Related Experiment Videos
Phenotypic characterization of three phylogenetically conserved stem-loop motifs in the mengovirus 3' untranslated
1Institute for Molecular Virology and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Insights
Investigating mengovirus's 3' untranslated region (UTR) revealed stem-loop structures crucial for viral RNA synthesis. Deleting stem III was lethal, while stem II deletions showed intermediate effects, with reversions restoring viral function.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- The 3' untranslated region (UTR) of cardioviruses contains conserved stem-loop structures.
- These structures are hypothesized to play critical roles in viral RNA replication and infectivity.
Purpose of the Study:
- To investigate the functional significance of three conserved stem-loop structures in the mengovirus 3' UTR.
- To determine the impact of deleting these stem-loops on viral RNA infectivity, RNA synthesis, and plaque formation.
Main Methods:
- Sequence alignment of cardiovirus genomes to identify conserved stem-loops in the mengovirus 3' UTR.
- Engineering deletions of individual stem-loops in mengovirus cDNAs and replicons.
- Evaluating RNA infectivity and plaque phenotype via transfection of viral transcripts into HeLa cells.
- Assessing RNA synthesis activity using luciferase assays in cellular extracts from replicon transfections.
- Isolation, amplification, sequencing, and genetic mapping of revertant viruses from plaque assays.
Main Results:
- Deletion of stem I had no significant impact on viral growth or luciferase expression.
- Deletion of stem III was lethal, completely abolishing RNA synthesis.
- Deletion of stem II (DeltaII) resulted in intermediate phenotypes with reduced RNA synthesis and plaque formation.
- Revertant viruses from DeltaII deletions acquired secondary mutations, including one in the polymerase (3D(pol)) and another in the 3' UTR, which restored viral function.
Conclusions:
- Stem III is essential for mengovirus viability and RNA synthesis.
- Stem II plays a significant role in efficient viral replication and infectivity.
- Reversion mutations can compensate for stem deletions, highlighting the complex interplay of regulatory elements in the 3' UTR and viral proteins.
Abstract:
An alignment of cardiovirus sequences led to the prediction of three conserved stem-loops in the 3' untranslated region (UTR) of mengovirus. Deletions of each stem were engineered in mengovirus cDNAs and also in mengovirus replicons, in which part of the viral capsid sequences were replaced with the firefly luciferase gene. The effect of deletion on RNA infectivity and plaque phenotype was evaluated after transfection of viral transcripts into HeLa cells or by luciferase assays of cellular extracts after transfection with RNA replicons. Stem I (mengovirus bases 7666 to 7687) was found to be dispensable for viral growth or exponential luciferase expression. Deletion of stem III (bases 7711 to 7721) was lethal to the virus, and the replicons were incapable of RNA synthesis. Deletion of stem II (DeltaII; bases 7692 to 7705) produced an intermediate phenotype, in that replicons had marginal RNA synthesis activity but transfection with genomic RNA usually failed to produce plaques after normal incubation times (31 h, 37 degrees C). In a few of the DeltaII transfections, however, plaques were observed after long incubation, especially if the cells received large amounts of RNA (3 microg per 3 x 10(6) cells). Viruses from two DeltaII-derived plaques were isolated and amplified. Their RNAs were converted into cDNA, sequenced, and mapped for genotype. Each maintained the DeltaII deletion and, in addition, had one or two reversion mutations, which were characterized by reverse genetics as responsible for the phenotypes. One reversion caused an amino acid change in the polymerase (3D(pol)), and the other was localized to the 3' UTR, upstream of stem I.