Global analysis of protein-RNA interactions in SARS-CoV-2-infected cells reveals key regulators of infection

The Castello lab, in collaboration with an international and multidisciplinary team, uncovered the interactions that SARS-CoV-2 RNA establishes with the host cell, many of which are fundamental for infection. These discoveries pave the way for the development of new therapeutic strategies for COVID-19 with broad-range antiviral potential. 

The genetic information of SARS-CoV-2 is encoded in an RNA molecule instead of DNA. The viral RNA is central to the SARS-CoV-2 life cycle, as it must be multiplied, translated, and packaged into new viral particles to produce the viral progeny. Despite the complexity of these processes, SARS-CoV-2 only encodes a handful of proteins able to engage with viral RNA. To circumvent this limitation SARS-CoV-2 hijacks cellular proteins and repurposes it for its own benefit. However, the identity of these proteins has remained unknown until now.
Researchers from the University of Oxford in collaboration with other labs across UK and Europe have developed a new approach to discover in a comprehensive manner the proteins that ‘stick’ to SARS-CoV-2 RNA in infected cells. With this method, authors uncovered that SARS-CoV-2 RNA hijacks more than a hundred cellular proteins, which appear to play critical roles in the viral life cycle. 
This work, published in Molecular Cell, identifies many potential therapeutic targets with hundreds of available drugs targeting them. In a proof-of-principle experiment, authors selected four drugs targeting four different cellular proteins. These drugs caused from moderate to strong effects in viral replication. 

‘These exciting results are only the beginning – said Alfredo Castello, one of the researchers that has led the work -. With hundreds of compounds that target these critical cellular proteins, it will be possible to identify novel antivirals. Our efforts, together with those of the scientific community, should focus now on testing these drugs in infected cells and animal models to uncover which ones are the best antivirals.’  

An unexpected observation of this study is that viruses from different origin such as SARS-CoV-2 and Sindbis, hijack a similar repertoire of cellular proteins. This discovery is very important, as cellular proteins with important and wide-spread roles in virus infection have potential as target for broad-spectrum antiviral treatments.

‘In this stage of the pandemic in which vaccines have proved their value – added Alfredo Castello – it becomes fundamental to develop new therapeutic approach to counteract emergent vaccine-resistant variants or novel pathogenic viruses with pandemic potential’.  
Professor Shabaz Mohammed adds: ‘These new methods to discover the interactors of viral RNA builds on nearly 6 years of joined effort between the Castello and Mohammed labs using Sindbis virus as discovery model. This pre-existent work allowed us to react rapidly at the beginning of the COVID-19 pandemic and study the interactions between SARS-CoV-2 and the host cell in a reduced timeframe. Our methodology will now be ready to respond rapidly to future viral threads.’  

The paper ‘Global analysis of protein-RNA interactions in SARS-CoV-2 infected cells reveals key regulators of infection’ is published in the journal Molecular Cell. The work was led by Dr Wael Kamel and Marko Noerenberg, postdoctoral researchers at Glasgow and Oxford, and Berati Cerikan, postdoctoral fellow at the University of Heidelberg.

Additional Information
The following videos have been posted on social media and explains this research in an accessible way:

When cellular proteins meet SARS-CoV-2 RNA: a story of protein-RNA interactions

I our recent work we comprehensively and systematically identify the complement of cellular RNA-binding proteins that are involved in SARS-CoV-2 infection. We discover that the cellular RNA-binding proteome (RBPome) is pervasively remodelled upon SARS-CoV-2 infection, affecting a broad range of RNA metabolism and antiviral pathways. We also apply a new method to uncover the composition of SARS-CoV-2 RNPs, revealing a dozens of cellular RBPs and seven viral proteins. Our study reveals a new universe of host-virus interactions awaiting to be characterised and with great potential for novel therapies againt COVID-19.

This work is a synergistic collaboration between the Castello, Mohammed, Bartenschlager, Martinez and Lilley labs. See full publication in BioRxiv below:

Global analysis of protein-RNA interactions in SARS-CoV-2 infected cells reveals key regulators of infection | bioRxiv

Interferons in SARS treatment – a doubled-edged sword

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal agent of coronavirus disease 2019 (COVID-19). It has caused a global pandemic that is posing a major threat to millions of people’s health. Part of the reason that causes this pandemic is the lack of pre-existent adaptive immunity among the population. Before successful development and deployment of effective vaccine, the first line of defence against SARS-CoV-2 in our body is our innate immunity. Once the cell detects atypical signatures related to viruses, such as double-stranded RNA, it secrets signalling proteins to alarm its surrounding cells, known as cytokines [1]. Secreted cytokines vary across cell types, and each has its own role in organizing the body’s defence against viruses. Some of the most important defences against viruses are the interferons (IFN), which recognise specific receptors in the surface of the cell as a key and a lock. The interaction of interferon with its receptor induces the expression of antiviral proteins in cytoplasm and nucleus of the cell to combat infection more effectively. Researchers has studied the functions of interferons for decades and developed recombinant proteins to be used in protein therapies, for example, to treat hepatitis C virus [2]. The current COVID-19 pandemic raises the important questions about the role of individual cytokines and, in particular interferons, in body’s response to SARS-CoV-2.

Schematic representation of the structure of interferons (illustration by David Goodsell)

As SARS-CoV-2 is a newly identified virus, researchers are still investigating the details about the importance of individual cytokines in COVID-19. However, we can infer their potential functions from its better studied relative SARS-CoV-1, the coronavirus that caused an outbreak in 2003. Cinatl and colleagues revealed that type I interferon (IFN-I), which includes IFN alpha and beta, can be used to effectively inhibit replication of SARS-CoV-1 in vitro [3]. In vivo studies using human ACE-2 transgenic macaques confirmed the anti-SARS effect of IFN-I. Haagmans and colleagues tested the effect of pegylated IFN-alpha injection on macaques infected with SARS-Cov-1 and they showed lower viral titres in both throat swab and lung homogenate, and better histopathology results [4]. The suppression of SARS-CoV-1 infection was even stronger when IFN was injected prior to the infection with the virus. Gao and colleagues conducted similar experiment in macaques using IFN-I nasal spray, which also showed protective effects [5]. The preliminary clinical studies on SARS-CoV-1 patients treated with IFN-I showed improvement in oxygen saturation and symptoms [6], but larger scale clinical trials were inconclusive due to the lack of patients as the outbreak quickly ceased by summer [7].  

Channappanavar et al., Cell Host Microb, 2016

Although IFN treatment sounds clinically promising for SARS-CoV-1 outbreak, we have also learnt that cytokines could be destructive when they go rampage. Typically, one week after SARS infection, patients that have arterial oxygen saturation (SO2) > 91% will recover within a week, while those that have SO2 < 91% enter a crisis phase that require access to ventilators, and have higher mortality rate. Cameron and colleagues discovered that patients that entered the crisis phase have naturally significantly higher levels of proinflammatory cytokines [8]. Channappanavar and colleagues studied further the IFN-induced tissular damage using human ACE-2 transgenic mouse model [9]. Surprisingly, mice lacking the receptor for IFN-I showed milder symptom (measured by body weight loss) and lower mortality rate when compared with wild-type mice after infected with lethal dose of SARS-CoV-1. Moreover, other studies have shown correlation between elevated levels of cytokines and severe symptoms in SARS-CoV-1 and MERS-CoV [10]. Can we thus conclude that interferons do more harm than good? Using transgenic mice model, Channappanavar and colleagues discovered that multiple cytokines, including IFN-I, showed a 24-hour delay in expression level after SARS-CoV-1 infection. Early intervention of IFN-I treatment cured SARS in infected mice and had substantially milder symptoms than mice lacking the IFN-I receptor. Therefore, it appears that IFN-I response at the right timing effectively contains the virus, but at the wrong time is responsible for severe symptoms in SARS-CoV-1 infection (see figure [9]). It remains unclear how our understanding of SARS-CoV-1 could help us combat SARS-CoV-2, but these discoveries could be beneficial in the development of effective treatment or even to understand the battle between our immune system and the virus and its consequences.

Writen by Honglin Chen, DPhil student


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How SARS-Cov-2 enters in the host cell?

The recent pandemic explosion of the “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2), firstly originated in the Hubei province of China and subsequently spread all over the word, reached officially over three million detected cases and caused the death of more than two hundred thousand people. Scientists have begun a race toward the understanding of the mechanisms at the basis of SARS-CoV-2 viral cycle and unique features. This virus belongs to the betacoronavirus genus, together with SARS-CoV-1 and Middle East respiratory syndrome (MERS-CoV), which caused outbreaks in the last two decades.

Researches across the globe are leading to the discovery of pivotal characteristics of SARS-Cov-2, some of which shared with its ‘cousins’ SARS-Cov-1 and MERS. One of these achievements is the documentation of the mechanism of entrance into the host cell. This involves the interaction between the transmembrane viral spike glycoprotein and the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell surface. The spike is a homotrimeric glycoprotein complex, in which each monomer is composed by two subunits. The S1 subunit comprises the receptor binding motif (RBM) and is responsible for the first attachment to the host receptor, while the S2 subunit mediates the membrane fusion, and requires a proteolytic cleavage executed by the host transmembrane serine protease 2 (TMPRSS2). Exciting biochemical and structural analysis of the virus-host interacting surfaces, revealed a very high binding affinity of these proteins and the occurrence of conformational changes in the complex which allow the entry of the virus. The interaction of the spike with its receptor is a key step in virus infection, which can be explained as the intimate relation of a key and its lock opening the doors to the intracellular environment. 

Advanced knowledge on the interaction between the virus and the target cells is extremely valuable and has been object of innovative studies aiming to develop vaccines and antiviral drugs, which could potentially be beneficial in our long-lasting fight against coronavirus, endowing us with potent tools and advantage in the battlefield.  

Written by Dr. Vincenzo Ruscica, Marie Sklodowska-Curie fellow


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