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As the name suggests, non-LTR retrotransposons lack the long terminal repeats characteristic of the LTR retrotransposons. Additionally, both LTR and non-LTR retrotransposons use distinct mechanisms of mobilization. Non-LTR retrotransposons are further divided into two classes - Long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), both of which occur abundantly in most mammals, including humans. Some of the active non-LTR retrotransposons in humans are L1...
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LTR retrotransposons are class I transposable elements with long terminal repeats flanking an internal coding region. These elements are less abundant in mammals compared to other class I transposable elements. About 8 percent of human genomic DNA comprises LTR retrotransposons. Some of the common examples of LTR retrotransposons are Ty elements in yeast and Copia elements in Drosophila.
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Retroviruses have a single-stranded RNA genome that undergoes a special form of replication. Once the retrovirus has entered the host cell, an enzyme called reverse transcriptase synthesizes double-stranded DNA from the retroviral RNA genome. This DNA copy of the genome is then integrated into the host’s genome inside the nucleus via an enzyme called integrase. Consequently, the retroviral genome is transcribed into RNA whenever the host’s genome is transcribed, allowing the...
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Reverse Genetics with a Full-Length Infectious cDNA Clone of Bovine Torovirus.

Makoto Ujike1,2, Yuka Etoh1, Naoya Urushiyama1

  • 1Laboratory of Veterinary Infectious Diseases, Faculty of Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino, Tokyo, Japan.

Journal of Virology
|November 24, 2021
PubMed
Summary

Researchers developed the first reverse genetics system for toroviruses (ToV), enabling manipulation of bovine ToV (BToV). Mutations in nonstructural proteins (NSPs) were found to improve the stability and replication of recombinant viruses carrying foreign genes like EGFP.

Keywords:
Tobaniviridaecoronavirushemagglutinin-esterasenonstructural proteinreverse genetic analysistorovirus

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Area of Science:

  • Virology
  • Molecular Biology
  • Genetics

Background:

  • Toroviruses (ToV), recently classified into the Tobaniviridae family, share historical links with coronaviruses (CoV).
  • Reverse genetics systems are crucial for studying viral mechanisms but were previously unavailable for ToVs.

Purpose of the Study:

  • To establish the first reverse genetics system for ToV using a bacterial artificial chromosome (BAC)-based infectious clone of bovine ToV (BToV).
  • To investigate the role of the hemagglutinin-esterase (HE) gene in BToV replication and to assess the stability of recombinant viruses expressing foreign genes.

Main Methods:

  • Construction of a full-length cDNA clone of BToV in a BAC for generating infectious BToV.
  • Rescue of recombinant BToV with genetic markers and modifications to the HE gene (full-length HEf, soluble HEs, HA-tagged HEf/HEs).
  • Generation of a recombinant virus (rEGFP) where HE was replaced by the enhanced green fluorescent protein (EGFP) gene.
  • Passaging of rEGFP to identify mutations conferring improved gene stability and replication.

Main Results:

  • A functional reverse genetics system for BToV was successfully established.
  • Recombinant BToV with HE gene modifications showed similar growth to wild-type (wt) BToV, indicating HE is not essential for replication in HRT18 cells.
  • The rEGFP virus exhibited reduced viral growth and instability, readily deleting the EGFP gene.
  • Variants of rEGFP accumulating two specific mutations in nonstructural proteins (NSPs) demonstrated enhanced EGFP expression, retention, and viral replication.
  • Introduction of these NSP mutations into rEGFP phenocopied the improved characteristics.

Conclusions:

  • The developed BAC-based reverse genetics system is the first for the Tobaniviridae family, offering a powerful tool for ToV research.
  • Mutations in NSPs appear to play a critical role in the acceptance and retention of exogenous genes (like HE or EGFP) in BToV.
  • These findings provide fundamental insights into BToV biology and pave the way for future studies on pathogenesis and vaccine development.