Identification of a Minimal Size Requirement for Termination of Vesicular Stomatitis Virus mRNA: Implications for the Mechanism of Transcription

Abstract

The nonsegmented negative-strand RNA (NNS) viruses have a single-stranded RNA genome tightly encapsidated by the viral nucleocapsid protein. The viral polymerase transcribes the genome responding to specific gene-start and gene-end sequences to yield a series of discrete monocistronic mRNAs. These mRNAs are not produced in equimolar amounts; rather, their abundance reflects the position of the gene with respect to the single 3-proximal polymerase entry site. Promoter-proximal genes are transcribed in greater abundance than more distal genes due to a localized transcriptional attenuation at each gene junction. In recent years, the application of reverse genetics to the NNS viruses has allowed an examination of the role of the gene-start and gene-end sequences in regulating mRNA synthesis. These studies have defined specific sequences required for initiation, 5 modification, termination, and polyadenylation of the viral mRNAs. In the present report, working with Vesicular stomatitis virus, the prototypic Rhabdovirus, we demonstrate that a gene-end sequence must be positioned a minimal distance from a gene-start sequence for the polymerase to efficiently terminate transcription. Gene-end sequences were almost completely ignored in transcriptional units less than 51 nucleotides. Transcriptional units of 51 to 64 nucleotides allowed termination at the gene-end sequence, although the frequency with which polymerase failed to terminate and instead read through the gene-end sequence to generate a bicistronic transcript was enhanced compared to the observed 1 to 3% for wild-type viral mRNAs. In all instances, failure to terminate at the gene end prevented initiation at the downstream gene start site. In contrast to this size requirement, we show that the sequence between the gene-start and gene-end signals, or its potential to adopt an RNA secondary structure, had only a minor effect on the efficiency with which polymerase terminated transcription. We suggest three possible explanations for the failure of polymerase to terminate transcription in response to a gene-end sequence positioned close to a gene-start sequence which contribute to our emerging picture of the mechanism of transcriptional regulation in this group of viruses.

Opposing Effects of Inhibiting Cap Addition and Cap Methylation on Polyadenylation during Vesicular Stomatitis Virus mRNA Synthesis

“…Earlier work showed that a polymerase must copy a minimum length of template in order to respond to an authentic gene end sequence (41). This length was Ͼ56 nt, which is reached by the cap-defective mutants despite their defect in the synthesis of full-length N mRNA.…”

Infectious Entry Pathway Mediated by the Human Endogenous Retrovirus K Envelope Protein

“…VSV-eGFP (enhanced green fluorescent protein) and VSV-EBOV (Ebola virus) were previously generated (35,37). All the viruses were propagated on BSRT7 cells in DMEM supplemented with 2% FBS and penicillinstreptomycin/kanamycin.…”

Viral transgenesis of embryonic cell cultures from the freshwater microcrustaceandaphnia

“…An infectious cDNA clone of VSV (Whelan et al,'95) engineered to express the marker gene, GFP, VSV-GFP has been previously described (Whelan et al, 2000;Cherry et al, 2005). The coding sequence of GFP, flanked by the essential VSV gene-start and -end sequences, was inserted between the 3′ leader regulatory region and the VSV N gene.…”

“…An infectious cDNA clone of the Indiana serotype of VSV (Whelan et al,'95) engineered to express green fluorescent protein (GFP), VSV-GFP (Whelan et al, 2000;Cherry et al, 2005), was propagated in baby hamster kidney (BHK-21) cells (American Type Culture Collection, Manassas, VA) in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 10% (v/v) FBS, 2 mM L-alanyl-L-glutamine (Glutamax, Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin. Initial infection was made with a multiplicity of infection of 0.01 in serum-free medium for 1 hr at 37 °C.…”

“…Genomic replication initiates at the 3′ end of the viral genome and requires ongoing translation to provide a source of the viral nucleocapsid protein that coats the viral genome (Patton et al,'84). Recently, infectious transgenic clones of VSV-containing additional open reading frames coding for a foreign reporter gene have been described (Whelan et al, 2000;Cherry et al, 2005). Here we present methods for the culture of embryonic Daphnia cells and the infection of these cultures with transgenic VSV, resulting in expression of a foreign transgene.…”