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Human cytomegalovirus forms phase-separated compartments at viral genomes to facilitate viral replication.

ABSTRACT: Human cytomegalovirus (HCMV) replicates its DNA genome in specialized replication compartments (RCs) in the host cell nucleus. These membrane-less organelles originate as spherical structures and grow in size over time. However, the mechanism of RC biogenesis has remained understudied. Using live-cell imaging and photo-oligomerization, we show that a central component of RCs, the UL112-113 proteins, undergo liquid-liquid phase separation (LLPS) to form RCs in the nucleus. We show that the self-interacting domain and large intrinsically disordered regions of UL112-113 are required for LLPS. Importantly, viral DNA induces local clustering of these proteins and lowers the threshold for phase separation. The formation of phase-separated compartments around viral genomes is necessary to recruit the viral DNA polymerase for viral genome replication. Thus, HCMV uses its UL112-113 proteins to generate RCs around viral genomes by LLPS to ensure the formation of a pro-replicative environment.

SUBMITTER: Caragliano E

PROVIDER: S-EPMC8924372 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Human cytomegalovirus forms phase-separated compartments at viral genomes to facilitate viral replication.

Caragliano Enrico E Bonazza Stefano S Frascaroli Giada G Tang Jiajia J Soh Timothy K TK Grünewald Kay K Bosse Jens B JB Brune Wolfram W

Cell reports 20220301 10

Human cytomegalovirus (HCMV) replicates its DNA genome in specialized replication compartments (RCs) in the host cell nucleus. These membrane-less organelles originate as spherical structures and grow in size over time. However, the mechanism of RC biogenesis has remained understudied. Using live-cell imaging and photo-oligomerization, we show that a central component of RCs, the UL112-113 proteins, undergo liquid-liquid phase separation (LLPS) to form RCs in the nucleus. We show that the self-i ...[more]

PMID: 35263605

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Project description:UnlabelledHuman cytomegalovirus (HCMV) counteracts host defenses that otherwise act to limit viral protein synthesis. One such defense is the antiviral kinase protein kinase R (PKR), which inactivates the eukaryotic initiation factor 2 (eIF2) translation initiation factor upon binding to viral double-stranded RNAs. Previously, the viral TRS1 and IRS1 proteins were found to antagonize the antiviral kinase PKR outside the context of HCMV infection, and the expression of either pTRS1 or pIRS1 was shown to be necessary for HCMV replication. In this study, we found that expression of either pTRS1 or pIRS1 is necessary to prevent PKR activation during HCMV infection and that antagonism of PKR is critical for efficient viral replication. Consistent with a previous study, we observed decreased overall levels of protein synthesis, reduced viral protein expression, and diminished virus replication in the absence of both pTRS1 and pIRS1. In addition, both PKR and eIF2α were phosphorylated during infection when pTRS1 and pIRS1 were absent. We also found that expression of pTRS1 was both necessary and sufficient to prevent stress granule formation in response to eIF2α phosphorylation. Depletion of PKR prevented eIF2α phosphorylation, rescued HCMV replication and protein synthesis, and reversed the accumulation of stress granules in infected cells. Infection with an HCMV mutant lacking the pTRS1 PKR binding domain resulted in PKR activation, suggesting that pTRS1 inhibits PKR through a direct interaction. Together our results show that antagonism of PKR by HCMV pTRS1 and pIRS1 is critical for viral protein expression and efficient HCMV replication.ImportanceTo successfully replicate, viruses must counteract host defenses that limit viral protein synthesis. We have identified inhibition of the antiviral kinase PKR by the viral proteins TRS1 and IRS1 and shown that this is a critical step in HCMV replication. Our results suggest that inhibiting pTRS1 and pIRS1 function or restoring PKR activity during infection may be a successful strategy to limit HCMV disease.

Dataset Information

Human cytomegalovirus forms phase-separated compartments at viral genomes to facilitate viral replication.

Publications

Human cytomegalovirus forms phase-separated compartments at viral genomes to facilitate viral replication.

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