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Kaposi’s sarcoma-associated herpesvirus induces specialised ribosomes to efficiently translate viral lytic mRNAs

ABSTRACT: Historically, ribosomes have been viewed as unchanged homogeneous macromolecular machines with no intrinsic regulatory capacity for mRNA translation. However, an emerging concept is that heterogeneity of ribosomal composition exists, which can exert a regulatory function or specificity in translational control. This is supported by recent discoveries identifying compositionally distinct ‘specialised ribosomes’ that actively regulate mRNA translation. Viruses lack their own translational machinery and impose a high translational demand on the host cell during replication. Here we explore the possibility that Kaposi’s sarcoma-associated herpesvirus (KSHV) can manipulate host ribosome biogenesis during infection to produce specialised ribosomes which preferentially translate viral transcripts. Quantitative proteomic analysis has identified changes in the stoichiometry and composition of precursor ribosomal complexes during the switch from latent to lytic KSHV replication. Intriguingly, we demonstrate the enhanced association of ribosomal biogenesis factors BUD23 and NOC4L, and a previously uncharacterised KSHV lytic protein, ORF11, with small ribosomal subunit precursor complexes during lytic KSHV infection. Notably, BUD23 depletion resulted in significantly reduced viral gene expression and progression through the lytic cascade, culminating in a dramatic reduction of infectious virion production. Importantly, ribosome profiling demonstrated that BUD23 is essential for the reduced association of ribosomes with KSHV uORFs in late lytic genes, required for the efficient translation of the main open reading frame. Together our results provide new mechanistic insights into KSHV-mediated manipulation of cellular ribosome composition inducing a population of specialised ribosomes to facilitate efficient translation of viral mRNAs.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Human Herpesvirus 8 Strain Rkshv.219

TISSUE(S): B Cell, Cell Suspension Culture

SUBMITTER: James Murphy

LAB HEAD: Adrian Whitehouse

PROVIDER: PXD032318 | Pride | 2022-12-21

REPOSITORIES: Pride

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Project description:The Kaposi's sarcoma associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi's sarcoma, primary effusion lymphoma (PEL), and some forms of multicentric Castleman's disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5´ ends. The position of these 5´-bound fragments correlated closely with the 5´-most exonintron junction of the pre-mRNA. We selected four candidates (BTG1, EGR1, ZFP36, and TNFSF9) and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation. HITS-CLIP was performed on TREx BCBL-Rta cells 20 hpi using antibodies against ORF57. Three biological replicates were performed.

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Kaposi’s sarcoma-associated herpesvirus induces specialised ribosomes to efficiently translate viral lytic mRNAs

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