title: Investigating the role of host TTR-RBPs during SFV4 and MHV-68 infection
Creators: name: Casswell, JC
Publisher: name: University of Liverpool Repository
Year: 2020

description: The stability and translation of messenger RNA (mRNA) in a cell is partially controlled by an array of turnover and translation regulatory RNA-binding proteins (TTR-RBPs). These proteins bind to the untranslated regions (UTRs) and poly(A) tail of mRNA and facilitate its processing, nuclear export, turnover and translation. Interestingly, a diverse range of viruses utilise different host TTR-RBPs to facilitate infection of host cells. Human antigen R (HuR) is a TTR-RBP which binds uracil rich sequences in mRNA, leading to increased mRNA stability and translation. Mammalian HuR has been shown to bind RNA sequences rich in uracil in the 3’UTR of a range of alphavirus genomes. Disruption of this interaction via deletion of the HuR binding sequences in the viral genome or knock-down of HuR by RNAi has been to shown to significantly decrease virus titre during Sindbis virus (SINV) infection of mammalian cells. Semliki Forest virus (SFV) genomic RNA also binds HuR via a uracil rich element (URE) and a conserved sequence element (CSE) at the 3’ of its 3’UTR. The main aim of this study was to investigate the significance of the URE and CSE of the SFV4 3’UTR during SFV4 infection of mammalian cells. This was achieved by engineering rSFV4 mutants with deletions of the URE and CSE from the SFV4 3’UTR and a rSFV4 with an additional four UREs in its 3’UTR. These rSFV4 mutants also expressed a reporter protein, either eGFP or Guassia luciferase for detection by confocal imaging and analysis of replication by luciferase assay, respectively. Akin to infection with SINV, this study identified that during infection of mammalian cells with SFV4, host HuR translocates from the nucleus to the cytoplasm. Infection of NIH/3T3 cells with rSFV4 lacking the URE in its 3’UTR resulted in significantly less HuR relocalising to the cytoplasm. A concurrent reduction in infectious virions, a reduction in viral RNA abundance, and a reduction in viral gene expression was also observed. In comparison, an addition of four UREs to the SFV4 3’UTR, increased the rate and amount of HuR relocalising to the cytoplasm post-infection, increased the concentration of infectious virions, increased viral RNA abundance and increased viral gene expression. These results suggest a significant role for the URE of the SFV4 3’UTR during infection in mammalian cells and suggest that the interaction between the URE of SFV4 and host HuR may, at least in part, be responsible for this effect. Mammalian cytoplasmic poly(A) binding protein (PABPC) protects mRNA transcripts from deadenylation and 3’ end degradation, facilitates mRNA nuclear export and mRNA translation. Infection of mammalian cells with the gammaherpesviruses of the Herpesviridae family results in the striking nuclear influx of PABPC from the cytoplasm. During lytic infection of mammalian cells with murine gammaherpevirus 68 (MHV-68) the viral protein muSOX elicits shut-off of host gene expression. This is achieved by the degradation of bulk host mRNA via an incompletely understood mechanism. The expression of muSOX on a transfected expression plasmid also results in the nuclear influx of PABPC suggesting a possible link between host shut-off and the nuclear influx of host PABPC. The secondary aim of this study was to investigate the role that PABPC may play during the host shut-off of gene expression elicited by the viral muSOX protein during MHV-68 infection. To investigate this, an MHV-68 mutant encoding muSOX with a premature stop codon (MHV-68 ORF37stop) was utilised. This study found that the nuclear influx of PABPC during infection of mammalian cells with MHV-68 occurred gradually over the course of lytic infection and did not occur during infection with MHV-68 ORF37stop virus. Since gradual nuclear influx of PABPC occurs in mammalian cells following a block in mRNA nuclear export, the possibility of MHV-68 causing a block in mRNA nuclear export was investigated. By separation of the nuclear and cytoplasmic fractions post-infection and analysis of host mRNA abundance by RT-qPCR, no block in mRNA nuclear export was detected during MHV-68 infection despite PABPC relocalising to the nucleus. The roles which TTR-RBPs play during infection of host cells with a range of viruses are still being fully elucidated. These results suggest that HuR plays a significant role during Semliki Forest virus infection of mammalian cells and proposes HuR and other TTR-RBPs as novel targets for antiviral drugs.