Regulation of CD4 T cell and HIV-1 gene expression by Sam68

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

Abstract

Humans have ~20,000 genes and over 50 trillion cells. However, the C. elegans worm has ~20,000 genes and ~1000 cells. Therefore, human complexity is not determined by the number of genes. Instead, human genes are highly regulated in terms of how many different proteins can be made from a single gene using processes such as alternative splicing and alternative polyadenylation. Also, genes are controlled in terms of when they are expressed by being transcribed into RNA and how efficiently this RNA is translated into protein. There is still little understanding of how RNA binding proteins control how many different proteins and how much of a specific protein is made from a gene. This proposal will determine how the cellular RNA binding protein Sam68 controls RNA abundance from specific genes, how efficiently this RNA is translated into protein, and how many different proteins can be made from that gene. We will analyse this in human CD4 T cells, which are an essential component of the immune system that protects the body from infections. We will determine how the location of Sam68 binding sites on an RNA controls its ability to regulate which proteins are expressed from a gene through alternative splicing. We will also analyse whether the genes that Sam68 regulates control important functions for the immune system. Sam68 is implicated in several types of cancer including breast, prostate, colorectal, cervical and renal cell carcinoma as well as the diseases fragile X-associated tremor/ataxia syndrome (FXTAS) and spinal muscular atrophy (SMA). Understanding how Sam68 controls gene expression may also help us understand how Sam68 regulates these diseases.

HIV infection causes AIDS and is a worldwide pandemic. Currently, there are approved antiviral drugs targeting four steps of the HIV life cycle. However, drug resistant strains of HIV are a problem and there is still a great need for new drug targets. One promising approach to develop novel antiviral drugs is to target cellular proteins. HIV has only nine genes and must highjack hundreds of cellular proteins to replicate. Sam68 regulates HIV gene expression but it is unclear how it does so. It is also unknown if HIV infection alters which cellular proteins are expressed by interacting with Sam68, especially if the cell and virus have to compete for it. Therefore, we will determine how HIV interacts with Sam68 to control cellular and viral gene expression. This may determine if Sam68 or other steps of HIV gene expression are potential drug targets.

Technical Summary

Sam68 is a cellular RNA binding protein that controls multiple steps of cellular gene expression and also regulates HIV-1 gene expression. To determine how Sam68 controls gene expression in primary human CD4 T cells, we will use RNA-seq to determine which RNAs change their abundance, alternative splicing, poly(A) site selection and translation efficiency upon T cell activation in a Sam68-dependent manner. We will then identify in vivo Sam68 RNA binding sites and use Bayesian networks to develop a predictive Sam68 target network. We will also determine if Sam68 controls the expression of functionally related proteins or pathways. Overall, these experiments will identify 1) the specific human CD4 T cell mRNAs that Sam68 regulates on a genome-wide level, 2) whether these mRNAs encode proteins that control related immunological functions, 3) the specific step(s) of gene expression that Sam68 regulates and 4) how the location of Sam68 RNA binding sites controls alternative splicing. This information can be used to understand how Sam68-regulated cellular RNAs control activated CD4 T cell function.

We will also characterise how HIV-1 infection of primary human CD4 T cells affects Sam68-regulated cellular RNAs and how Sam68 regulates HIV-1 gene expression. This will allow two important analyses. First, we will determine if HIV-1 infection regulates cellular gene expression by altering Sam68 activity or availability, which could alter the expression of immunologically active proteins. Second, we will compare how Sam68 regulates cellular and HIV-1 RNAs. It will be interesting to determine if HIV-1 RNAs interact with Sam68 in an identical manner to cellular mRNAs, if a subset of cellular mRNAs are regulated more similarly to HIV-1 RNAs than other cellular mRNAs, or if HIV-1 RNAs interact with Sam68 differently than cellular mRNAs to control viral gene expression, which may indicate that Sam68 is a potential antiretroviral target.

Planned Impact

The primary objective of this proposal is to determine how Sam68 controls CD4 T cell and HIV-1 gene expression. This research will have several different types of impact. First, the experiments in this grant will determine how Sam68 regulates CD4 T cell gene expression, which could provide insight into how CD4 T cells help control infection and are abnormally regulated in autoimmune diseases. Second, this proposal characterises how Sam68 regulates HIV-1 gene expression and replication, which could lead to new insights into HIV-1 post-transcriptional control mechanisms and novel antiretroviral treatments. It also characterises how HIV-1 infection may alter cellular gene expression by inhibiting the interaction between Sam68 and cellular RNAs. The development of new anti-HIV drugs remains a critical priority for combating the AIDS pandemic and targeting cellular factors is an exciting strategy for drug development. Maraviroc, which inhibits viral entry into the cell by binding CCR5, is the first approved antiretroviral that targets a host protein. Small molecules targeting other cellular RNA binding proteins that regulate HIV-1 replication, such as the SR protein SRSF1 and the RNA helicase DDX3, have been developed and inhibit HIV replication in vitro. This proposal may validate Sam68 as a potential antiretroviral drug target and, by increasing our understanding of HIV-1 gene expression, identify other novel antiretroviral drug targets. We will engage KCL's Business and Innovation department to evaluate the commercial potential of any discoveries that we make and, if warranted, help foster collaboration with pharmaceutical partners to develop novel therapeutics. Third, characterizing how Sam68 controls cellular gene expression at a genome wide level, and developing a predictive Sam68 target network, may allow the development of new insight and therapies into other diseases that Sam68 has been implicated in. These diseases include fragile X-associated tremor/ataxia syndrome (FXTAS), spinal muscular atrophy (SMA) and several types of cancer such as breast, prostate, colorectal, cervical and renal cell carcinoma. Fourth, this grant will employ two postdoctoral associates and they will receive training in cutting edge molecular biology and bioinformatics technology. The proposed RNA-seq and iCLIP experiments are at the forefront of molecular biology and the integration of the different types of data via the bioinformatics approaches will teach the postdocs how to answer complex biological questions that require an interdisciplinary team. They will also learn how to apply the molecular biology of how Sam68 regulates gene expression to virology and immunology scientific questions. These skills will be useful for a range of future career options including academic and pharmaceutical. Therefore, the beneficiaries of this research will potentially include: 1) scientists studying CD4 T cell gene expression and function, 2) scientists and companies studying HIV-1 gene expression and who are developing novel anti-HIV therapeutics, 3) scientists and companies who are studying FXTAS, SMA or several types of cancer in which Sam68 is upregulated as well as the patients who have these diseases and 4) the two postdoctoral associates who will receive further training in molecular biology, immunology, virology and bioinformatics.
 
Description Identification of Compounds with Anti-Human Cytomegalovirus Activity 
Organisation St George's University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution My lab collaborated by providing reagents and technical expertise.
Collaborator Contribution Our partners did all of the experiments for the project.
Impact PMID: 27956134
Start Year 2014
 
Description Regulation of HIV-1 RNA nuclear export 
Organisation University of Wisconsin-Madison
Country United States 
Sector Academic/University 
PI Contribution I contributed reagents and technical advice.
Collaborator Contribution They did all of the experiments.
Impact 10.1371/journal.ppat.1005565 10.1128/JVI.01897-14
Start Year 2011
 
Description Regulation of gene expression in T and B cells in critically ill patients with sepsis 
Organisation King's College London
Department Department of Immunobiology
Country United Kingdom 
Sector Academic/University 
PI Contribution I collaborated to provide technical assistance on RNA extraction for genome wide analysis of gene expression.
Collaborator Contribution They did all of the experiments.
Impact PMID: 28296810
Start Year 2016
 
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, Chad Swanson presented a poster on the Regulation of HIV-1 Gag expression by SR proteins. He was also a co-author for the talk by Luis Apolonia 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, Charlotte Mahiet, presented a poster on Regulation of HIV-1 Gag expression by SR proteins. Chad Swanson also attended this conference. This led to substantial research discussions and new experimental ideas.

In 2016, a postdoc in the Swanson lab, Charlotte Mahiet, 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, Chad Swanson was a co-author for a talk by his collaborator Nathan Sherer 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. Chad Swanson was also the senior author on a poster presented by Jonathon Sumner on how cis-acting RNA sequences regulate HIV-1 Envelope and Vpu translation. Chad Swanson 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, Laura Hidalgo, presented a poster on "Regulation of CD4 T cell and HIV-1 gene expression by Sam68". Chad Swanson also attended this conference. This led to substantial research discussions and new experimental ideas.
Year(s) Of Engagement Activity 2014,2015,2016,2018
 
Description Erasmus+ 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I have had two Erasmus+ students do projects in my lab.
Year(s) Of Engagement Activity 2015,2017
 
Description Genetics Society Summer Studentship 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Soo Oh recieved a Genetics Society Summer Studentship to work on a project analysing how SR proteins regulate HIV-1 gene expression in 2015. Kieran Killington recieved a Genetics Society Summer Studentship in 2017 to work on a project analysing how Sam68 and other cellular proteins differentially phosphorylated upon HIV-1 entry regulate HIV-1 gene expression.
Year(s) Of Engagement Activity 2015,2017
 
Description Microbiology Society Annual Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact In 2017, I gave a talk at the Microbiology Society Annual Conference entitled "Regulation of human immunodeficiency virus type 1 (HIV-1) Gag expression and virion production by SR proteins". In addition, Irati Antzin-Anduetza, a PhD student in my lab, gave a talk on "Altering the CG content of the HIV-1 gag sequence attenuates viral replication". Finally, Jonathan Sumner, gave an talk on "Cis-acting RNA sequences regulate HIV-1 envelope and Vpu translation", which is a project that I am collaborating on with Stuart Neil's lab.
Year(s) Of Engagement Activity 2017
 
Description National Student Association for Medical Research (NSAMR) Taster Month 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact In 2016, I gave a talk about the benefits of doing a research based intercalated BSc at a NSAMR Taster Month presentation at King's College London. I also hosted a student in my lab for one day where she shadowed members of my lab to see what it is like to do laboratory based research. In 2017, I hosted a student for three days in my lab.
Year(s) Of Engagement Activity 2016,2017
 
Description Seminar at GSK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I presented my lab's research on how the HIV-1 genomic RNA is regulated.
Year(s) Of Engagement Activity 2017
 
Description The Virus Within: Hearing HIV 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I collaborated with the composer Benjamin Oliver to create a lecture-recital performance called The Virus Within: Hearing HIV. For the performance, we collaborated to create a three movement composition representing the HIV replication cycle and the "shock and kill" potential cure treatment. I presented a scientific introduction for each movement and then professional musicians played each movement. We then had a question and answer session and the musicians played all three movements through continously. We also created text for the public engagement section of my KCL website describing the science that the music was based upon.
Year(s) Of Engagement Activity 2018
 
Description Translation UK conference 
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 Translation UK conference in 2011, 2013, 2014 and 2016. In 2014, the postdoc in my lab who is studying how SR proteins regulate HIV-1 gene expression, Charlotte Mahiet, also attended this conference. This conference has given us an opportunity to present our data on how SR proteins, Sam68 and specific cis-acting elements in the HIV-1 RNA regulate HIV-1 gene expression and virion production to the UK research community who are studying translational control mechanisms. This has led to helpful feedback on the experiments and established preliminary collaborations.
Year(s) Of Engagement Activity 2011,2013,2014,2016
 
Description Wellcome Trust Vacation Scholarship 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact James Scott was awarded a Wellcome Trust Vacation Scholarship to work in my lab.
Year(s) Of Engagement Activity 2016
 
Description Work experience students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact In 2015 I arranged for an A-level student visit my lab for a day as part of her work experience. In 2016 I organised an A-level student to spend a week visiting my lab. Both students learned what it is like to be a professional scientist and do molecular biology bench work.
Year(s) Of Engagement Activity 2015,2016