Targeting transfer RNA-derived fragments during KSHV infection

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus required for the development of Kaposi's sarcoma (KS). There are no specific KSHV antivirals or vaccines and current treatments for KSHV-associated diseases are not targeted, relying on rebuilding the immune system and using cytotoxic agents. As KS is an AIDS-defining disease, controlling HIV/AIDS using immune reconstitution has been investigated as a KS treatment. However, even with effective Antiretroviral Therapy (ART) and well-controlled HIV infection, many patients still develop progressive KS. HIV-KS patients can also exhibit a worsening of symptoms upon ART, developing KS-associated immune reconstitution inflammatory syndrome, now a major contributor to KSHV-associated deaths. Consequently, anti-KSHV therapies are urgently needed.

Like all herpesviruses KSHV has two distinct life cycles, a persistence life-long infection (latency) and infectious productive cycle (lytic replication). Lytic replication plays an important part in KSHV pathogenesis. Therefore, it is essential to study the molecular mechanisms which regulate lytic replication. Moreover, inhibiting KSHV lytic replication may provide an opportunity to develop novel antiviral strategies to inhibit KS formation.

We have exciting data demonstrating that KSHV manipulates a recently classified group of non-coding RNAs, termed transfer RNA-derived small RNA fragments (tRFs), to enhance lytic replication. tRFs are derived from precursor or mature tRNAs and are implicated in multiple biological pathways, functioning as regulators of gene expression. Not surprisingly abnormal tRF levels are identified in a wide range of human diseases, including cancer, metabolic and neurological diseases and infections. However, it remains to be determined how tRF biogenesis is regulated and how tRF dysregulation contributes to disease. Therefore, KSHV manipulation of tRF levels presents an novel opportunity to examine how tRF dysregulation impacts gene expression and how this leads to disease. Moreover, reversing the effect of KSHV-mediated tRF dysregulation reduces virus replication, suggesting it has potential as a novel antiviral strategy.

Five structural categories of tRFs have been reported and our preliminary results demonstrate that KSHV specifically dysregulates two distinct tRF subtypes. Firstly, we observe a specific downregulation of 3'-tRFs, and due to their small size and association with Argonaute proteins we believe they may function in a similar manner to miRNAs to regulate gene expression during infection. We will identify mRNA targets of these dysregulated 3'-tRFs and then assess their role in virus replication. In contrast, we observe an upregulation of a second tRF subset, i-tRFs. i-tRFs fail to associate with Argonaute proteins and instead contain conserved RNA binding protein (RBP) motifs, suggesting they function as RBP sponges. We will identify cellular RBPs which bind these upregulated i-tRFs and assess what effect RBP sequestration has on virus replication and transcriptome-wide gene expression. Moreover, we will investigate novel mechanisms of how KSHV manipulates tRF levels during infection. Finally, we show that reversing the effect of tRF dysregulation has a detrimental effect on virus replication. Therefore, we will optimise the best approach to reverse tRF dysregulation in KSHV-infected cell lines and a 3D tumour model to validate this approach as a novel antiviral strategy.

In summary, this project will identify novel ways KSHV can manipulate the host cell to enhance its own replication and provide a better understanding of how tRFs regulate gene expression. This will impact on our understanding of the emerging role of tRFs in cell and developmental processes, the development of human disease and provide new strategies for therapeutic intervention of an important human pathogen.

Non-coding RNAs (ncRNAs) play diverse roles in a multitude of cellular processes, functioning as critical regulators of gene expression. This is reinforced by ncRNA dysregulation being implicated in the development and progression of a wide range of human diseases. We have exciting data demonstrating that the oncogenic herpesvirus, KSHV, manipulates a recently identified class of ncRNAs, termed transfer RNA-derived small RNA fragments (tRFs), to enhance its own replication.

We provide evidence that KSHV specifically dysregulates two distinct tRF subgroups, namely 3'-tRFs and i-tRFs. Notably, reversing KSHV-mediated tRF dysregulation is detrimental for KSHV lytic replication, which may provide a novel antiviral strategy.

We will examine the role of these two distinct tRF subsets in virus replication. Firstly examining the role of downregulated 3'-tRFs in regulating mRNA levels in a similar fashion to miRNAs. Secondly, we will assess the role of upregulated i-tRFs as RNA binding protein (RBP) sponges. The role of these target mRNAs and RBPs will then be further investigated in the context of virus replication. In addition, we will examine virus-induced mechanisms which lead to tRF dysregulation during infection, particularly focussing on KSHV-mediated manipulation of RNA modifying enzymes. Finally, we will assess and optimise the best approach to reverse tRF dysregulation in KSHV producer cell lines and a 3D tumour model.

This project will therefore determine how KSHV-mediated manipulation of tRFs can regulate gene expression and identify novel mechanisms of how tRF biogenesis is regulated during infection. It will also assess whether reversing tRF levels is a viable antiviral therapeutic approach. Together it will provide valuable information on tRFs as emerging regulators of gene expression with diverse biological functions.