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Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain.

ABSTRACT: Long interspersed nuclear element-1 (L1) is a retrotransposable element that autonomously replicates in the human genome, resulting in DNA damage and genomic instability. Activation of L1 in senescent cells triggers a type I interferon response and age-associated inflammation. Two open reading frames encode an ORF1 protein functioning as messenger RNA chaperone and an ORF2 protein providing catalytic activities necessary for retrotransposition. No function has been identified for the conserved, disordered N-terminal region of ORF1. Using microscopy and NMR spectroscopy, we demonstrate that ORF1 forms liquid droplets in vitro in a salt-dependent manner and that interactions between its N-terminal region and coiled-coil domain are necessary for phase separation. Mutations disrupting blocks of charged residues within the N-terminus impair phase separation, whereas some mutations within the coiled-coil domain enhance phase separation. Demixing of the L1 particle from the cytosol may provide a mechanism to protect the L1 transcript from degradation.

SUBMITTER: Newton JC

PROVIDER: S-EPMC8390800 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain.

Newton Jocelyn C JC Naik Mandar T MT Li Grace Y GY Murphy Eileen L EL Fawzi Nicolas L NL Sedivy John M JM Jogl Gerwald G

Biophysical journal 20210331 11

Long interspersed nuclear element-1 (L1) is a retrotransposable element that autonomously replicates in the human genome, resulting in DNA damage and genomic instability. Activation of L1 in senescent cells triggers a type I interferon response and age-associated inflammation. Two open reading frames encode an ORF1 protein functioning as messenger RNA chaperone and an ORF2 protein providing catalytic activities necessary for retrotransposition. No function has been identified for the conserved, ...[more]

PMID: 33798566

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Project description:The interactions between viruses and actin cytoskeleton have been widely studied. We showed that rotaviruses remodel microfilaments in intestinal cells and demonstrated that this was due to the VP4 spike protein. Microfilaments mainly occur in the apical domain of infected polarized enterocytes and favor the polarized apical exit of viral progeny. The present work aims at the identification of molecular determinants of actin-VP4 interactions. We used various deletion mutants of VP4 that were transfected into Cos-7 cells and analyzed interactions by immunofluorescence confocal microscopy. It has been established that the C-terminal part of VP4 is embedded within viral particles when rotavirus assembles. The use of specific monoclonal antibodies demonstrated that VP4 is expressed in different forms in infected cells: classically as spike on the outer layer of virus particles, but also as free soluble protein in the cytosol. The C terminus of free VP4 was identified as interacting with actin microfilaments. The VP4 actin binding domain is unable to promote microfilament remodeling by itself; the coiled-coil domain is also required in this process. This actin-binding domain was shown to dominate a previously identified peroxisomal targeting signal, located in the three last amino acids of VP4. The newly identified actin-binding domain is highly conserved in rotavirus strains from species A, B, and C, suggesting that actin binding and remodeling is a general strategy for rotavirus exit. This provides a novel mechanism of protein-protein interactions, not involving cell signaling pathways, to facilitate rotavirus exit.IMPORTANCE Rotaviruses are causal agents of acute infantile viral diarrhea. In intestinal cells, in vitro as well as in vivo, virus assembly and exit do not imply cell lysis but rely on an active process in which the cytoskeleton plays a major role. We describe here a novel molecular mechanism by which the rotavirus spike protein VP4 drives actin remodeling. This relies on the fact that VP4 occurs in different forms. Besides its structural function within the virion, a large proportion of VP4 is expressed as free protein. Here, we show that free VP4 possesses a functional actin-binding domain. This domain, in coordination with a coiled-coil domain, promotes actin cytoskeleton remodeling, thereby providing the capacity to destabilize the cell membrane and allow efficient rotavirus exit.

Dataset Information

Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain.

Publications

Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain.

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