Dataset Information

Efficient plasmid-based rescue of T7 RNA polymerase-driven calicivirus reverse genetics systems in mammalian cells using vaccinia virus RNA capping enzymes C4PR_LIV

ABSTRACT: Calicivirus reverse genetics systems have typically relied on either in vitro transcribed RNA or plasmid-based rescue with enzyme supplementation. Plasmid based calicivirus rescue relies on supplementation with Fowl Pox T7 RNA polymerase which has cost and labour considerations. Here we present a novel system integrating vaccinia capping enzymes D1R and D12L encoded on plasmids in a rescue system for Murine Norovirus (MNV). Addition of D1R, D12L and T7 expressing plasmids increases viral rescue tires of MNV in both BSR-T7 cells and transgenic BSR-T7CD300LF cells and viral polyprotein abundance. This system offers an opportunity to increase viral rescue efficiency for MNV, other caliciviruses and permit higher throughput experimentation such as mutagenic screening.

INSTRUMENT(S):

ORGANISM(S): Murine Norovirus Mesocricetus Auratus (golden Hamster)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Leandro Neves

LAB HEAD: Dr Ed Emmott

PROVIDER: PXD074707 | Pride | 2026-05-13

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2024-12-18-decoys-contam-20241211_UP000189706_10036_onePTNperGeneplus_MNVproteome_UP000109015_pp-full-partial_vp1_orf2-vp2_vf1.fasta	Fasta
	20250420_BRHT-EVOSEP30_CPR_LeXN_SA_BSRT7-MNV_BS_Mock_N1_S6-A1_1_7689.d.zip	Other
	20250420_BRHT-EVOSEP30_CPR_LeXN_SA_BSRT7-MNV_BS_Mock_N1_S6-A1_1_7689_calibrated.mzML	Mzml
	20250420_BRHT-EVOSEP30_CPR_LeXN_SA_BSRT7-MNV_BS_Mock_N2_S6-A7_1_7683.d.zip	Other
	20250420_BRHT-EVOSEP30_CPR_LeXN_SA_BSRT7-MNV_BS_Mock_N2_S6-A7_1_7683_calibrated.mzML	Mzml

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Project description:Rare cases of non-hepatotropic virus (NHV) infection in humans can cause severe hepatitis and even acute liver failure. Clinically relevant animal models of NHV-induced hepatitis are limited, contributing to the incomplete understanding of pathological mechanisms. Murine norovirus (MNV) elicits hepatosplenomegaly in mice lacking the antiviral immune effector Signal Transducer and Activator of Transcription-1 (STAT1), providing a model to investigate mechanisms of NHV-induced hepatic pathology. STAT1-sufficient and -deficient (Stat1Het, Stat1KO) littermates infected intravenously (i.v.) with MNV strain CR6 were assessed for hepatic inflammation and viral burden. Cell types and molecular pathways associated with hepatic pathology in CR6-infected Stat1KO mice were identified by flow cytometry and RNAseq of liver tissue. The relative importance of hematopoietic vs non-hematopoietic expression of STAT1 in restricting CR6 replication and maintaining tissue homeostasis was assessed in bone marrow chimeras. MNV CR6 Stat1KO mice developed severe hepatitis with patchy hepatocellular necrosis and localized enrichment of CR6-infected myeloid cells, particularly macrophages. Gene set enrichment analysis (GSEA) of hepatic biopsies isolated from CR6-infected Stat1KO mice suggested dysregulated myeloid cell activation and indicated similarities between murine and human hepatic pathologies. STAT1 expression in hematopoietic cells was protective against hepatic viral dissemination, but hematopoietic STAT1-deficiency permitted persistent hepatic MNV infection, facilitating dysregulated myeloid cell activation and hepatic fibrosis. These results demonstrate that the role of STAT1 extends beyond restricting MNV dissemination and suggest that STAT1-dependent regulation of myeloid cell activation prevents acute hepatic necroinflammation and secondary fibrosis. This model of MNV-induced hepatitis may prove valuable in elucidating mechanisms of rare clinical complications.

Dataset Information

Efficient plasmid-based rescue of T7 RNA polymerase-driven calicivirus reverse genetics systems in mammalian cells using vaccinia virus RNA capping enzymes C4PR_LIV

Dataset's files

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