Reconstitution of hepatitis C virus protease activities in yeast.

The hepatitis C virus (HCV) protease genes (NS2/3 and NS3) were expressed in yeast with their natural substrates fused to a ligand-dependent transcriptional activator, the retinoic acid receptor (RARbeta). RARbeta can activate transcription in yeast cells in response to retinoic acids. We hypothesized that cis-cleavage at the NS2-3 or NS3-4A junctions by the appropriate HCV proteases would release RARbeta, thereby activating transcription of a reporter gene.

Primer-dependent synthesis by poliovirus RNA-dependent RNA polymerase (3D(pol)).

Properties of poliovirus RNA-dependent RNA polymerase (3D(pol)) including optimal conditions for primer extension, processivity and the rate of dissociation from primer-template (k(off)) were examined in the presence and absence of viral protein 3AB. Primer-dependent polymerization was examined on templates of 407 or 1499 nt primed such that fully extended products would be 296 or 1388 nt, respectively. Maximal primer extension was achieved with low rNTP concentrations (50-100 microM) using pH 7 and low (<1 mM) MgCl(2) and KCl (<20 mM) concentrations.

Determination of the mutation rate of poliovirus RNA-dependent RNA polymerase.

The fidelity of poliovirus RNA-dependent RNA polymerase (3D(pol)) was determined using a system based on the fidelity of synthesis of the alpha-lac gene which codes for a subunit of beta-galactosidase. Synthesis products are screened for mutations by an alpha-complementation assay, in which the protein product from alpha-lac is used in trans to complement beta-galactosidase activity in bacteria that do not express alpha-Lac. Several polymerases have been analyzed by this approach allowing comparisons to be drawn.

Inhibition of influenza A virus replication by compounds interfering with the fusogenic function of the viral hemagglutinin.

Several compounds that specifically inhibited replication of the H1 and H2 subtypes of influenza virus type A were identified by screening a chemical library for antiviral activity. In single-cycle infections, the compounds inhibited virus-specific protein synthesis when added before or immediately after infection but were ineffective when added 30 min later, suggesting that an uncoating step was blocked. Sequencing of hemagglutinin (HA) genes of several independent mutant viruses resistant to the compounds revealed single amino acid changes that clustered in the stem region of the HA trimer in and near the HA2 fusion peptide.

Refined X-ray crystallographic structure of the poliovirus 3C gene product.

The X-ray crystallographic structure of the recombinant poliovirus 3C gene product (Mahoney strain) has been determined by single isomorphous replacement and non-crystallographic symmetry averaging and refined at 2.1 A resolution. Poliovirus 3C is comprised of two six-stranded antiparallel beta-barrel domains and is structurally similar to the chymotrypsin-like serine proteinases.

Poliovirus protein 3AB forms a complex with and stimulates the activity of the viral RNA polymerase, 3Dpol.

Poliovirus protein 3B (also known as VPg) is covalently linked to the 5' ends of both genomic and antigenomic viral RNA. Genetic and biochemical studies have implicated protein 3AB, the membrane-bound precursor to VPg, in the initiation of genomic RNA synthesis. We have purified 3AB to near homogeneity following thrombin cleavage of purified glutathione S-transferase-3AB.

Purification, properties, and mutagenesis of poliovirus 3C protease.

Poliovirus protease 3C, type 1 Mahoney strain, was expressed in Escherichia coli under phage T7 promoter control and purified to homogeneity from resolubilized inclusion bodies. The renatured protein was as enzymatically active as the protease found in the soluble portion of the bacterial lysate. Proteolytic activity was assayed using as substrate either [35S]methionine-labeled recombinant poliovirus proteins 2C3AB or a truncated version of 3ABC, or synthetic peptide 16-mers corresponding to the cleavage sites at 2C/3A and 3A/3B.

Purification and properties of poliovirus RNA polymerase expressed in Escherichia coli.

A cDNA clone encoding the RNA polymerase of poliovirus has been expressed in Escherichia coli under the transcriptional control of a T7 bacteriophage promoter. The poliovirus enzyme was designed to contain only a single additional amino acid, the N-terminal methionine. The recombinant enzyme has been purified to near homogeneity, and polyclonal antibodies have been prepared against it.

DNA probes for mycobacteria. I. Isolation of DNA probes for the identification of Mycobacterium tuberculosis complex and for mycobacteria other than tuberculosis (MOTT).

Traditional methods used in identifying mycobacteria such as acid-fast bacillus stains and culture are often time-consuming, insensitive and non-specific. The isolation of DNA probes, coupled to a non-radioactive, e.g.

In vitro splicing of influenza viral NS1 mRNA and NS1-beta-globin chimeras: possible mechanisms for the control of viral mRNA splicing.

In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation.

A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription.

We propose a mechanism for the priming of influenza viral RNA transcription by capped RNAs in which specific 5'-terminal fragments are cleaved from the capped RNAs by a virion-associated endonuclease. These fragments would serve as the actual primers for the initiation of transcription by the initial incorporation by the initial incorporation of a G residue at their 3' end. We show that virions and purified viral cores contain a unique endonuclease that cleaves RNAs containing a 5' methylated cap structure (m7GpppXm) preferentially at purine residues 10 to 14 nucleotides from the cap, generating fragments with 3'-terminal hydroxyl groups.

Both the 7-methyl and the 2'-O-methyl groups in the cap of mRNA strongly influence its ability to act as primer for influenza virus RNA transcription.

The ability of eukaryotic mRNAs to serve as primers for influenza virus RNA transcription depends on the presence of a 5'-terminal methylated can structure, the absence of which eliminates essentially all priming activity [Plotch, S. J., Bouloy, M.

Identification of the RNA region transferred from a representative primer, beta-globin mRNA, to influenza mRNA during in vitro transcription.

Capped eukaryotic mRNAs strongly stimulate influenza viral RNA transcription in vitro and donate their cap and also additional nucleotides to the viral transcripts (1). To identify which bases of a given primer mRNA are transferred, we synthesized influenza viral mRNA using a primer rabbit globin mRNA (enriched in beta-globin mRNA) which had been labeled in vitro to high specific activity with 125I. We show that during transcription the same 125I-labeled oligonucleotides were transferred to the 5' termini of each of the eight viral mRNA segments.

RNA primers and the role of host nuclear RNA polymerase II in influenza viral RNA transcription.

Influenza viral RNA transcription in the infected cell is inhibited by alpha-amanitin, a specific inhibitor of the host nuclear RNA polymerase II. Because viral RNA transcription in vitro catalysed by the virion-associated transcriptase is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by RNA polymerase II. In addition, because we did not detect any capping and methylating enzymes in virions, we have proposed that the 5' terminal methylated cap found on in-vivo viral messenger RNA (mRNA) is derived from the putative primer RNAs.

Transfer of 5'-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro.

We have recently demonstrated that globin mRNAs are effective primers for influenza viral RNA transcription in vitro catalyzed by the virion transcriptase [Bouloy, M., Plotch, S. J.

Absence of detectable capping and methylating enzymes in influenza virions.

In the presence of Mg(2+) and a specific dinucleotide primer (ApG or GpG), the influenza virion transcriptase synthesizes the eight discrete segments of complementary RNA (cRNA) containing polyadenylic acid (Plotch and Krug, J. Virol. 21:24-34, 1977).

Globin mRNAs are primers for the transcription of influenza viral RNA in vitro.

Because influenza viral RNA transcription in vitro is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by the host cell, specifically by RNA polymerase II, thereby explaining the alpha-amanitin sensitivity of viral RNA transcription in vivo. Here, we identify such primer RNAs, initially in reticulocyte extracts, where they are shown to be globin mRNAs. Purified globin mRNAs very effectively stimulated viral RNA transcription in vitro, and the resulting transcripts directed the synthesis of all the nonglycosylated virus-specific proteins in micrococcal nuclease-treated L cell extracts.

Segments of influenza virus complementary RNA synthesized in vitro.

In the presence of Mg(2+) and a specific primer, ApG or GpG, the influenza WSN virion transcriptase synthesizes large, polyadenylic acid-containing complementary RNA (cRNA) (Plotch and Krug, J. Virol., 21:24-34, 1977).

Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA.

The influenza virion transcriptase is capable of synthesizing in vitro complementary RNA (cRNA) that is similar in several characteristics to the cRNA synthesized in the infected cell, which is the viral mRNA. Most of the in vitro cRNA is large (approximately 2.5 X 10(5) to 10(6) daltons), similar in size to in vivo cRNA.