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Cooperative assembly of p97 complexes involved in replication termination.

ABSTRACT: The p97 ATPase extracts polyubiquitylated proteins from diverse cellular structures in preparation for destruction by the proteasome. p97 functions with Ufd1-Npl4 and a variety of UBA-UBX co-factors, but how p97 complexes assemble on ubiquitylated substrates is unclear. To address this, we investigated how p97 disassembles the CMG helicase after it is ubiquitylated during replication termination. We show that p97^Ufd1-Npl4 recruitment to CMG requires the UBA-UBX protein Ubxn7, and conversely, stable Ubxn7 binding to CMG requires p97^Ufd1-Npl4. This cooperative assembly involves interactions between Ubxn7, p97, Ufd1-Npl4, and ubiquitin. Another p97 co-factor, Faf1, partially compensates for the loss of Ubxn7. Surprisingly, p97^{Ufd1-Npl4-Ubxn7} and p97^{Ufd1-Npl4-Faf1} also assemble cooperatively on unanchored ubiquitin chains. We propose that cooperative and substrate-independent recognition of ubiquitin chains allows p97 to recognize an unlimited number of polyubiquitylated proteins while avoiding the formation of partial, inactive complexes.

SUBMITTER: Kochenova OV

PROVIDER: S-EPMC9633789 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Cooperative assembly of p97 complexes involved in replication termination.

Kochenova Olga V OV Mukkavalli Sirisha S Raman Malavika M Walter Johannes C JC

Nature communications 20221103 1

The p97 ATPase extracts polyubiquitylated proteins from diverse cellular structures in preparation for destruction by the proteasome. p97 functions with Ufd1-Npl4 and a variety of UBA-UBX co-factors, but how p97 complexes assemble on ubiquitylated substrates is unclear. To address this, we investigated how p97 disassembles the CMG helicase after it is ubiquitylated during replication termination. We show that p97<sup>Ufd1-Npl4</sup> recruitment to CMG requires the UBA-UBX protein Ubxn7, and conv ...[more]

PMID: 36329031

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Project description:Chikungunya virus (CHIKV), an enveloped positive-sense RNA virus, is a member of the alphaviruses and cause fever and arthralgia in humans. We performed genome-wide CRISPR/Cas9-based screens and identified Y-box binding protein 1 (YBX1) as an essential cellular factor for CHIKV. Deficiency of YBX1 inhibited CHIKV RNA replication and impaired virus production. Upon CHIKV infection, YBX1 showed a striking re-localization to viral replication complexes (vRCs), where it co-localized with CHIKV nsP3 and dsRNA intermediates. YBX1 directly interacted with CHIKV nsP3, and mutation of the YBX1-binding motif in CHIKV nsP3 suppressed viral replication in host cells. Furthermore, YBX1 bound to viral RNA and increased the viral RNA-binding activity of CHIKV nsP3. Consistently, the RNA-binding activity of YBX1, as well as the ability of nsP3 to bind to YBX1, was required for efficient CHIKV replication. In addition to CHIKV, YBX1 was also essential for replication of all examined alphaviruses including the prototypic alphavirus. Our findings suggest that YBX1 acts as a scaffold for assembly of chikungunya vRCs and an important factor for replication of multiple alphaviruses, which may serve as a potential target for the development of anti-alphavirus therapies.IMPORTANCEAlphaviruses are a group of mosquito-transmitted, enveloped, positive-strand RNA viruses in the Togaviridae family. Most alphaviruses are important pathogens that continue to cause human disease ranging from severe and potentially fatal neurological disease to chronic arthritic disease on a global scale. Here, we found that YBX1 promotes binding of CHIKV genomic RNA to nsP3, which is a key component of the replication complex, and is therefore pivotal for CHIKV replication. Deficiency of YBX1 results in reduced replication of multiple alphaviruses, including arthritogenic and encephalitic alphaviruses. These findings suggest that YBX1 is an important cellular factor for multiple alphaviruses and a potential target for preventing alphavirus infections.

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Cooperative assembly of p97 complexes involved in replication termination.

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Cooperative assembly of p97 complexes involved in replication termination.

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