Restriction of HIV and coronavirus infections by the innate immunity protein Shiftless

Project Information

RIBOSFL

Grant agreement ID: 101023196

DOI

10.3030/101023196

Project closed

EC signature date 18 March 2021

Start date 1 May 2021

End date 30 April 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 174 806,40

EU contribution

€ 174 806,40

Coordinated by

MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Germany

Periodic Reporting for period 1 - RIBOSFL (Restriction of HIV and coronavirus infections by the innate immunity protein Shiftless)

Reporting period: 2021-05-01 to 2023-04-30

Summary of the context and overall objectives of the project

In the recent past, there has been an alarming increase in the emergence of novel pathogenic viruses like SARS-CoV-2, while relatively older viruses like HIV-1 remain without a cure. Additionally, rapid development of resistance to current therapeutics have led to a continuous need for new antiviral therapeutic strategies. To this end, –1 programmed ribosomal frameshifting (–1PRF) has gained attention as a new therapeutic target. What is –1PRF? Let us first go back to the central dogma of life- while DNA stores genetic information, which is transcribed to messenger RNAs (mRNAs), ribosomes ‘translate’ the information encoded in mRNAs to make chains of amino acids called proteins. A reading frame is simply the division of nucleotides into non-overlapping, consecutive triplets called codons. If the ribosome shifts forward or backward by 1 or 2 nucleotides, the amino acid sequence will radically change and will very often lead to the appearance of a premature stop codon. PRF is present across all domains of life but majority of the reported instances of PRF have been found in viruses. In the simplest terms, –1PRF is the backward movement of the ribosome by 1 nucleotide on the slippery site of an mRNA, when the ribosome is hindered by nearby mRNA secondary structures. C19orf66, also known as Shiftless (SFL) functions against a plethora of viruses by affecting viral RNA stability (in Dengue virus), formation of the viral replication organelle (in Hepatititis C virus), targeting viral proteins for degradation (in Zika virus) and inhibiting ribosomal frameshifting (in HIV-1 and many other viruses). For the project RIBOSFL, we will focus only on the frameshift inhibitory function of SFL. In the context of HIV-1, SFL is thought to bind simultaneously to the stem-loop and a non-canonical rotated state of a ribosome in the process of undergoing frameshifting on the slippery site. This leads to ribosomal stalling. SFL is then, believed to recruit release factors to the ribosome to terminate translation at the slippery site and prevent further translation in the –1 frame. Although SFL has mainly been studied as a frameshift inhibitor in the context of HIV-1 and Japanese Encephalitis Virus, SFL is also an effective frameshift inhibitor for SARS-CoV-2. Since SFL has many modes of antiviral action, it is possible that SFL can increase specificity for viral mRNAs by employing a combination of strategies.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The exact mechanism of action of SFL as a modulator of translation is not known. This knowledge is crucial in order to eventually utilize SFL as an antiviral therapeutic. As a first step, we cloned and purified SFL. During the process of protein purification, we noted that SFL exists as a chain of molecules, called a multimer. We focused on a 36 amino acid comprising region (termed Required for Antiviral Activity (RAA)) that is essential for suppression of –1PRF and HIV infection and is missing from SFL short (SFLS), an inactive splice variant of SFL. We showed that SFL is capable of multimerization and the RAA region is essential for this function. We also confirmed that the RAA region is necessary for ribosome association and binding to the HIV RNA. Collectively, our results showed that the multimerization of SFL is important for these binding functions. We carried out specialized experiments, called crosslinking mass spectrometry, to identify the interacting partners of SFL on ribosomes. The interacting partners could be divided into 2 groups: a. ribosomal proteins and b. stress granule proteins. Stress granules are membraneless RNA-protein organelles, which are formed under stress. After the stress is removed, mRNAs can undergo translation, degradation or further storage. A recent publication with the Kaposi’s Sarcoma Herpesvirus (KSHV) has indicated that SFL is present in stress granules during KSHV infection. This indicates that SFL in stress granules is also likely to play a role in HIV infection (PMID: 36326276). We are the first group to study SFL in an in vitro (outside a biological system with defined and measured components) translation system. Our in vitro studies indicate that SFL is a general translation inhibitor (for all mRNAs) in addition to a specific inhibitor of –1PRF (details will be available in our future publications). Additionally, our in vitro translation experiments have allowed us to form translation complexes which we will soon use to study the structure of SFL-bound ribosomes.

While benchwork is the first step in understanding a scientific concept, it is equally important to communicate one’s results to other scientists and laymen. Within our institute, I have discussed my research with other scientists at formal and informal meetings. I have attended a number of conferences to discuss my data with other researchers such as two Marie Curie Alumni Association meetings, the 28th tRNA Conference and the SPP1923 symposium on the innate sensing and restriction of retroviruses. In September 2023, I will attend the ‘Protein Synthesis and Translational Control’ meeting. So far, I have already published one scientific paper (https://pubmed.ncbi.nlm.nih.gov/35891432) and I am working on more scientific papers and review articles on Shiftless. I have written an article about ribosomal frameshifting and Shiftless for the journal ‘Resonance’ that is published by the Indian Academy of Sciences for undergraduate students and teachers. I spoke to the BSc and MSc students of St. Xavier’s College, Mumbai in January 2023 and integrated MSc students of the UM-DAE Centre for Excellence in Basic Sciences, Mumbai in January 2022 about my academic journey and current work regarding SFL. In addition, working on RIBOSFL has also inspired me to work on scientific engagement outside the realm of SFL. I use my Twitter account to discuss my work and science in general. I am a mentor for the CyberMentor Programme which pairs female high school students with female scientists in a bid to encourage women to remain in the STEM subjects. I also helped organize Pint of Science, Göttingen 2023 where scientists discuss their work with non-scientists in an informal setting. I have also participated in a webinar hosted by Euraxess India to help prospective MSCA postdoctoral fellowship applicants with their applications.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

RIBOSFL has come a long way in characterizing SFL as has been enumerated in the previous section. During the course of RIBOSFL, we have also been creating a human in vitro translation system, which will be crucial to study all other translation inhibitors, such as antivirals and anticancer drugs. It is very important to develop peptide mimetics (a molecule that biologically mimics active regions of important biomolecules) of relevant regions of SFL once we have a better understanding of their structure-function relationship. Additionally, it would be interesting to develop pharmaceutical strategies specifically to express molecules like SFL in the human body in response to viral infection.