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

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.

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.

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.