KSHV-induced translation of lncRNAs - role of micropeptides in virus replication

KSHV is an oncogenic virus required for the development of Kaposi's sarcoma (KS) and several lymphoproliferative diseases. KS is the most common adult HIV-associated cancer and amongst the most common childhood cancers in sub-Saharan Africa. There are no specific KSHV antivirals or vaccines, therefore it is essential to study the molecular mechanisms which regulate KSHV replication to fully understand KSHV pathogenesis. Here we highlight a novel role of long non-coding RNAs (lncRNAs) in KSHV biology. LncRNAs are critical regulators of gene expression, playing key roles in KSHV persistence, immune evasion and lytic replication. Until recently, lncRNAs were thought to function solely by their RNA sequence and structure, however, we provide exciting data showing that KSHV induces the translation of small open reading frames (smORFs) within cytoplasmic-localised lncRNAs, producing micropeptides. ORFquant analysis of Ribo-seq datasets highlight significant ribosome occupancy and translation of novel smORFs in cellular lncRNAs during KSHV lytic replication. This is reinforced using polysome profiling, showing lncRNAs associate with translating ribosomes and FLAG-tagged mini-reporter constructs containing the novel smORFs, which are expressed in various subcellular localisations. Importantly, micropeptide overexpression enhances KSHV lytic replication and infectious virion production. Together, these data suggest that KSHV induces the translation of micropeptides from lncRNAs, enhancing KSHV lytic replication. These findings provide a unique opportunity to assess how lncRNA-mediated translation of micropeptides is regulated during infection and how micropeptide function impacts virus replication and/or the host response. We therefore now wish to exploit these findings and our scientific objectives are underpinned by the following defined stages: We have prioritised 10 micropeptides for detailed characterisation. We will firstly examine their temporal expression and subcellular localisation during the course of KSHV lytic replication, utilising CRISPR/Cas9 tagging approaches and the generation of specific Affimers-binding reagents to the prioritised micropeptides. Our preliminary analysis suggests that micropeptide overexpression can enhance KSHV lytic replication. However, to definitely discriminate between the role of the parental lncRNA and translated micropeptide, we will generate CRISPR/Cas9 mutants which either (i) specifically disrupt the start codon of the lncRNA-smORF, (ii) create a frameshift near the start codon or (iii) inactivate lncRNA function at the genomic level. This will confirm that the micropeptides are the functional unit of the translated lncRNA. We will characterise the role of the prioritised micropeptides in KSHV lytic replication by mapping their protein interactions, assessing the impact of their expression on host and viral gene expression and providing mechanistic insights based on their subcellular localisation. Our preliminary analysis highlights an enrichment in the m6A status of the translated lncRNA during KSHV lytic replication. We will therefore assess the role of m6A methylation in the translational efficiency of lncRNAs during infection, by identifying the m6A reader proteins which bind the m6A-enriched lncRNAs and investigate their role in recruitment of KSHV-induced specialised ribosomes. Together, these scientific objectives will determine how KSHV manipulates host cell lncRNA translation to enhance its own replication and provide a better understanding of how micropetides regulate virus replication. This may provide new strategies for therapeutic intervention of an important human pathogen. The project will have wide implications by providing valuable information on the diverse biological functions of micropeptides, impacting our understanding of their role in cell and developmental processes. This will be essential in understanding how lncRNA translation and micropeptide dysregulation contributes to disease.