Infection Medicine
Infection Medicine
Edinburgh Medical School
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EASTBIO PhD projects available at Infection Medicine

A number of BBSRC EASTBIO PhD projects are currently available at Infection Medicine.

Project 1: How do the HUSH complex and REAF/RPRD2 control endogenous retroelements?

Projec supervisors: ,

Project description

The aim of this project is to understand how the recently identified antiviral HUSH complex and the antiviral protein REAF/RPRD2 can inhibit endogenous retroelements. Our preliminary data/working hypothesis suggest both act via epigenetic and post-transcriptional mechanisms, respectively. Genome embedded endogenous retroelements are evolutionary and functionally related to extant retroviruses, and so they are often susceptible to innate immune factors that can inhibit viruses. Equally, as major constituents of the genome, they are regulated by specific epigenetic means. When these processes are deregulated, this can lead to inflammatory autoimmune disease due to aberrant retroelement expression. We and others have identified the HUSH complex and REAF/RPRD2 as able to repress the expression and mobility of endogenous retroelements, while both are also known to affect Human Immunodeficiency Virus (HIV) replication. However, the underlying molecular mechanism of inhibition is still not understood. This project will use a range of molecular and cellular techniques to define how the HUSH complex and REAF/RPRD2 can control active human endogenous retroelements: LINE-1 and Alu retrotransposons. There will be particular emphasis on understanding protein-RNA interactions, and post-translational modifications. Full training will be given in a range of molecular approaches, cell culture, affinity proteomics, and confocal microscopy. The project will also use a range of sequencing technologies such as RNA-seq, ChIP-Seq and/or CLIP-Seq. Understanding endogenous retroelement control in this manner will allow new insights regarding the role of genome elements in autoimmune disease.

References

  1. Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators. Liu N, Lee CH, Swigut T, Grow E, Gu B, Bassik MC, Wysocka J. Nature. 2018. 553(7687):228-232.
  2. RNase H2, mutated in Aicardi-Goutières syndrome, promotes LINE-1 retrotransposition. Benitez-Guijarro M, Lopez-Ruiz C, Tamauskaite Z, Murina O, Mohammad MM, Williams TC, Fluteau A, Sanchez L, Vilar-Astasio R., Garcia-Cañadas M, Cano D, Kempen M-J, Sanchez-Pozo A, Heras SR, Jackson A, Reijns M, Garcia-Perez JL. EMBO Journal.2018. 37(15).
  3. HIV-1 Vpr accessory protein interacts with REAF and mitigates its associated anti-viral activity. Gibbons JM, Marno KM, Pike R, Lee WY J, Jones CE, Fu RM, Bryan A, Ogunkolade BW, Pardieu C, Warnes G, Rowley PA, Sloan RD, McKnight Á. bioarXiv preprint server. 2018. doi: https://doi.org/10.1101/408161

Project 2: The role of secreted tumour suppressor SPZ1 in Papillomavirus E7-mediated oncogenesis

Supervisors: Professor Jürgen Haas, Dr Kate Cushieri

Project description

Currently, 112 different non-human papillomavirus (PV) types are known, which have been isolated from a broad range of 54 host species including cattle, canines, horses, rodents, bats, dolphins and primates. Bovine PVs (BPV) are associated with cancer, similar to the high risk types of the >100 different known Human Papillomaviruses (HPV). The diverse PVs possess a highly conserved genome structure, but only the core proteins E1, E2, L2 and L1 are present in all PV genomes characterized by now. Most PVs, however, encode 8 proteins. PVs that cause cancer encode several oncogenes which cause cellular transformation, including E7 which interacts and inhibits the tumor suppressor Retinoblastoma (pRb) protein1. pRb binds to  and suppresses the transcription factor E2F which controls the expression of proteins involved in cell cycle control and cellular proliferation2. High risk E7 leads to destabilization and degradation of pRb via a LxCxE motif. In our previous work we found that the cellular protein SPZ1 interacts with E7 derived from high risk but not low risk HPV and binds adjacent to the LxCxE motif in E7. SPZ1 is a helix-loop-helix domain protein reported to be involved in MAP kinase signalling, transcriptional regulation and proliferation3. We found that SPZ1 is secreted via a non-classical secretion pathway and induces proliferation in human keratinocytes. If coexpressed with high risk E7, however, it inhibits E7-induced cellular proliferation of keratinocytes. In pulse-chase experiments with cycloheximide SPZ1 leads to an increased abundance of tumour suppressor pRb in E7 transformed cells, but also inhibits the degradation of E7. Thus, SPZ1 inhibits E7 by fixing and accumulating it in a non-functional form, and is able to  either switch on or off cell cycle control depending on the cellular context.

In this Ph.D. project, we will further investigate how the secreted tumour suppressor protein SPZ1 inhibits cellular transformation, and whether recombinant soluble SPZ1 can be used to treat PV-caused cancers. In particular, we will test if SPZ1 acts via Rb, whether it affects the degradation pRB and thereby controls the cell cycle, whether SPZ1 forms a heterotrimeric complex with pRB and E7, whether other cellular proteins are involved and whether SPZ1 secretion via the non-classical pathway can be blocked or enhanced. Furthermore, we will also test whether SPZ1 is effective against other cancers in which pRB is mutated but which are not caused by PV E7. A broad range of different molecular, biochemical and cellular methods will be applied, including cutting edge technologies such as genome-wide RNAi screens and CRISPR/Cas9 gene editing.

References

  1. Giarre M, Caldeira S, Malanchi I, Ciccolini F, Leao MJ, Tommasino M. Induction of pRb degradation by the human papillomavirus type 16 E7 protein is essential to efficiently overcome p16INK4a-imposed G1 cell cycle Arrest. Journal of virology 2001; 75(10): 4705-12.
  2. Klingelhutz AJ, Roman A. Cellular transformation by human papillomaviruses: lessons learned by comparing high- and low-risk viruses. Virology 2012; 424(2): 77-98.
  3. Hsu SH, Hsieh-Li HM, Huang HY, Huang PH, Li H. bHLH-zip transcription factor Spz1 mediates mitogen-activated protein kinase cell proliferation, transformation, and tumorigenesis. Cancer Res 2005; 65(10): 4041-50.

Project 3: Chloride ion-mediated inhibition of animal and human viruses as a broadly active antiviral mechanism

Project supervisors: Dr Sandeep Ramalingam, Professor Jürgen Haas

Project description

Treatment with hypertonic saline is recommended in patients with upper respiratory tract infections bronchiolitis and cystic fibrosis1. It is commonly believed that this effect is due to the mucolytic activity of hypertonic saline.

We recently published evidence that sodium chloride (NaCl) and other chloride salts have a direct, dose-dependent inhibitory effect on viruses2. NaCl was broadly inhibitory against human and animal viruses, including DNA and RNA viruses as well as enveloped and non-enveloped viruses In a pilot randomised controlled trial (RCT) done in parallel we showed that hypertonic saline nasal irrigation and gargling was effective in reducing the duration of the common cold in adults by two days.

Viral inhibition by NaCl was chloride ion (Cl-) dependent (since inhibition is reversed by chloride channels inhibitors). NaCl neither had a direct effect on virus particles nor on viral attachment and uptake. The inhibition required the conversion of Cl- to hypochlorous acid (HOCl) which was observed within 2-6 hours after infection. In myeloid cells myeloperoxidase (MPO) converts Cl- to HOCl, but the enzyme which converts Cl- to HOCl in non-myeloid cells is currently unknown.

HSV-1 which causes oral, genital infections as well as infections of the braincan be effectively treated by acyclovir/its derivatives. But resistant viruses can develop. In our experiments, Cl- mediated inhibition of HSV-1 was seen in various cell types and cell lines with few exceptions. Interestingly, we identified two neuroblastoma cells lines, SH-N-SH (parental cell line) and SH-SY5Y (mutant subclone derived from SH-N-SH), in which HSV-1 showed a differential sensitivity to salt inhibition, indicating that the genetic background of the host cell plays an important role in NaCl susceptibility. Interestingly, acyclovir had no effect on replication in all neuroblastoma cell lines tested even at high concentrations, whereas chloride was able to inhibit HSV-1 in most cell lines.

The aim of this PhD project is to further investigate the mechanism by which Cl- blocks viral replication, to test whether it can be a therapeutic agent against animal/human viruses. In particular, we will test at which stage the block of replication occurs, which cellular proteins and enzymes (e.g. ion channels, myeloperoxidase and superoxide dismutase) are involved. We will also investigate the significance of the viral/host genetic background for chloride-mediated viral inhibition. By comparing the whole genome sequence of the SH-N-SH and SH-SY5Y cell line pairs we will try to identify the gene(s) responsible for chloride sensitivity. We will also perform a genome-wide RNA interference screen to identify the gene(s) responsible. Furthermore, we will test whether chloride and acyclovir are effective in different types of differentiated glioma cells derived from induced pluripotent stem cells (iPSC). We will also generate cell lines by CRISPR/Cas9 gene editing in which the gene(s) responsible for chloride sensitivity will be knocked-out or overexpressed. In summary, a broad range of different molecular, biochemical and cellular methods will be applied, including cutting edge technologies such as genome-wide RNAi screens and CRISPR/Cas9 gene editing.

References

  1. Ramalingam S, Graham C, Dove J, Morrice L, Sheikh A. A pilot, open labelled, randomised controlled trial of hypertonic saline nasal irrigation and gargling for the common cold. Sci Rep. 2019;9(1):1015.
  2. Ramalingam S, Cai B, Wong J, et al. Antiviral innate immune response in non-myeloid cells is augmented by chloride ions via an increase in intracellular hypochlorous acid levels. Sci Rep. 2018;11;8(1):13630.
  3. Griffiths SJ, Koegl M, Boutell C, et al. A systematic analysis of host factors reveals a Med23-interferon-lambda regulatory axis against Herpes simplex virus Type 1 replication. PLoS pathogens 2013; 9(8): e1003514.

The application date for these positions has now passed. 

 

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