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Visualization of feline calicivirus replication in real-time with recombinant viruses engineered to express fluorescent reporter proteins.

ABSTRACT: Caliciviruses are non-enveloped, icosahedral viruses with a single-stranded, positive sense RNA genome. Transposon-mediated insertional mutagenesis was used to insert a transprimer sequence into random sites of an infectious full-length cDNA clone of the feline calicivirus (FCV) genome. A site in the LC gene (encoding the capsid leader protein) of the FCV genome was identified that could tolerate foreign insertions, and two viable recombinant FCV variants expressing LC fused either to AcGFP, or DsRedFP were recovered. The effects of the insertions on LC processing, RNA replication, and stability of the viral genome were analyzed, and the progression of a calicivirus single infection and co-infection were captured by real-time imaging fluorescent microscopy. The ability to engineer viable recombinant caliciviruses expressing foreign markers enables new approaches to investigate virus and host cell interactions, as well as studies of viral recombination, one of the driving forces of calicivirus evolution.

SUBMITTER: Abente EJ

PROVIDER: S-EPMC2855553 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Visualization of feline calicivirus replication in real-time with recombinant viruses engineered to express fluorescent reporter proteins.

Abente Eugenio J EJ Sosnovtsev Stanislav V SV Bok Karin K Green Kim Y KY

Virology 20100204 1

Caliciviruses are non-enveloped, icosahedral viruses with a single-stranded, positive sense RNA genome. Transposon-mediated insertional mutagenesis was used to insert a transprimer sequence into random sites of an infectious full-length cDNA clone of the feline calicivirus (FCV) genome. A site in the LC gene (encoding the capsid leader protein) of the FCV genome was identified that could tolerate foreign insertions, and two viable recombinant FCV variants expressing LC fused either to AcGFP, or ...[more]

PMID: 20137802

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Project description:Background: Microglia activation contributes to chronic pain and to the adverse effects of opiate use such as analgesic tolerance and opioid-induced hyperalgesia. Both mu opioid receptor (MOR) encoded by Oprm1/OPRM1 gene and toll like receptor 4 (TLR4) have been reported to mediate these morphine effects and a current question is whether microglia express the Oprm1 transcript and MOR protein. The aim of this study was to characterize Oprm1-MOR expression in naive murine and human microglia, combining transcriptomics datasets previously published by other groups with our own imaging study using the Cx3cr1-eGFP-MOR-mCherry reporter mouse line. Methods: We analyzed microglial Oprm1/OPRM1 expression obtained from transcriptomics datasets, focusing on ex vivo studies from adult wild-type animals and adult post-mortem human cerebral cortex. Oprm1, as well as co-regulated gene sets were examined. The expression of MOR in microglia was also investigated using our novel fluorescent Cx3cr1-eGFP-MOR-mcherry reporter mouse line. We determined whether CX3cR1-eGFP positive microglial cells expressed MOR-mCherry protein by imaging various brain areas including the Frontal Cortex, Nucleus Accumbens, Ventral Tegmental Area, Central Amygdala, and Periaqueductal Gray matter, as well as spinal cord. Results: Oprm1 expression was found in all 12 microglia datasets from mouse whole brain, in 7 out of 8 from cerebral cortex, 3 out of 4 from hippocampus, 1 out of 1 from striatum, and 4 out of 5 from mouse or rat spinal cord. OPRM1 was expressed in 16 out of 17 microglia transcriptomes from human cerebral cortex. In Cx3cr1-eGFP-MOR-mCherry mice, the percentage of MOR-positive microglial cells ranged between 35.4 and 51.6% in the different brain areas, and between 36.8 and 42.4% in the spinal cord. Conclusion: The comparative analysis of the microglia transcriptomes indicates that Oprm1/OPRM1 transcripts are expressed in microglia. The investigation of Cx3cr1-eGFP-MOR-mCherry mice also shows microglial expression of MOR proteinin the brain and spine. These results corroborate functional studies showing the actions of MOR agonists on microglia and suppression of these effects by MOR-selective antagonists or MOR knockdown.

Dataset Information

Visualization of feline calicivirus replication in real-time with recombinant viruses engineered to express fluorescent reporter proteins.

Publications

Visualization of feline calicivirus replication in real-time with recombinant viruses engineered to express fluorescent reporter proteins.

Similar Datasets

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