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

Abstract

Historically, ribosomes were viewed as unchanged homogeneous macromolecular machines with no regulatory capacity for mRNA translation. An emerging concept is that heterogeneity of ribosomal composition exists, exerting 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 high translational demands on the host during replication. We explore the possibility that KSHV manipulates ribosome biogenesis producing specialised ribosomes which preferentially translate viral transcripts. Quantitative proteomic analysis identified changes in the stoichiometry and composition of precursor ribosomal complexes during the switch from latent to lytic replication. We demonstrate the enhanced association of ribosomal biogenesis factors BUD23 and NOC4L, and the KSHV ORF11 protein, with small ribosomal subunit precursor complexes during lytic replication. BUD23 depletion resulted in significantly reduced viral gene expression, culminating in dramatic reduction of infectious virion production. Ribosome profiling demonstrated BUD23 is essential for reduced association of ribosomes with KSHV uORFs in late lytic genes, required for the efficient translation of the downstream coding sequence. Results provide mechanistic insights into KSHV-mediated manipulation of cellular ribosome composition inducing a population of specialised ribosomes facilitating efficient translation of viral mRNAs.

Nucleolin Regulates the Expression of Kaposi’s Sarcoma-Associated Herpesvirus’ Latency-Associated Nuclear Antigen through G-Quadruplexes in the mRNA

“…The synthesis and initial assembly of ribosomal RNA (rRNA) and ribosomal proteins into ribosomal subunits takes place in the nucleolus. KSHV actively modifies this ribosomal assembly process, leveraging it to support its replication needs [68,69]. During its lytic phase, KSHV disrupts normal ribosome biogenesis in the nucleolus, leading to the creation of specialized ribosomes, uniquely modified for efficient translation of viral mRNAs, especially those crucial for the virus's late lytic cycle.…”

HOP/STIP1 is required for KSHV lytic replication

“…TREx BCBL-1-RTA cells were maintained in RPMI 1640 medium, 10% v/v FBS, 1% v/v penicillin/streptomycin, 1% v/v glutamax, 100 μg/mL hygromycin B (GA7834, Glentham Life Sciences) at 37°C with 5% CO2. To generate stable cell lines expressing shRNA for HOP (or shNT control) or overexpressing GFP and Hop-GFP, TREx BCBL-1-RTA cells were lentivirally transduced with the shRNA or overexpression plasmids (Murphy et al 2023). Cells were maintained in the TREx BCBL-1-RTA medium with additional 3 μg/mL puromycin (P7255, Sigma-Aldrich) at 37°C with 5% CO 2 .…”

m6A regulates the stability of cellular transcripts required for efficient KSHV lytic replication

“…Beads were rotated for 1 hour at 4°C, washed 3 times with immunoprecipitation buffer and incubated with lysates overnight at 4°C. The following day, beads were washed 3 times in immunoprecipitation buffer and analysed at the University of Bristol Proteomics facility for tandem mass tagging (TMT) coupled with liquid chromatography and mass spectrometry analysis (LC-MS/MS) as previously described [66]. Data was outputted as a Microsoft Excel spreadsheet containing the results of a Sequest search against the Uniprot Human Database.…”