Detection of virus-specific RNA-dependent RNA polymerase activity in extracts from cells infected with lymphocytic choriomeningitis virus: in vitro synthesis of full-length viral RNA species

A Tacaribe virus in vitro system for RNA synthesis was established and found in large part to faithfully reproduce RNA synthesis in vivo. Similar to influenza virus and bunyavirus in vitro systems, this system was also highly dependent on added oligonucleotides. Of the eight tested, only three were active, in the order GpC > CpG > ApApC. Determination of the 5' ends of the transcripts suggested that the oligonucleotides were acting as primers. In particular, whereas stimulation with CpG (complementary to positions + 1 and +2 of the template) led to RNAs whose 5' ends were at position + 1 as expected, GpC stimulation led to transcripts whose 5' ends were at position -1 rather than at position +2, as GpC is complementary to positions +2 and +3 of the template. This finding suggests a model for the initiation of genome replication in which pppGpC is first made on the template at positions +2 and +3 but slips backwards on the template so that the 5' end is at position -1 before elongation can continue.CAGCATAA-5', with a T7 promoter (underliieid) fused to nt -1 to 19 of the TAC S antigenome with a single extra G 1370 on July 6, 2020 by guest

Section: Resultsmentioning

confidence: 99%

Section: Vol 66 1992mentioning

confidence: 99%

Tacaribe arenavirus RNA synthesis in vitro is primer dependent and suggests an unusual model for the initiation of genome replication

Garcin

Kolakofsky

1992

“…L protein mediates the synthesis of two RNA species: mRNA terminating in the intergenic region and noncapped genomic or antigenomic RNA representing a full-length copy of the genome (10,35). The central domain of the protein from amino acid residues 1000 to 1500 harbors the RNA-dependent RNA polymerase (RdRp) (8,11,19,29,47). Enzymatic functions residing in the N terminus and C terminus of the L protein are unknown.…”

mentioning

confidence: 99%

An N-Terminal Region of Lassa Virus L Protein Plays a Critical Role in Transcription but Not Replication of the Virus Genome

Lelke

Brunotte

Busch

et al. 2010

The central domain of the 200-kDa Lassa virus L protein is a putative RNA-dependent RNA polymerase. Nand C-terminal domains may harbor enzymatic functions important for viral mRNA synthesis, including capping enzymes or cap-snatching endoribonucleases. In the present study, we have employed a large-scale mutagenesis approach to map functionally relevant residues in these regions. The main targets were acidic (Asp and Glu) and basic residues (Lys and Arg) known to form catalytic and binding sites of capping enzymes and endoribonucleases. A total of 149 different mutants were generated and tested in the Lassa virus replicon system. Nearly 25% of evolutionarily highly conserved acidic and basic side chains were dispensable for function of L protein in the replicon context. The vast majority of the remaining mutants had defects in both transcription and replication. Seven residues (Asp-89, Glu-102, Asp-119, Lys-122, Asp-129, Glu-180, and Arg-185) were selectively important for mRNA synthesis. The phenotype was particularly pronounced for Asp-89, Glu-102, and Asp-129, which were indispensable for transcription but could be replaced by a variety of amino acid residues without affecting genome replication. Bioinformatics disclosed the remote similarity of this region to type IIs endonucleases. The mutagenesis was complemented by experiments with the RNA polymerase II inhibitor ␣-amanitin, demonstrating dependence of viral transcription from the cellular mRNA pool. In conclusion, this paper describes an N-terminal region in L protein being important for mRNA, but not genome synthesis. Bioinformatics and cell biological experiments lend support to the hypothesis that this region could be part of a cap-snatching enzyme.

“…For most or all positive-strand RNA viruses of eukaryotes examined to date, the viral RNA-dependent RNA replication complex copurifies with membrane extracts from infected cells (4,11,16,21,45,46,55). In some cases, negative-strand RNA synthesis activity can be solubilized from such membrane extracts (55,64), but in vitro studies with positive-strand RNA viruses as varied as picornaviruses and nodaviruses suggest that membrane association is important for synthesis of positivestrand RNA (3,47,64).…”

mentioning

confidence: 99%

Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis

Restrepo-Hartwig

Ahlquist

1996

197

106

The helicase-like 1a and polymerase-like 2a proteins of brome mosaic virus (BMV) are required for viral RNA replication in vivo, are present in membrane-bound viral RNA polymerase extracts, and share conservation with the many other members of the alphavirus-like superfamily. To better understand BMV RNA replication and BMV-host interactions, we used confocal microscopy and double-label immunofluorescence to determine and compare the sites of 1a, 2a, and nascent viral RNA accumulation in BMV-infected barley protoplasts. 1a and 2a showed nearly complete colocalization throughout infection, accumulating in defined cytoplasmic spots usually adjacent to or surrounding the nucleus. These spots grew throughout infection and by 16 h postinoculation often assumed a vesicle-like appearance. The BMV RNA replication complex incorporated 5-bromouridine 5-triphosphate into RNA in vitro and in vivo, allowing immunofluorescent detection of nascent RNA. The cytoplasmic sites of BMV-specific RNA synthesis coincided with the sites of 1a and 2a accumulation, and at the resolution of confocal microscopy, all sites of 1a and 2a accumulation were sites of BMV RNA synthesis. Double-label immunofluorescence detection of selected subcellular markers and 1a or 2a showed that BMV replication complexes were tightly associated with markers for the endoplasmic reticulum but not the medial Golgi or later compartments of the cellular secretory pathway. Defining this association of BMV RNA replication complexes with endoplasmic reticulum markers should assist in identifying and characterizing host factors involved in BMV RNA replication.