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. 2023 Jun 20;15(6):1402.
doi: 10.3390/v15061402.

The Roles of the 5' and 3' Untranslated Regions in Human Astrovirus Replication

Nicole Wildi  1   2 , Torsten Seuberlich  1
Affiliations

The Roles of the 5' and 3' Untranslated Regions in Human Astrovirus Replication

Nicole Wildi et al. Viruses. .

Abstract

Astroviruses are small nonenveloped single-stranded RNA viruses with a positive sense genome. They are known to cause gastrointestinal disease in a broad spectrum of species. Although astroviruses are distributed worldwide, a gap in knowledge of their biology and disease pathogenesis persists. Many positive-sense single-stranded RNA viruses show conserved and functionally important structures in their 5' and 3' untranslated regions (UTRs). However, not much is known about the role of the 5' and 3' UTRs in the viral replication of HAstV-1. We analyzed the UTRs of HAstV-1 for secondary RNA structures and mutated them, resulting in partial or total UTR deletion. We used a reverse genetic system to study the production of infectious viral particles and to quantify protein expression in the 5' and 3' UTR mutants, and we established an HAstV-1 replicon system containing two reporter cassettes in open reading frames 1a and 2, respectively. Our data show that 3' UTR deletions almost completely abolished viral protein expression and that 5' UTR deletions led to a reduction in infectious virus particles in infection experiments. This indicates that the presence of the UTRs is essential for the life cycle of HAstV-1 and opens avenues for further research.

Keywords: astrovirus; replication; reverse genetics; translation; untranslated region.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematics of the human astrovirus type 1 (HAstV-1) genome organization, showing the RNA-dependent RNA polymerase knockout (RdRp ko) site used to generate a replication-incompetent control mutant from the wild type (wt) (a) and the HAstV-1 virus rescue system (b). (c) Schematics of the HAstV-1 dual-reporter replicons. Two nonlabeled arrows pointing to regions in the ORF1a where the first 45 nt of the ORF1a are placed twice. iv, in vitro; UTR, untranslated region; ORF, open reading frame; VPg, viral protein linked to the genome; GFP, green fluorescent protein; nluc, nanoluciferase; fluc, firefly luciferase.
Figure 2
Figure 2
Secondary RNA structures predicted for the 5′ (a) and 3′ (b) UTRs of HAstV-1 and design of UTR deletion mutants. The deleted nucleotides are shown in red. The green boxes indicate the start codon of ORF1a, and the orange boxes indicate the stop codon of ORF2. These structure models were generated using mfold [51] and modified using CorelDraw X6 (version 16.0.0.707). SL, stem-loop; s2m, stem-loop 2 motif.
Figure 3
Figure 3
Translation of viral proteins and viral RNA replication in HAstV-1 UTR deletion mutants. (a) BSR-T7 cells transfected with wt HAstV-1 and deletion mutants. The HAstV capsid protein is labeled in red and cellular nuclei in blue. Scale bar = 20 µm. (b) Images from the in situ hybridization for antigenomic HAstV-1 RNA of BSR-T7 cells transfected with wt HAstV-1 and deletion mutants. Red dots and arrows indicate RNA probe hybridization. Scale bar = 50 µm, 40× magnification.
Figure 4
Figure 4
Compromised viral particle assembly and release of HAstV-1 UTR deletion mutants. (a) Immunofluorescence images of CaCo-2 cells infected with supernatants of transfected BSR-T7 samples. The HAstV-1 capsid protein is stained red, and nuclei are stained blue. Scale bar = 20 µm. (b) Titration of HAstV-1 5′ UTR mutants (after BSR-T7 cell transfection = P0) on CaCo-2 cells. The titers (quantified as the 50% tissue culture infectious dose (TCID50)/mL) were normalized to those of wt HAstV-1. Data for the 3′ UTR and HAstV-1 RdRp ko mutants are not shown because these mutants did not infect CaCo-2 cells. The error bars indicate standard deviations from three independent experiments. *** p < 0.001. (c) Virus titers of HAstV-1 mutants on CaCo-2 cells in sequential passages (n = 5). P0, after BSR-T7 cell transfection; P1–P5, after CaCo-2 cell infection (n = 2), ns = nonsignificant.
Figure 5
Figure 5
HAstV-1 UTR deletions compromise viral protein expression. Fluc (a) and nluc (b) activity at 72 h after transfection (hpt; left) and over time (2–72 h, right). *** p < 0.01. The experiments were conducted in triplicate. The error bars indicate standard deviations (n = 9). fluc, firefly luciferase; nluc, nanoluciferase; RLUs, relative luminescence units.
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Grants and funding

This research was supported by the Swiss Federal Food Safety and Veterinary Office.