Regulation of HIV-1 and Ebola virus replication by CpG dinucleotides, ZAP and TRIM25

Lead Research Organisation: King's College London
Department Name: Immunology Infection and Inflam Diseases

Abstract

Human immunodeficiency virus type I (HIV-1) is a retrovirus whose worldwide spread has caused the acquired immune deficiency syndrome (AIDS) pandemic. In 2016, ~37 million people globally were living with HIV, ~1.8 million new people became infected and ~1 million people died from AIDS-related illnesses (http://www.unaids.org/en/resources/fact-sheet). Overall, ~76 million people have been infected with HIV since the epidemic started and ~35 million people have died from AIDS-related illnesses. HIV-1 primarily infects CD4 T cells and, when these cells die, the immune system is compromised allowing opportunistic infections and other diseases such as cancer to develop. Once a person is infected, the virus cannot be eliminated but its replication can be inhibited with anti-HIV drugs. Currently, there are approved anti-HIV drugs targeting four steps of the viral replication cycle and ~21 million people worldwide are currently accessing these drugs. These drugs can control the infection indefinitely, but do not cure a person. However, drug resistant strains of HIV are an increasing problem. Therefore, it is necessary to continue to develop new anti-HIV therapies.

Ebola virus (EBOV) is a highly pathogenic virus that causes haemorrhagic fevers in humans with mortality rates up to 90%. The 2014-2016 West African epidemic caused a global emergency when it spread from rural Guinea to major population centres in that country and neighbouring Liberia and Sierra Leone. By the time the outbreak was declared over, EBOV had infected over 28,000 people and killed more than 11,000 (http://www.who.int/mediacentre/factsheets/fs103/en/). Although there are promising vaccines for EBOV, there is currently no effective therapy for an infected person. Therefore, studies of the virus-host interactions at the cellular level are essential for the development of novel EBOV therapeutics. The high mortality of EBOV necessitates its study in containment level 4 laboratories, which hampers progress in the understanding how the virus replicates. However, we have established a safe, non-infectious system to analyse Ebola virus biology.

In this proposal, we will characterise how cellular proteins inhibit HIV-1 and Ebola virus replication when a specific pattern in the virus is sensed by a human cell. There are four nucleotide bases in all RNA sequences, including viral genomes: adenosine (A), cytosine (C), guanosine (G) and uracil (U). The abundance of a specific pattern of RNA sequence in a virus genome, C followed by a G, has been proposed to be recognised by the human cell and inhibits viral replication. However, it is unclear how this CG pattern inhibits the virus. In this grant, we will determine how the CG RNA sequence interacts with cellular proteins and how these proteins inhibit viral replication. In particular, we will characterise how the antiviral proteins ZAP and TRIM25 inhibit HIV-1 and Ebola virus in the context of the safe, non-infectious system.

Technical Summary

Synonymous genome recoding can inhibit RNA virus replication by introducing large numbers of synonymous mutations in viral open reading frames. This is a potential new approach to develop novel vaccines. However, whether these mutations inhibit viral mRNA translation efficiency or have a different deleterious mechanism of action has been controversial. Recently, it has become clear that increasing the abundance of CpG dinucleotides using synonymous mutations is sufficient to attenuate picornavirus and influenza A virus replication. Furthermore, we and others have shown that HIV-1 replication is inhibited by increasing the CpG abundance. Therefore, CpG dinucleotides in RNA may be recognised by cellular proteins to inhibit viral replication.

The cellular antiviral protein ZAP has been shown to inhibit HIV-1 replication and bind regions of the HIV-1 genome containing CpG dinucleotides. ZAP has also been shown to inhibit Ebola virus (EBOV) replication. Recent progress in EBOV reverse genetics has yielded a safe, non-infectious tetracistronic transcription- and replication-competent virus-like particle (trVLP) system that allows modelling of multiple rounds of the EBOV replication cycle in standard containment level 2 laboratories. We are using this trVLP system to study how cellular proteins regulate EBOV replication. In this proposal, we use HIV-1 and EBOV trVLPs as model systems to characterise how CpGs inhibit RNA virus replication. We will explore how ZAP and its cofactor TRIM25 inhibit HIV-1 and EBOV trVLP replication in a CpG-dependent manner. In addition, other cellular proteins may regulate the response to CpG-containing viral RNA and we will use proteomics approaches to identify and characterise these. Overall, this proposal will increase our understanding of how CpG dinucleotides inhibit HIV-1 and EBOV trVLP replication and the cellular antiviral proteins that restrict these viruses. This may allow the development of novel vaccines and antiviral therapies.

Planned Impact

The primary objective of this proposal is to determine how the interaction of CpG dinucleotides in RNA virus genomes with cellular proteins, such as ZAP, inhibits viral replication. This research will have several beneficiaries. First, the experiments in this grant will determine how CpG dinucleotides, ZAP, TRIM25 and other cellular proteins regulate HIV-1 and Ebola virus replication. These viruses impact people around the world and further understanding of how these pathogens interact with host proteins will be of great interest to scientists and the general public. To communicate our research to the scientific community, we will publish in the highest impact journals possible and ensure that these papers are open access. We will also present our research in university/institute seminar series and national/international conferences. To communicate our findings to the general public, we will undertake specific public engagement activities that are described in detail in the Pathways to Impact.

Second, the underlying mechanisms of how CpG dinucleotides in RNA virus genomes inhibit viral replication may impact the development of RNA virus vaccines and may lead to new antiviral therapies. Potential beneficiaries include those in academia and the commercial private sector that are developing new vaccine and antiviral therapeutic strategies. We will engage with KCL's Business and Innovation Department to evaluate the commercial potential of any discoveries we make and, if warranted, help foster collaboration with pharmaceutical partners. This may help contribute to the UK's health and wealth.

Third, this grant will employ two postdoctoral associates and they will receive training in cutting edge molecular virology technologies, including transcriptomic and proteomic techniques. These skills will be useful for a range of future career options including academic, pharmaceutical or other employment sectors.

Fourth, we are both very active in teaching KCL students studying virology and immunology. We also host multiple KCL and external BSc and MSc students in our labs undertaking wet bench research projects as well as pre-university students on work experience projects. These activities help inspire the next generation of scientists and teach critical thinking skills, which is an essential skill is almost all employment sectors.

Therefore, the beneficiaries of this research will potentially include: 1) scientists and companies studying the innate immune response to viral infections and who are developing novel vaccine strategies 2) scientists and companies studying HIV-1 and Ebola virus and who are developing novel antiviral therapeutics 3) the general public who are interested in how cutting edge scientific research develops a further understanding of pathogen-host interactions and improves human health 4) the two postdoctoral associates who will receive further training in molecular virology and 5) pre-university, BSc and MSc students with an interest in studying virology.
 
Description Blizard Institute HIV Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact I attended the Blizard Institute HIV Symposium in 2014, 2015, 2016, 2018 and 2019. In 2014, I was an invited speaker presenting our latest data on how SR proteins regulate HIV-1 gene expression. This highlighted to the regional HIV community how SR proteins are important for HIV-1 gene expression and provided useful feedback on our research. In 2018 I was also an invited speaker and I presented our research on how CpG dinucleotides and ZAP inhibit HIV-1 replication. In 2019, a postdoc in a collaborator's lab presented our data on how KHNYN is a cofactor for ZAP to inhibit retroviral replication.
Year(s) Of Engagement Activity 2014,2015,2016,2018,2019
 
Description Cambridge RNA club 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact I presented my lab's work on ZAP, TRIM25 and KHNYN at the Cambridge RNA Club in November 2020. This led to new experimental ideas and potential collabororations.
Year(s) Of Engagement Activity 2020
 
Description Cold Spring Harbor Retrovirus Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact In 2014, I presented a poster on the Regulation of HIV-1 Gag expression by SR proteins. I was also a co-author for the talk titled The hitchhikers' guide to the viral particle. Both led to substantial research discussions and new experimental ideas.

In 2015, a postdoc in the Swanson lab presented a poster on Regulation of HIV-1 Gag expression by SR proteins. I also attended this conference. This led to substantial research discussions and new experimental ideas.

In 2016, a postdoc in the Swanson lab presented a talk on how SRSF10, TRA2A, and TRA2B control translation of the p40Gag isoform and a poster on how Rescue of HIV-1 Gag expression by SRSF4 and SRSF6 links different non-permissive conditions for HIV-1 gene expression. In addition, I was a co-author for a talk by a collaborator on how HIV-1 and M-PMV mRNA nuclear export pathways program RNA genomes for remarkably distinct cytoplasmic trafficking behaviors. This talk included unpublished data on how Sam68 regulates HIV-1 gene expression. I was also the senior author on a poster presented by a PhD student on how cis-acting RNA sequences regulate HIV-1 Envelope and Vpu translation. I attended this conference and these oral and poster presentations led to many research discussions and new experimental ideas.

In 2018, a postdoc in the Swanson lab presented a poster on "Regulation of CD4 T cell and HIV-1 gene expression by Sam68". A PhD student in the Swanson lab presented how CpG dinucleotides inhibit HIV-1 replication. I also attended this conference. Attendence at this conference led to substantial research discussions and new experimental ideas.

In 2019, a PhD student in the Swanson lab presented a talk on how KHNYN is a novel cofactor for ZAP to inhibit retroviral replication. I presented a poster at this conference on how CpG dinucleotides inhibit HIV-1 replication using ZAP-dependent and independent mechanisms. This led to substantial research discussionsm new experimental ideas and collaborations.

In 2020, a postdoctoral fellow in the Swanson lab presented a talk on how specific domains in ZAP are required for its antiviral activity against HIV-1. Attendence at this conference led to substantial research discussions and new experimental ideas.
Year(s) Of Engagement Activity 2014,2015,2016,2018,2019,2020
 
Description EMBO Pathogen Immunity and Signalling Workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I presented a flash talk and poster during this conference on how ZAP and its cofactor KHNYN inhibited retroviral replication. This led to excellent feedback on our research and initiated new collaborations.
Year(s) Of Engagement Activity 2019
 
Description RNA UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact I attended the RNA UK 2016 conferece and presented our latest research on how SR proteins regulate HIV-1 gene expression. I also attended the RNA UK 2020 conference and presented our latest research on how CpG dinucleotides, ZAP and its cofactor KHNYN inhibit HIV-1 replication. These presentations led to useful feedback on our research.
Year(s) Of Engagement Activity 2016,2020
 
Description The Virus Within: Hearing HIV 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact In 2017-2018, I collaborated with Dr Benjamin Oliver at University of Southampton to create The Virus Within: Hearing HIV, which was funded by the MRC Engagement in Science Activities Seed Fund. This project used contemporary music to enable audiences to develop an understanding of how HIV impacts human health, how the virus infects a cell and replicates its genes, and how cutting-edge research aims to move from lifelong treatment to curing an infected person. The Virus Within: Hearing HIV is a three-movement piece that uses innovative and compelling musical structures to depict the biological processes involved in HIV replication and how 'Shock and Kill' treatments could provide a cure for HIV. The first movement, 'Integration', focuses on HIV reverse transcription and integration. The second movement, 'Gene Expression', musically embodies HIV gene expression in a single cell. The third movement, 'Shock and Kill', shows how HIV-1 gene expression could be reactivated and the infected cells killed. The composition was performed by Workers Union Ensemble and presented as a lecture-recital performance at Guy's Chapel in February 2018. To start the concert, I gave a contextual overview on how HIV currently impacts human health. The three movements were then performed individually, preceded by a presentation from myself explaining the biological processes. During the interval, the audience wrote down questions which were discussed in a Q&A. The concert concluded with an uninterrupted performance of Hearing HIV, which allowed the audience to hear the composition as a concert work and reaffirmed the scientific ideas. Feedback from the audience showed a new appreciation for HIV from both the lecture and music. The audience also learned about how basic research on HIV has led to antiviral drug development and could lead to a cure. This performance was recorded and uploaded to YouTube and I have posted a description of the project and links to each movement on my KCL website. We then did two further performances of a modified version of The Virus Within: Hearing HIV on 16th March 2019 at Turner Sims Southampton as part of the University of Southampton Science and Engineering Day.
Year(s) Of Engagement Activity 2017,2018,2019