Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs

Mihalič, Filip; Simonetti, Leandro; Giudice, Girolamo; Sander, Marie Rubin; Lindqvist, Richard; Peters, Marie Berit Akprioro; Benz, Caroline; Kassa, Eszter; Badgujar, Dilip; Inturi, Raviteja; Ali, Muhammad; Krystkowiak, Izabella; Sayadi, Ahmed; Andersson, Eva; Aronsson, Hanna; Söderberg, Ola; Dobritzsch, Doreen; Petsalaki, Evangelia; Överby, Anna K.; Jemth, Per; Davey, Norman E.; Ivarsson, Ylva

doi:10.1038/s41467-023-38015-5

Cited by 24 publications

(12 citation statements)

References 120 publications

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“…In astroviruses, the NTD, followed by transmembrane helices, represents a unique combination to target the nonstructural polyprotein to the correct position within the infected cell. We explored the power of molecular mimicry used by many viruses 26 to identify the potential residues responsible for ER targeting that were predicted using the ELM web server (http://elm.eu.org/) 27 . The search revealed the presence of conserved di-arginine motifs (Fig 6A), which were defined by two consecutive arginine residues (RR) or with a single residue insertion (RXR).…”

Section: Resultsmentioning

confidence: 99%

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

Ali,

Noyvert,

Hankinson

et al. 2024

Preprint

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An essential aspect of viral replication is the anchoring of 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. In this study, we provide 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. Through mutational analysis of the N-terminal domain in replicon and reverse genetics systems, we identified di-arginine motifs responsible for the perinuclear ER retention and formation of functional RCs. Our findings highlight the intricate virus-ER interaction mechanism employed by astroviruses, potentially leading to the development of novel antiviral intervention strategies.Author SummaryHuman astroviruses are a significant cause of acute gastroenteritis, accounting for up to 9% of cases in young children. Immunocompromised individuals and infants experience more critical symptoms, such as severe and persistent diarrhea, as well as sporadic systemic and even fatal diseases. To date, no drugs have been developed to protect against astrovirus infection. Our study provides the first evidence that the integrity of the N-terminal domain of nsP1a is essential for establishing early replication. Central to this process, the di-arginine motifs in the N-terminal domain are responsible for ER retention, the formation of functional replication complexes, and viral replication. Therefore, selectively targeting N-terminal domain-mediated ER retention could be a promising therapeutic strategy to effectively control astrovirus infection.

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Section: Resultsmentioning

confidence: 99%

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

Ali,

Noyvert,

Hankinson

et al. 2024

Preprint

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“…None of the five mutations found in the C-terminal domain interfere with the previously described short linear motifs found in this region, which interact with amphiphysin-SH3 (which is recruited by the virus to promote viral RNA replication) [88] and the G3BP-NTF2 domains (which are hijacked to block stress granule formation) [89]. Given the high density of short linear motifs in the disordered regions of viral proteins and the fact that the C-terminal disorder domain of Nsp3 acts as an interaction hub for host factors, it is possible that the T337I, T338M, D372E, L461P, and P471S mutations create new binding sites for host proteins or destroy existing ones [90]. Alternatively, these mutations might not have significant effects on the function of the protein and might be consequences of random drift.…”

Section: Resultsmentioning

confidence: 99%

The evolutionary and molecular history of a chikungunya virus outbreak lineage

Krambrich,

Mihalič,

Gaunt

et al. 2024

Preprint

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In 2018–2019, Thailand experienced a nationwide spread of chikungunya virus (CHIKV), with approximately 15,000 confirmed cases of disease reported. Here, we investigated the evolutionary and molecular history of the East/Central/South African (ECSA) genotype to determine the origins of the 2018–2019 CHIKV outbreak in Thailand. This was done using newly sequenced clinical samples from travellers returning to Sweden from Thailand in late 2018 and early 2019 and previously published genome sequences. Our phylogeographic analysis showed that before the outbreak in Thailand, the Indian Ocean lineage (IOL) found within the ESCA, had evolved and circulated in East Africa, South Asia, and Southeast Asia for about 15 years. In the first half of 2017, an introduction occurred into Thailand from another South Asian country, most likely Bangladesh, which subsequently developed into a large outbreak in Thailand with export to neighbouring countries. Based on comparative phylogenetic analyses of the complete CHIKV genome and protein modelling, we also identified amino acid substitutions that may be associated with immune evasion, increased spread, and virulence. We identified several mutations in the E1/E2 spike complex, such as E1 K211E and E2 V264A, which are highly relevant as they may lead to changes in vector competence, transmission efficiency and pathogenicity of the virus. A number of mutations (E2 G205S, Nsp3 D372E, Nsp2 V793A), that emerged shortly before the outbreak of the virus in Thailand in 2018 may have altered antibody binding and recognition due to their position. This study not only improves our understanding of the factors contributing to the epidemic in Southeast Asia, but also has implications for the development of effective response strategies and the potential development of new vaccines.

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“…For example, except for certain key positions, such interactions appear robust to changes in the amino acid sequence increasing the possibility for permissive neutral substitutions, which may allow new functions to evolve upon further mutation and selection (Hultqvist et al, 2017). In addition, interactions of this type are often hijacked by viral proteins, (Mihalic et al, 2023) potentially imposing additional selection pressure. While we here attempted to pinpoint historical amino acid substitutions shaping the affinity between the p53TAD binding motif and MDM2, as done for other short motifs, (Laursen et al, 2021) conformational heterogeneity among the bound conformations of p53TAD is also likely contributing to the variation in affinity.…”

Section: Discussionmentioning

confidence: 99%

Evolution of affinity between p53 transactivation domain and MDM2 across the animal kingdom demonstrates high plasticity of motif‐mediated interactions

et al. 2023

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The interaction between the transcription factor p53 and the ubiquitin ligase MDM2 results in the degradation of p53 and is well-studied in cancer biology and drug development. Available sequence data suggest that both p53 and MDM2-family proteins are present across the animal kingdom. However, the interacting regions are missing in some animal groups, and it is not clear whether MDM2 interacts with, and regulates p53 in all species. We used phylogenetic analyses and biophysical measurements to examine the evolution of affinity between the interacting protein regions: a conserved 12-residue intrinsically disordered binding motif in the p53 transactivation domain (TAD) and the folded SWIB domain of MDM2. The affinity varied significantly across the animal kingdom. The p53TAD/MDM2 interaction among jawed vertebrates displayed high affinity, in particular for chicken and human proteins (K D around 0.1 μM). The affinity of the bay mussel p53TAD/ MDM2 complex was lower (K D = 15 μM) and those from a placozoan, an arthropod, and a jawless vertebrate were very low or non-detectable (K D > 100 μM). Binding experiments with reconstructed ancestral p53TAD/ MDM2 variants suggested that a micromolar affinity interaction was present in the ancestral bilaterian animal and was later enhanced in tetrapods while lost in other linages. The different evolutionary trajectories of p53TAD/ MDM2 affinity during speciation demonstrate high plasticity of motifmediated interactions and the potential for rapid adaptation of p53 regulation during times of change. Neutral drift in unconstrained disordered regions may underlie the plasticity and explain the observed low sequence conservation in TADs such as p53TAD.Filip Mihalič and Emma Åberg contributed equally to this study.

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Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs

Cited by 24 publications

References 120 publications

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

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

The evolutionary and molecular history of a chikungunya virus outbreak lineage

Evolution of affinity between p53 transactivation domain and MDM2 across the animal kingdom demonstrates high plasticity of motif‐mediated interactions

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