Generation of Reporter-Expressing New World Arenaviruses: A Systematic Comparison

Fénéant, Lucie; Leske, Anne; Günther, Karla; Groseth, Allison

doi:10.3390/v14071563

Cited by 3 publications

(1 citation statement)

References 85 publications

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“…Furthermore, the effect of insertion on viral replication in cultured cells was minimal. The self-cleaving 2A peptide is used to express foreign genes in segmented RNA viruses [ 30 , 36 , 37 ]. For MRV, as one genomic segment codes for one protein gene, the upstream (5′ end) or downstream (3′ end) of the viral gene can be the site for insertion of a gene cassette for bicistronic expression.…”

Section: Discussionmentioning

confidence: 99%

Cytocidal Effect of Irradiation on Gastric Cancer Cells Infected with a Recombinant Mammalian Orthoreovirus Expressing a Membrane-Targeted KillerRed

Shirasaka,

Yamada,

Etoh

et al. 2024

Pharmaceuticals

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The outcomes of unresectable gastric cancer (GC) are unfavorable even with chemotherapy; therefore, a new treatment modality is required. The combination of an oncolytic virus and photodynamic therapy can be one of the promising modalities to overcome this. Mammalian orthoreovirus (MRV) is an oncolytic virus that has been used in clinical trials for several cancers. In this study, we developed and evaluated a recombinant MRV strain type 3 Dearing (T3D) that expresses membrane-targeting KillerRed (KRmem), a phototoxic fluorescent protein that produces cytotoxic reactive oxygen species upon light irradiation. KRmem was fused in-frame to the 3′ end of the σ2 viral gene in the S2 segment using a 2A peptide linker, enabling the expression of multiple proteins from a single transcript. RNA electrophoresis, Western blotting, and immunofluorescence analyses confirmed functional insertion of KRmem into the recombinant virus. The growth activity of the recombinant virus was comparable to that of the wild-type MRV in a cultured cell line. The recombinant virus infected two GC cell lines (MKN45P and MKN7), and a significant cytocidal effect was observed in MKN45P cells infected with the recombinant virus after light irradiation. Thus, recombinant MRV-expressing KRmem has the potential to serve as a novel treatment tool for GC.

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Section: Discussionmentioning

confidence: 99%

Cytocidal Effect of Irradiation on Gastric Cancer Cells Infected with a Recombinant Mammalian Orthoreovirus Expressing a Membrane-Targeted KillerRed

Shirasaka,

Yamada,

Etoh

et al. 2024

Pharmaceuticals

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Alternative translation contributes to the generation of a cytoplasmic subpopulation of the Junín virus nucleoprotein that inhibits caspase activation and innate immunity

Bostedt,

Fénéant,

Leske

et al. 2024

J Virol

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The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP 53kD , NP 47kD , and NP 40kD . While both NP 47kD and NP 40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP 53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP 40kD plays the predominant role. In contrast to full-length NP (i.e., NP 65kD ), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP 53kD , NP 47kD , and NP 40kD all retain robust interferon antagonistic and 3′−5′ exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection. IMPORTANCE A limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP 47kD and NP 40kD ) are known to be produced by caspase cleavage, while, here, we show that NP 53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP 40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.

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Engineering recombinant replication-competent bluetongue viruses expressing reporter genes for in vitro and non-invasive in vivo studies

Utrilla-Trigo,

Jiménez-Cabello,

Marín-López

et al. 2024

Microbiol Spectr

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The use of replication-competent viruses that encode a traceable fluorescent or luciferase reporter protein has significantly contributed to the in vitro and in vivo study of viral infections and the development of novel therapeutic approaches. In this work, we have generated rBTV that express fluorescent or luminescence proteins to track BTV infection both in vitro and in vivo . Despite the availability of vaccines, BTV and other related orbivirus are still associated with a significant impact on animal health and have important economic consequences worldwide. Our studies may contribute to the advance in orbivirus research and pave the way for the rapid development of new treatments, including vaccines.

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Generation of Reporter-Expressing New World Arenaviruses: A Systematic Comparison

Cited by 3 publications

References 85 publications

Cytocidal Effect of Irradiation on Gastric Cancer Cells Infected with a Recombinant Mammalian Orthoreovirus Expressing a Membrane-Targeted KillerRed

Cytocidal Effect of Irradiation on Gastric Cancer Cells Infected with a Recombinant Mammalian Orthoreovirus Expressing a Membrane-Targeted KillerRed

Alternative translation contributes to the generation of a cytoplasmic subpopulation of the Junín virus nucleoprotein that inhibits caspase activation and innate immunity

Engineering recombinant replication-competent bluetongue viruses expressing reporter genes for in vitro and non-invasive in vivo studies

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