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Cited by 11Viral gastroenteritis research and epidemiologyAnimal Virus Infections StudiesVirology and Viral Diseases

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The astrovirus N-terminal nonstructural protein anchors replication complexes to the perinuclear ER membranes

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Ali, Hashim, Noyvert, David, Hankinson, Jacqueline, Lindsey, Gemma, Lulla, Aleksei, Lulla, Valeria·2024·PLOS Pathogens
DOI 10.1371/journal.ppat.1011959PMC PMC11271882

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Abstract

An essential aspect of positive-sense RNA virus replication is anchoring the replication complex (RC) to cellular membranes. Positive-sense RNA viruses employ diverse strategies, including co-translational membrane targeting through signal peptides and co-opting cellular membrane trafficking components. Often, N-terminal nonstructural proteins play a crucial role in linking the RC to membranes, facilitating the early association of the replication machinery. Astroviruses utilize a polyprotein strategy to synthesize nonstructural proteins, relying on subsequent processing to form replication-competent complexes. This study provides evidence for the perinuclear ER membrane association of RCs in five distinct human astrovirus strains. Using tagged recombinant classical human astrovirus 1 and neurotropic MLB2 strains, we establish that the N-terminal domain guides the ER membrane association. We identified di-arginine motifs responsible for the perinuclear ER retention and formation of functional RCs through mutational analysis of the N-terminal domain in replicon and reverse genetics systems. In addition, we demonstrate the association of key components of the astrovirus replication complex: double-stranded RNA, RNA-dependent RNA polymerase, protease, and N-terminal protein. Our findings highlight the intricate virus-ER interaction mechanism employed by astroviruses, potentially leading to the development of novel antiviral intervention strategies.

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Figures

Fig 1 Analysis of the role of the N-terminal putative helicase in astrovirus replication. (A) Schematic representation of the astrovirus genome, location of N-terminal domain (NTD), and putative Walker A motif, aligned for several astrovirus genomes. (B) Predicted domain organization of the astrovirus nonstructural polyprotein. SPase, signal peptidase; Pro, protease domain; VPg, viral protein genome-linked. C- and N-terminal ends of polyprotein are indicated. (C) The activity of HAstV1-based Renilla luciferase-expressing replicon was measured in Huh7.5.1 cells at 24 hpt. RdRp knock-out mutant (GDD→GNN) is used as replication-deficient control. (D) The virus titer was measured in Caco2 cells after electroporation of BSR cells with in vitro transcribed T7 HAstV1 RNA. (E-F) The effect of guanidine hydrochloride (GuHCl) on the activity of mCherry-expressing astrovirus (E) and enterovirus (F) replicons; hpt, hours post-transfection. The mean cell confluence is provided for each time point. Data are mean ± SEM. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.1, ns, nonsignificant, using one-way ANOVA (C), Student’s t-test (D) or two-way ANOVA (E-F), against wt (C-D) or untreated control (E-F).
Fig 1 Analysis of the role of the N-terminal putative helicase in astrovirus replication. (A) Schematic representation of the astrovirus genome, location of N-terminal domain (NTD), and putative Walker A motif, aligned for several astrovirus genomes. (B) Predicted domain organization of the astrovirus nonstructural polyprotein. SPase, signal peptidase; Pro, protease domain; VPg, viral protein genome-linked. C- and N-terminal ends of polyprotein are indicated. (C) The activity of HAstV1-based Renilla luciferase-expressing replicon was measured in Huh7.5.1 cells at 24 hpt. RdRp knock-out mutant (GDD→GNN) is used as replication-deficient control. (D) The virus titer was measured in Caco2 cells after electroporation of BSR cells with in vitro transcribed T7 HAstV1 RNA. (E-F) The effect of guanidine hydrochloride (GuHCl) on the activity of mCherry-expressing astrovirus (E) and enterovirus (F) replicons; hpt, hours post-transfection. The mean cell confluence is provided for each time point. Data are mean ± SEM. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.1, ns, nonsignificant, using one-way ANOVA (C), Student’s t-test (D) or two-way ANOVA (E-F), against wt (C-D) or untreated control (E-F).CC BY · PubMed Central
Fig 2 Design and characterization of HA-tagged astroviruses. (A) Schematic representation of astrovirus ORF1a polyprotein with HA-tagged NTD domain. The amino acid positions are indicated for each astrovirus strain (top panel). Predicted membrane topology for HA-tag within nsP1a polyprotein (bottom panel). Predicted molecular weight is provided for HAstV1-HA and MLB2-HA SPase-cleaved N-terminal protein. (B) Multistep growth curves of MLB2 and MLB2-HA viruses in Huh7.5.1 cells. Cells were infected at MOI 0.1 (passage 2), and virus titer was measured from the extracellular fractions in triplicates. Data are mean ± SEM. (C) Titers of rescued (passage 0) and passaged (passage 1 in Caco2 cells) HAstV1 and HAstV1-HA viruses. (D) Huh7.5.1 cells were infected at MOI 0.1 (passage 2), harvested at indicated hpi and analyzed by western blotting with anti-CP and anti-HA antibodies. (E) Caco2 cells were infected at MOI 1 (passage 0), harvested at indicated hpi and analyzed by western blotting with anti-HA antibodies. (F) The 24 hpi samples from (D) and (E) were analyzed using Bis-Tris acrylamide gel electrophoresis. (G-H) Representative confocal images of fixed and permeabilized infected cells visualized for MLB2 (G) or HAstV1 (H) CP (green) and HA-tag (magenta). Nuclei were stained with Hoechst (blue). Scale bars are 25 μm (G-H).
Fig 2 Design and characterization of HA-tagged astroviruses. (A) Schematic representation of astrovirus ORF1a polyprotein with HA-tagged NTD domain. The amino acid positions are indicated for each astrovirus strain (top panel). Predicted membrane topology for HA-tag within nsP1a polyprotein (bottom panel). Predicted molecular weight is provided for HAstV1-HA and MLB2-HA SPase-cleaved N-terminal protein. (B) Multistep growth curves of MLB2 and MLB2-HA viruses in Huh7.5.1 cells. Cells were infected at MOI 0.1 (passage 2), and virus titer was measured from the extracellular fractions in triplicates. Data are mean ± SEM. (C) Titers of rescued (passage 0) and passaged (passage 1 in Caco2 cells) HAstV1 and HAstV1-HA viruses. (D) Huh7.5.1 cells were infected at MOI 0.1 (passage 2), harvested at indicated hpi and analyzed by western blotting with anti-CP and anti-HA antibodies. (E) Caco2 cells were infected at MOI 1 (passage 0), harvested at indicated hpi and analyzed by western blotting with anti-HA antibodies. (F) The 24 hpi samples from (D) and (E) were analyzed using Bis-Tris acrylamide gel electrophoresis. (G-H) Representative confocal images of fixed and permeabilized infected cells visualized for MLB2 (G) or HAstV1 (H) CP (green) and HA-tag (magenta). Nuclei were stained with Hoechst (blue). Scale bars are 25 μm (G-H).CC BY · PubMed Central
Fig 3 Astrovirus replication sites are associated with the perinuclear ER membranes. (A) Caco2 cells were infected with HAstV1, HAstV4 and VA1 astroviruses. (B) Huh7.5.1 cells were infected with MLB1, MLB2 and VA1 astroviruses. (A-B) Representative confocal images of fixed and permeabilized cells visualized for dsRNA (magenta) and ER (calnexin, green). Nuclei were stained with Hoechst (blue). Scale bars are 25 μm. Intensity profiles of dsRNA (magenta), calnexin (green) and nuclear staining (blue) were obtained using ImageJ software, along a straight line shown on the merged image crossing the representative cell. Quantification of co-localization of dsRNA with calnexin. The Pearson correlation coefficient was calculated for 12 images in each experiment.
Fig 3 Astrovirus replication sites are associated with the perinuclear ER membranes. (A) Caco2 cells were infected with HAstV1, HAstV4 and VA1 astroviruses. (B) Huh7.5.1 cells were infected with MLB1, MLB2 and VA1 astroviruses. (A-B) Representative confocal images of fixed and permeabilized cells visualized for dsRNA (magenta) and ER (calnexin, green). Nuclei were stained with Hoechst (blue). Scale bars are 25 μm. Intensity profiles of dsRNA (magenta), calnexin (green) and nuclear staining (blue) were obtained using ImageJ software, along a straight line shown on the merged image crossing the representative cell. Quantification of co-localization of dsRNA with calnexin. The Pearson correlation coefficient was calculated for 12 images in each experiment.CC BY · PubMed Central
Fig 4 Astrovirus replication sites demonstrate perinuclear ER localization. (A) Caco2 cells were infected with HAstV1, HAstV4 and VA1 astroviruses. (B) Huh7.5.1 cells were infected with MLB1, MLB2 and VA1 astroviruses. (A-B) Representative confocal images of fixed and permeabilized cells visualized for dsRNA (magenta) and nuclear lamin (green). Nuclei were stained with Hoechst (blue). Scale bars are 25 μm. Intensity profiles of dsRNA (magenta), lamin (green) and nuclear staining (blue) were obtained using ImageJ software, along a straight line shown on the merged image crossing the representative cell. Quantification of co-localization of dsRNA with lamin. The Pearson correlation coefficient was calculated for 12 images in each experiment.
Fig 4 Astrovirus replication sites demonstrate perinuclear ER localization. (A) Caco2 cells were infected with HAstV1, HAstV4 and VA1 astroviruses. (B) Huh7.5.1 cells were infected with MLB1, MLB2 and VA1 astroviruses. (A-B) Representative confocal images of fixed and permeabilized cells visualized for dsRNA (magenta) and nuclear lamin (green). Nuclei were stained with Hoechst (blue). Scale bars are 25 μm. Intensity profiles of dsRNA (magenta), lamin (green) and nuclear staining (blue) were obtained using ImageJ software, along a straight line shown on the merged image crossing the representative cell. Quantification of co-localization of dsRNA with lamin. The Pearson correlation coefficient was calculated for 12 images in each experiment.CC BY · PubMed Central
Fig 5 Astrovirus NTD is associated with RNA replication sites. (A) Caco2 cells were infected with HAstV1-HA. (B) Huh7.5.1 cells were infected with MLB2-HA. (A-B) Representative confocal images of fixed and permeabilized cells visualized for HA-tag (NTD, magenta), dsRNA (green), ER (calnexin, green), and lamin (green). Nuclei were stained with Hoechst (blue). Scale bars are 25 μm. Intensity profiles of NTD (magenta), dsRNA (green), ER (green), lamin (green), and nuclear staining (blue) were obtained using ImageJ software, along a straight line shown on the merged image crossing the representative cell. Quantification of co-localization of NTD with dsRNA, calnexin, and lamin. The Pearson correlation coefficient was calculated for 12 images in each experiment. Data are mean ± SEM. **** p < 0.0001, * p < 0.05, ns, nonsignificant, using one-way ANOVA test against wt.
Fig 5 Astrovirus NTD is associated with RNA replication sites. (A) Caco2 cells were infected with HAstV1-HA. (B) Huh7.5.1 cells were infected with MLB2-HA. (A-B) Representative confocal images of fixed and permeabilized cells visualized for HA-tag (NTD, magenta), dsRNA (green), ER (calnexin, green), and lamin (green). Nuclei were stained with Hoechst (blue). Scale bars are 25 μm. Intensity profiles of NTD (magenta), dsRNA (green), ER (green), lamin (green), and nuclear staining (blue) were obtained using ImageJ software, along a straight line shown on the merged image crossing the representative cell. Quantification of co-localization of NTD with dsRNA, calnexin, and lamin. The Pearson correlation coefficient was calculated for 12 images in each experiment. Data are mean ± SEM. **** p < 0.0001, * p < 0.05, ns, nonsignificant, using one-way ANOVA test against wt.CC BY · PubMed Central
Fig 6 Astrovirus replication sites are tightly associated with dsRNA, NTD, protease and RdRp, the capsid protein is dispersed throughout the cytoplasm. (A) Coomassie-stained SDS-PAGE profile of purified proteins from E. coli (left-to-right): folded domains of VA1 capsid protein (CP), MLB2 and HAstV1 protease and HAstV1 RdRp. Predicted molecular weight is given for each purified protein domain. (B) The indicated cell line was infected with MLB2 (top) or VA1 (bottom) and stained for dsRNA (magenta) and virus-specific capsid protein (green). Nuclei were stained with Hoechst (blue). (C) The indicated cell line was infected with HAstV1 or MLB2 and stained for dsRNA (magenta) and virus-specific nonstructural proteins (green). Nuclei were stained with Hoechst (blue). The quantification of co-localization of dsRNA with indicated virus protein is shown on the right (B-C). The Pearson correlation coefficient was calculated for 12 images in each experiment (B-C). (D-E) Proximity ligation assay (PLA) for key RC components in astrovirus-infected cells. Caco2 (D) and Huh7.5.1 (E) cells were infected with the viruses indicated on the left of each panel, followed by PLA staining using pairs of rabbit (Rb) and mouse (Ms) derived antibodies indicated on top of each panel. A representative experiment of two independent repeats is shown. Scale bars are 25 μm (B-E).
Fig 6 Astrovirus replication sites are tightly associated with dsRNA, NTD, protease and RdRp, the capsid protein is dispersed throughout the cytoplasm. (A) Coomassie-stained SDS-PAGE profile of purified proteins from E. coli (left-to-right): folded domains of VA1 capsid protein (CP), MLB2 and HAstV1 protease and HAstV1 RdRp. Predicted molecular weight is given for each purified protein domain. (B) The indicated cell line was infected with MLB2 (top) or VA1 (bottom) and stained for dsRNA (magenta) and virus-specific capsid protein (green). Nuclei were stained with Hoechst (blue). (C) The indicated cell line was infected with HAstV1 or MLB2 and stained for dsRNA (magenta) and virus-specific nonstructural proteins (green). Nuclei were stained with Hoechst (blue). The quantification of co-localization of dsRNA with indicated virus protein is shown on the right (B-C). The Pearson correlation coefficient was calculated for 12 images in each experiment (B-C). (D-E) Proximity ligation assay (PLA) for key RC components in astrovirus-infected cells. Caco2 (D) and Huh7.5.1 (E) cells were infected with the viruses indicated on the left of each panel, followed by PLA staining using pairs of rabbit (Rb) and mouse (Ms) derived antibodies indicated on top of each panel. A representative experiment of two independent repeats is shown. Scale bars are 25 μm (B-E).CC BY · PubMed Central
Fig 7 Di-arginine motifs in NTD are required for virus replication. (A) Schematic representation of astrovirus nsP1a polyprotein with di-arginine motifs. The amino acid positions are indicated for each astrovirus strain. (B-C) 3D model for wt HAstV1 (B) and wt MLB2 (C) NTD predicted with AlphaFold 3. The positions of the di-arginine motifs, the inserted HA-tag (insertion position only), and the transmembrane (TM) helix are indicated. (D) Schematic representation of astrovirus replicon with introduced R-to-A mutations. The mutant annotation is shown on the left. (E-F) Relative luciferase activities of HAstV1 (E) and MLB2 (F) based replicons in Huh7.5.1 cells at 24 hours post-transfection. Data are mean ± SD. **** p < 0.0001, using one-way ANOVA test against wt. RdRp knock-out mutant (GDD→GNN) is used as replication-deficient control. (G-H) Titers (infectious units per ml) of recombinant viruses after RNA electroporation of BSR (HAstV1) or Huh7.5.1 (MLB2) cells followed by titration on Caco2 (HAstV1, G) or Huh7.5.1 (MLB2, H) cells.
Fig 7 Di-arginine motifs in NTD are required for virus replication. (A) Schematic representation of astrovirus nsP1a polyprotein with di-arginine motifs. The amino acid positions are indicated for each astrovirus strain. (B-C) 3D model for wt HAstV1 (B) and wt MLB2 (C) NTD predicted with AlphaFold 3. The positions of the di-arginine motifs, the inserted HA-tag (insertion position only), and the transmembrane (TM) helix are indicated. (D) Schematic representation of astrovirus replicon with introduced R-to-A mutations. The mutant annotation is shown on the left. (E-F) Relative luciferase activities of HAstV1 (E) and MLB2 (F) based replicons in Huh7.5.1 cells at 24 hours post-transfection. Data are mean ± SD. **** p < 0.0001, using one-way ANOVA test against wt. RdRp knock-out mutant (GDD→GNN) is used as replication-deficient control. (G-H) Titers (infectious units per ml) of recombinant viruses after RNA electroporation of BSR (HAstV1) or Huh7.5.1 (MLB2) cells followed by titration on Caco2 (HAstV1, G) or Huh7.5.1 (MLB2, H) cells.CC BY · PubMed Central
Fig 8 Di-arginine motifs define the efficient expression and perinuclear ER localization of NTD. (A) Schematic representation of CAG promoter containing mammalian expression vector used to over-express HA-tagged NTD in Huh7.5.1 cells. (B-C) Huh7.5.1 cells were electroporated with pCAG plasmids expressing wt and mutant NTD-HA and analyzed by western blotting with anti-HA and anti-tubulin antibodies. Lysates obtained from infected cells were used as a control. (D-E) Huh7.5.1 cells were electroporated with pCAG plasmids expressing wt and mutant NTD-HA and analyzed by immunofluorescence. Representative confocal images of fixed and permeabilized cells visualized for HA-tag (NTD, magenta) and ER (calnexin, green). Nuclei were stained with Hoechst (blue). Scale bars are 25 μm. Averaged intensity profiles of NTD (magenta), ER (green) and nuclear staining (blue) were obtained using ImageJ software, along a straight line spanning from the nucleus to the plasma membrane of 17–20 transfected cells. The changes in the overlap signal between NTD-HA and ER were compared using Manders coefficient (M2).
Fig 8 Di-arginine motifs define the efficient expression and perinuclear ER localization of NTD. (A) Schematic representation of CAG promoter containing mammalian expression vector used to over-express HA-tagged NTD in Huh7.5.1 cells. (B-C) Huh7.5.1 cells were electroporated with pCAG plasmids expressing wt and mutant NTD-HA and analyzed by western blotting with anti-HA and anti-tubulin antibodies. Lysates obtained from infected cells were used as a control. (D-E) Huh7.5.1 cells were electroporated with pCAG plasmids expressing wt and mutant NTD-HA and analyzed by immunofluorescence. Representative confocal images of fixed and permeabilized cells visualized for HA-tag (NTD, magenta) and ER (calnexin, green). Nuclei were stained with Hoechst (blue). Scale bars are 25 μm. Averaged intensity profiles of NTD (magenta), ER (green) and nuclear staining (blue) were obtained using ImageJ software, along a straight line spanning from the nucleus to the plasma membrane of 17–20 transfected cells. The changes in the overlap signal between NTD-HA and ER were compared using Manders coefficient (M2).CC BY · PubMed Central
Fig 9 The proposed mechanism of NTD-directed formation of astrovirus replication complex. The translation of astrovirus nonstructural polyprotein begins with the NTD, that is anchored to ER membranes through cooperative interactions of charged arginine residues. Non-structural proteins including viral RdRp, protease, and NTD, form the replication complex in tight association with perinuclear ER membranes and other virus/host proteins. Newly synthesized viral genomic and subgenomic RNAs (gRNA and sgRNA) are used for translation, replication, and/or packaging.
Fig 9 The proposed mechanism of NTD-directed formation of astrovirus replication complex. The translation of astrovirus nonstructural polyprotein begins with the NTD, that is anchored to ER membranes through cooperative interactions of charged arginine residues. Non-structural proteins including viral RdRp, protease, and NTD, form the replication complex in tight association with perinuclear ER membranes and other virus/host proteins. Newly synthesized viral genomic and subgenomic RNAs (gRNA and sgRNA) are used for translation, replication, and/or packaging.CC BY · PubMed Central

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Metadata

Authors
Ali, H., Noyvert, D., Hankinson, J., Lindsey, G., Lulla, A., & Lulla, V.
Affiliations
University of Cambridge (GB)
Corresponding author
Lulla, Valeria
ORCID
Venue
PLOS Pathogens
Publisher
Public Library of Science (PLoS)
Published
2024-07-15
Volume
20
Issue
7
Pages
e1011959
ISSN
  • 1553-7366 (ISSN-L)
  • 1553-7374
DOI
10.1371/journal.ppat.1011959
PMC
PMC11271882
OpenAlex
W4400647539
Cited by
11
References
52
Citations by year
2026: 4 · 2025: 7
FWCI
3.80
Type
journal article
Language
en
Update notices
new_version (2024-07-25)
Open access
Licence
Locations
  • PLoS Pathogens (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • UCL Discovery (University College London) (submittedVersion, cc-by)
  • Apollo (University of Cambridge) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Wellcome Trust (220620/Z/20/Z)
  • Isaac Newton Trust
  • Medical Research Foundation (MR/T000376/1)
  • Studentship from Elisabeth Mann Fund
Topics
  • Health Sciences › Medicine › Infectious Diseases › Viral gastroenteritis research and epidemiology
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
  • Health Sciences › Medicine › Epidemiology › Virology and Viral Diseases
Keywords
astrovirus, viral replication complex, ER retention signal, polyprotein processing, RNA-dependent RNA polymerase, double-stranded RNA, antiviral drug targets
MeSH terms
Mamastrovirus, Endoplasmic Reticulum, Humans, Intracellular Membranes, Virus Replication, Viral Nonstructural Proteins, Astroviridae Infections

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