Traditionally, ribosomes are considered static, machine-like structures that have a role in translating cellular mRNA into protein, without any regulatory control. However, an emerging hypothesis suggests heterogeneity in ribosome composition exists, allowing these so-called ‘specialised ribosomes’ to translate specific mRNAs. We explore the possibility that viruses can induce the formation of specialised ribosomes during infection to preferentially translate their own mRNA over host cellular mRNA.
Preliminary research has shown an increased association of cellular proteins (NOC4L, NOP14 and EMG1) and one viral protein (ORF11) with pre-ribosomal subunits during their biogenesis in KSHV lytic replication. Chapters 3 and 4 of this thesis have investigated the roles of the cellular protein EMG1, and the viral protein ORF11 during KSHV lytic replication. Despite increased association between EMG1 and pre-ribosomes, total EMG1 transcript and protein levels decreased during KSHV lytic replication, suggesting an active recruitment of EMG1 to pre-ribosomes complexes during biogenesis. In contrast, viral protein production, lytic replication and infectious virion production are reduced during EMG1 knockdown, suggesting it is essential for KSHV replication. Furthermore, GFP co-immunoprecipitations demonstrated that a GFP-tagged ORF11 associated the pre-40S ribosomal complex independently of other viral factors, and we hypothesise ORF11 orchestrates the modification of ribosomes via indirect interactions with EMG1 for preferential translation of KSHV mRNA.
Chapter 5 explores a secondary role for ORF11 in the formation of KSHV-induced paraspeckle-like condensates. Paraspeckles are nuclear-localised membraneless organelles composed of several core paraspeckles proteins around a ncRNA scaffold, with putative roles in RNA processing, RNA retention and splicing. These novel condensates differ from canonical paraspeckles in structure, size and contents, with their formation essential for successful viral replication. TMT-LC/MS uncovered protein interaction profiles in these condensates that are distinct from canonical paraspeckle-associated proteins thus suggesting a unique role for these structures in driving KSHV lytic replication and virion production.
