CCR4‐NOT differentially controls host versus virus poly(a)‐tail length and regulates HCMV infection

Unlike most RNA and DNA viruses that broadly stimulate mRNA decay and interfere with host gene expression, human cytomegalovirus (HCMV) extensively remodels the host translatome without producing an mRNA decay enzyme. By performing a targeted loss‐of‐function screen in primary human fibroblasts, we here identify the host CCR4‐NOT deadenylase complex members CNOT1 and CNOT3 as unexpected pro‐viral host factors that selectively regulate HCMV reproduction. We find that the scaffold subunit CNOT1 is specifically required for late viral gene expression and genome‐wide host responses in CCR4‐NOT‐disrupted cells. By profiling poly(A)‐tail lengths of individual HCMV and host mRNAs using nanopore direct RNA sequencing, we reveal poly(A)‐tails of viral messages to be markedly longer than those of cellular mRNAs and significantly less sensitive to CCR4‐NOT disruption. Our data establish that mRNA deadenylation by host CCR4‐NOT is critical for productive HCMV replication and define a new mechanism whereby herpesvirus infection subverts cellular mRNA metabolism to remodel the gene expression landscape of the infected cell. Moreover, we expose an unanticipated host factor with potential to become a therapeutic anti‐HCMV target. Synopsis The multi‐subunit cellular deadenylase complex CCR4‐NOT controls poly(A)‐tail length, regulating mRNA decay and translation. HCMV requires CCR4‐NOT activity for productive infection, though its own mRNAs are less susceptible to CCR4‐NOT degradation. HCMV requires CCR4‐NOT complex subunits CNOT1 and CNOT3 for efficient replication and gene expression. Chemical inhibition of a CCR4‐NOT nuclease phenocopies CNOT1/3 knockdown. Specific CCR4‐NOT subunits are upregulated by HCMV infection. HCMV mRNAs have long poly(A)‐tails that are less sensitive to CCR4‐NOT‐disruption than those of cellular mRNAs. The multi‐subunit cellular deadenylase complex CCR4‐NOT controls poly(A)‐tail length, regulating mRNA decay and translation. HCMV requires CCR4‐NOT activity for productive infection, though its own mRNAs are less susceptible to CCR4‐NOT degradation.