Detection of Protein Interactions During Virus Infection by Bimolecular Fluorescence Complementation

Becker, Stefan; Einem, Jens von

doi:10.1007/978-1-62703-601-6_3

Cited by 3 publications

(2 citation statements)

References 31 publications

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“…The BiFC assay was performed as previously described (Becker and von Einem, 2013). Therefore, MRC- 5 cells were first transfected by electroporation as described in section immunocytochemistry with the DNA of conditional expression vectors expressing either YC-VPS4A-FLAG, YC-VPS4A-FLAG-V13D, or YC- VPS4A-FLAG-V64D fusion proteins.…”

Section: Methodsmentioning

confidence: 99%

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly

Butt,

Fischer,

Rep

et al. 2024

Preprint

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The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across beta-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.

show abstract

Section: Methodsmentioning

confidence: 99%

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly

Butt,

Fischer,

Rep

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…The BiFC assay was performed as previously described [ 105 ]. Therefore, MRC-5 cells were first transfected by electroporation as described in section immunocytochemistry with the DNA of conditional expression vectors expressing either YC-VPS4A-FLAG, YC-VPS4A-FLAG-V13D, or YC-VPS4A-FLAG-V64D fusion proteins.…”

Section: Methodsmentioning

confidence: 99%

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly

Butt,

Fischer,

Rep

et al. 2024

PLoS Pathog

Self Cite

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The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across β-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.

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A transcription-independent role for HIF-1α in modulating microprocessor assembly

Li,

Wang,

Ruan

et al. 2024

Nucleic Acids Research

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Microprocessor is an essential nuclear complex responsible for the initial RNase-mediated cleavage of primary miRNA, which is a tightly controlled maturation process that requires the proper assembly of Drosha and DGCR8. Unlike previously identified mechanisms directly targeting the enzymatic subunit Drosha, current knowledge about the biological ways of controlling miRNA nuclear maturation through DGCR8 is less addressed. In this study, we unveiled that the microprocessor assembly is governed by a master gene regulator HIF-1α irrespective of its canonical transcriptional activity. First, a widespread protein binding of HIF-1α with DGCR8 instead of Drosha was observed in response to biological stimulations. Similar protein interactions between their corresponding orthologues in model organisms were also observed. After dissecting the essential protein domains, we noticed that HIF-1α suppresses microprocessor assembly via binding to DGCR8. Furthermore, our results showed that HIF-1α hijacks monomeric DGCR8 thus reducing its dimer formation prior to microprocessor assembly, and consequently, the suppressed microprocessor formation and nuclear processing of primary miRNA were demonstrated. In conclusion, here we unveiled the mechanism of how microprocessor assembly is regulated by HIF-1α, which not only demonstrates a non-transcriptional function of nuclear HIF-1α but also provides new molecular insights into the regulation of microprocessor assembly through DGCR8.

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Detection of Protein Interactions During Virus Infection by Bimolecular Fluorescence Complementation

Cited by 3 publications

References 31 publications

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly

A transcription-independent role for HIF-1α in modulating microprocessor assembly

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