Biology subjects

Mottram, T. J.

Publications and source records attributed to Mottram, T. J..

4 recordsLinked to original sources

bioRxiv2023

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

The epitranscriptomic modification N6-methyladenosine (m6A) is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposis sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m6A in cellular transcripts upregulated during KSHV lytic replication. Results show that m6A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent-lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NF{kappa}B signalling. Overall, this work highlights the central importance of m6A in modulating cellular gene expression to influence viral infection. Author SummaryChemical modifications on mRNA, such as m6A, are functionally linked to all stages of mRNA metabolism and regulate a variety of biological processes. As such, m6A modification offers unique possibilities for viruses to modulate both viral and host gene expression. m6A has been identified on transcripts encoded by a wide range of viruses and studies to investigate m6A function have highlighted distinct roles in virus life cycles. In addition, cellular transcripts undergoing differential m6A status during infection may also be important for virus replication. In this study we investigate the impact of differential m6A modification in host transcripts during KSHV lytic replication, by identifying transcripts with altered methylation profiles between latent and lytic replication programmes. We show that increased m6A content in one of these cellular mRNAs, GPRC5A, enhances its stability and correlates with increased abundance during KSHV lytic replication. Moreover, the importance of GPRC5A is demonstrated by depletion studies, showing that GPRC5A enhances KSHV lytic replication by inhibiting cell signalling pathways.

microbiology↗

bioRxiv2023

Pseudouridine prevalence in Kaposi's sarcoma associated herpesvirus transcriptome reveals an essential mechanism for viral replication

Pseudouridylation is a prevalent RNA modification shown to occur in tRNAs, rRNAs, snoRNAs and most recently mRNAs and lncRNAs. Emerging evidence suggests that this dynamic RNA modification is implicated in altering gene expression by regulating RNA stability, modulating translation elongation and modifying amino acid substitution rates. However, the role of pseudouridylation in infection is poorly understood. Here we demonstrate that Kaposis sarcoma-associated herpesvirus (KSHV) manipulates the pseudouridylation pathway to enhance replication. We show the pseudouridine synthases (PUS), PUS1 and PUS7 are essential for efficient KSHV lytic replication, supported by the redistribution of both PUS1 and PUS7 to viral replication and transcription complexes. We present a comprehensive analysis of KSHV RNA pseudouridylation, revealing hundreds of modified RNAs at single-nucleotide resolution. Notably, we further demonstrate that pseudouridylation of the KSHV-encoded polyadenylated nuclear RNA (PAN) plays a significant role in the stability of PAN RNA and in the association of the KSHV ORF57 protein. Our findings reveal a novel and essential role of pseudouridine modification in the KSHV replication cycle.

microbiology↗

bioRxiv2022

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

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 Kaposis 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.

microbiology↗

bioRxiv2021

CircHIPK3 dysregulation of the miR-30c/DLL4 axis is essential for KSHV lytic replication

Non-coding RNA (ncRNA) regulatory networks are emerging as critical regulators of gene expression. These intricate networks of ncRNA:ncRNA interactions modulate multiple cellular pathways and impact the development and progression of multiple diseases. Herpesviruses, including Kaposis sarcoma-associated herpesvirus, are adept at utilising ncRNAs, encoding their own as well as dysregulating host ncRNAs to modulate virus gene expression and the host response to infection. Research has mainly focused on unidirectional ncRNA-mediated regulation of target protein-coding transcripts; however, we have identified a novel host ncRNA regulatory network essential for KSHV lytic replication in B cells. KSHV-mediated upregulation of the host cell circRNA, circHIPK3, is a key component of this network, functioning as a competing endogenous RNA of miR-30c, leading to increased levels of the miR-30c target, DLL4. Dysregulation of this network highlights a novel mechanism of cell cycle control during KSHV lytic replication in B cells. Importantly, disruption at any point within this novel ncRNA regulatory network has a detrimental effect on KSHV lytic replication, highlighting the essential nature of this network and potential for therapeutic intervention.

microbiology↗