recent studies found that hnRNP A1 relocates from nucleus to cytoplasm in Sindbis virus (SV)-infected cells.

In vitro synthesis of Sindbis virus genomic and subgenomic RNAs: influence of nsP4 mutations and nucleoside triphosphate concentrations.

Two positive-strand mRNAs are made in Sindbis virus-infected cells, the genomic (G) RNA and the subgenomic (SG) RNA. In mosquito cells infected with wild-type (wt) Sindbis virus, the latter is made in excess over the former; however, in cells infected with SVpzf or SVcpc more G RNA is made than SG RNA. Use was made of in vitro systems to investigate the effects of the SVpzf and SVcpc mutations on the synthesis of SG and G RNAs.

hnRNP A1 interacts with the 5' untranslated regions of enterovirus 71 and Sindbis virus RNA and is required for viral replication.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is involved in pre-mRNA splicing in the nucleus and translational regulation in the cytoplasm. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here we demonstrate that hnRNP A1 not only is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5' untranslated region (UTR) of enterovirus 71 (EV71) and regulates IRES-dependent translation but also binds to the 5' UTR of Sindbis virus (SV) and facilitates its translation.

Synthesis of genomic and subgenomic RNA in mosquito cells infected with two Sindbis virus nsP4 mutants: influence of intracellular nucleoside triphosphate concentrations.

Cells infected with Sindbis virus (SV) make two positive-strand RNAs, a genomic-length RNA (G) RNA and a subgenomic (SG) RNA. In cells infected with SVstd, and in general in cells infected with wt alphaviruses, more SG RNA is made than G RNA. How the balance between synthesis of G RNA and SG RNA is regulated is not known.

Viral protein synthesis is required for Enterovirus 71 to induce apoptosis in human glioblastoma cells.

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 24 h after infection with EV71 but not with UV-inactivated EV71.

Distinct sites on the Sindbis virus RNA-dependent RNA polymerase for binding to the promoters for the synthesis of genomic and subgenomic RNA.

Sindbis virus-infected cells make two positive-strand RNAs, a genomic (G) RNA and a subgenomic (SG) RNA. Here we report the amino acid sequence in nonstructural protein 4 (nsP4), the viral RNA-dependent RNA polymerase, that binds to the promoter for the synthesis of G RNA. In addition, using a cell-free system that makes both G and SG RNA, we show that specific amino acid changes in nsP4 that abolish the synthesis of SG RNA have no effect on the synthesis of G RNA.

Antiviral therapy targeting viral polymerase.

Viral DNA and RNA polymerases are enzymes, which are responsible for copying the genetic materials of viruses and are therefore central components in the life cycles of viruses. The polymerases are essentially required for the replication of viruses. The reverse transcriptase (RT) of the retroviruses and the hepadnaviruses is the sole viral enzyme required for the synthesis of DNA from viral RNA.

A cell-free system for the synthesis of Sindbis virus subgenomic RNA: importance of the concentration of the initiating NTP.

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 infected with recombinant vaccinia viruses expressing the SV nonstructural proteins, P123 and nsP4 (the nsP2 coding region in P123 contained a mutation which results in more rapid than normal processing of P123). Our data indicate that the SG RNA transcript is of the expected size, of positive polarity, and is initiated at the expected site.

Identification of genes involved in the host response to enterovirus 71 infection.

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.

A mutant of Sindbis virus which is able to replicate in cells with reduced CTP makes a replicase/transcriptase with a decreased Km for CTP.

We reported earlier the isolation and characterization of a Sindbis virus mutant, SV(PZF), that can grow in mosquito cells treated with pyrazofurin (PZF), a compound that interferes with pyrimidine biosynthesis (Y. H. Lin, P.

Identification of the amino acid sequence in Sindbis virus nsP4 that binds to the promoter for the synthesis of the subgenomic RNA.

A gel mobility-shift assay was used to demonstrate the binding of the Sindbis virus transcriptase to the promoter for the synthesis of subgenomic (SG) RNA. The assay made use of a P15 fraction (the cell fraction that is pelleted at 15,000 x g) from cells infected with recombinant vaccinia virions expressing various Sindbis virus nonstructural proteins (nsPs) and a (32)P-labeled 24-mer oligoribonucleotide representing the minimal sequence with SG promoter activity. By itself, nsP4, the viral RNA-dependent RNA polymerase, did not bind to the SG promoter; rather, all four nsPs were required for the binding of the transcriptase to the promoter.

Alphaviruses and apoptosis.

Many reports have indicated that infection with SV or SFV induces apoptosis both in cultured cells and in the CNS of mice. In general, the ability of virus strains to induce apoptosis correlates with their neurovirulence, although both apoptosis and neurovirulence are age dependent, i.e.

The 3C protease activity of enterovirus 71 induces human neural cell apoptosis.

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.

Restriction of a Sindbis virus mutant in BHK cells and relief of the restriction by the addition of adenosine.

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 phenotype.