A Vesiculovirus Showing a Steepened Transcription Gradient and Dominant trans -Repression of Virus Transcription

Abstract

bVesicular stomatitis virus (VSV) is a prototype nonsegmented, negative-sense virus used to examine viral functions of a broad family of viruses, including human pathogens. Here we demonstrate that S 2 VSV, an isolate with a small plaque phenotype compared to other Indiana strain viruses, has a transcription defect resulting in an altered pattern and rapid decline of transcription. The S 2 VSV transcription gradient is dominant over the wild-type transcription in a coinfection. This is the first characterization of an altered gradient of transcription not dependent on RNA template sequence or host response and may provide insight into new approaches to viral attenuation. V esicular stomatitis virus (VSV) is a prototype nonsegmented, negative-strand (NNS) RNA virus with robust growth kinetics and is highly genetically tractable in laboratory conditions. Because of this, VSV has frequently been used to probe fundamental questions about virus replication. Specifically, the RNA transcription and replication strategy used by NNS viruses was first described and extensively characterized with VSV (1,21,(24)(25)(26)33).VSV transcription requires the negative-sense RNA genome and three viral proteins. The large polymerase subunit (L) carries out the enzymatic functions of the polymerase, including transcription, capping, methylation, and polyadenylation (5, 23, 33). The phosphoprotein (P) is a required cofactor for the polymerase and interacts with both L and the nucleocapsid protein (N). N encapsidates the viral genome, allowing the RNA-dependent RNA polymerase (RdRp) to access the RNA and preventing degradation or detection by the host cell. The VSV polymerase complex consists of an oligomer of P for each molecule of L (8,14,15,30). Transcription of viral mRNAs occurs in an obligate sequential stop-start manner beginning with the N gene near the 3= end of the genome. Reinitiation of transcription between genes is not completely efficient, leading to a gradient of mRNA transcription along the genome. The mRNAs that are transcribed from regions closest to the 3= promoter are transcribed in highest abundance, while genes further from the 3= end are transcribed in lower abundance.Here we show that an isolate of VSV previously demonstrated to be attenuated has a steeper gradient of transcription that is dominant over wild-type VSV. This suggests that the ability of the polymerase to reinitiate transcription is dependent not only on the cis-acting genomic sequence but also on the protein components of the polymerase complex.S 2 VSV was originally isolated from an undifferentiated VSVIndiana pool as a small plaque strain and clonally selected five times to ensure phenotype stability (39). This small plaque isolate was shown to be 1,000 times less lethal than wild-type virus in animal models but still able to induce a robust interferon response (39). Further characterization showed that S 2 VSV transcribes more small viral mRNA species (12S to 18S; N, P, M and G mRNA) but less of the largest viral RNA species (40S; replication...

Investigating Macrophages Plasticity Following Tumour–Immune Interactions During Oncolytic Therapies

“…The thin dotted line depicts VSV detection level which, according to Hodges et al. (2012) is about cells. b The percentages of M1 and M2 cells on three different days: , and .…”

“…In this case, the presence of the oncolytic virus that is detected for up to 4 days after the last injection (assuming that detection threshold is Hodges et al. 2012), leads to a slow-down in tumour growth. This slow-down in tumour growth is also the result of a large T cell population (Fig.…”

“…Therefore, we choose such that VSV particles persist in the system until day (i.e., 3–4 days after their second inoculation), above a detection level of at least 100 PFU/ml (Hodges et al. 2012). This leads to (so and for when the VSV is introduced in the system).…”

The molecular basis of the pathogenicity of Newcastle disease virus in chickens

“…However, the work investigating the steepened transcription gradient of an attenuated vesicular stomatitis virus (VSV) is consistent with our findings, in that the steepened gradient of Meredith/02 transcription is also associated with reduced pathogenicity. 202 As hypothesized in the VSV study, it is also possible that there is a mutation in the Meredith/02 polymerase complex that prevents it from efficiently reinitiating transcription after the initial nucleocapsid protein mRNA has been transcribed. …”

“…199 The transcription gradient has also been found to vary between different strains of the same virus in the case of vesicular stomatitis virus, another negative-sense RNA virus. 202 In this case, a small plaque phenotype, attenuated strain was found to have a steepened transcription gradient and less viral mRNA transcription overall, when compared with the wild type virus.…”