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:
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 . 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 . 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.
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 . 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 . 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 . The preliminary clinical studies on SARS-CoV-1 patients treated with IFN-I showed improvement in oxygen saturation and symptoms , but larger scale clinical trials were inconclusive due to the lack of patients as the outbreak quickly ceased by summer .
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 . Channappanavar and colleagues studied further the IFN-induced tissular damage using human ACE-2 transgenic mouse model . 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 . 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 ). 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
 Kumar, H., Kawai, T. and Akira, S., 2011. Pathogen recognition by the innate immune system. International reviews of immunology, 30(1), pp.16-34.
 Feld, J.J. and Hoofnagle, J.H., 2005. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature, 436(7053), pp.967-972.
 Cinatl, J., Morgenstern, B., Bauer, G., Chandra, P., Rabenau, H. and Doerr, H.W., 2003. Treatment of SARS with human interferons. The Lancet, 362(9380), pp.293-294.
 Haagmans, B.L., Kuiken, T., Martina, B.E., Fouchier, R.A., Rimmelzwaan, G.F., Van Amerongen, G., van Riel, D., De Jong, T., Itamura, S., Chan, K.H. and Tashiro, M., 2004. Pegylated interferon-α protects type 1 pneumocytes against SARS coronavirus infection in macaques. Nature medicine, 10(3), pp.290-293.
 Gao, H., Zhang, L.L., Wei, Q., Duan, Z.J., Tu, X.M., Yu, Z.A., Deng, W., Zhang, L.P., Bao, L.L., Zhang, B. and Tong, W., 2005. Preventive and therapeutic effects of recombinant IFN-alpha2b nasal spray on SARS-CoV infection in Macaca mulata. Zhonghua shi yan he lin chuang bing du xue za zhi= Zhonghua shiyan he linchuang bingduxue zazhi= Chinese journal of experimental and clinical virology, 19(3), pp.207-210.
 Loutfy, M.R., Blatt, L.M., Siminovitch, K.A., Ward, S., Wolff, B., Lho, H., Pham, D.H., Deif, H., LaMere, E.A., Chang, M. and Kain, K.C., 2003. Interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study. Jama, 290(24), pp.3222-3228.
 Stockman, L.J., Bellamy, R. and Garner, P., 2006. SARS: systematic review of treatment effects. PLoS medicine, 3(9).
 Cameron, M.J., Ran, L., Xu, L., Danesh, A., Bermejo-Martin, J.F., Cameron, C.M., Muller, M.P., Gold, W.L., Richardson, S.E., Poutanen, S.M. and Willey, B.M., 2007. Interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome. Journal of virology, 81(16), pp.8692-8706.
 Channappanavar, R., Fehr, A.R., Vijay, R., Mack, M., Zhao, J., Meyerholz, D.K. and Perlman, S., 2016. Dysregulated type I interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in SARS-CoV-infected mice. Cell host & microbe, 19(2), pp.181-193.
 Channappanavar, R. and Perlman, S., 2017, July. Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology. In Seminars in immunopathology (Vol. 39, No. 5, pp. 529-539). Springer Berlin Heidelberg.
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
Hoffmann et al., 2020, Cell 181, 271-280 April 16, 2020
Walls et al., 2020, Cell 181, 281-292, April 16, 2020
RBPs are key key drivers of gene expression by controlling RNA fate. However, our knowledge about RBPs in plants is very limited. I our recent work, we report an improved RNA interactome capture approach to discover RNA-binding proteins (RBPs) in plant leaves . Using this ‘plant-adapted RNA interactome capture’ (ptRIC) we have identified hundreds novel RBPs, including many enzymes and proteins from the photosynthetic apparatus. ptRIC did not only allowed the generation of the deepest ‘RBPome’ of plant tissue to date, but also opens the possibility to study how the RBPome remodels in response to environmental, physiological and pathological cues.
This work is an interdisciplinary collaborative effort between the Castello lab (Department of Biochemistry) and Preston lab (Department of Plant Sciences) at the University of Oxford. Find out more about this work here:
There are many things that we do not yet understand about the new coronavirus that has driven us all inside and stretched health systems across the globe to breaking point. One of its most fundamental aspects that remains obscure is how many people are actually infected. While official case numbers rise to 2 million, a frequently repeated maxim is that the confirmed cases represent just ‘the tip of the iceberg’. Part of the reason that the number of confirmed cases is so much lower than the actual number of cases is that many people are asymptomatic or present(ed) mild symptoms and have not been tested. Indeed, since testing capacity has been limited across the globe, only people who experience severe illness, or even who end up in hospital, are usually tested for SARS-Cov2 (the virus responsible for COVID-19) with RT-PCR assays. A proposed implication of these asymptomatic or mild-symptom infections is that we could be close to reaching, or even have already reached, a state of ‘herd immunity’. In this state, the immunized population acts as a firewall that protects those individuals that have not been exposed to the virus, disrupting the host-to-host transmission chain.
“Herd immunity is a form of indirect protection from an infectious disease that occurs when a large percentage of a population has become immune to the pathogen. Immunized individuals act as firewalls that disrupt pathogen spreading from host to host. Herd immunity can be reached when the pathogen has infected a large proportion of the population (typically 2/3) or by the generalised use of effective vaccines.”
This is a very enticing prospect, and one that has received a lot of attention in recent weeks. If not backed up by sufficient evidence, however, taking actions based on the principle of herd immunity has the potential to do more harm than good. Slackening of public health measures too early could potentially lead to catastrophic second, and even third, waves of infections, and an overconfidence in herd immunity could detract from ongoing efforts to generate a vaccine or an effective antiviral. Even if herd immunity is achieved through natural infection, we do not yet have convincing evidence that this immunity will keep the virus at bay for long enough and consistently enough to end this pandemic. Before we make any decisions based on this principle, it will, therefore, be important to understand precisely what proportion of the population has been infected, and whether infection confers long-term protection.
“Implementation of systematic serological tests is critical to determine the proportion of the population that has been exposed to the virus.”
The first necessary step will be to develop an accurate serology test to detect antibodies against SARS-CoV2 in our blood. Antibodies are one of our most effective tools in fighting viral infection and are generated as a result of a highly specific adaptive immune response. Hence, the presence of antibodies against SARS-Cov2 is in an unequivocal sign that someone has been infected and recovered from the virus. Once such a test is systematically applied, we will have a better idea on how close we are to threshold for herd immunity to be effective. These analyses will only provide information about the proportion of people exposed to the virus, but will not answer two critical questions: are these antibodies effective at combating the virus, and can they provide long-term protection?
One way in which antibodies can stop being protective is if the virus mutates into a form that is no longer recognised. Unlike other viruses, SARS-CoV2 has a relatively low mutation rate due to a proof-reading enzyme encoded in its genome, which corrects errors introduced by the RNA polymerase during replication. While this does not mean that the virus never mutates, comparisons made between common cold coronavirus strains from 30 years ago and those in circulation today indicate that changes in the viral genome are minimal, and that these do not substantially affect the viral proteins known to be the targets of antibodies. This is important and heartening evidence that a vaccine designed against this virus is likely to confer lasting protection. However, it also poses another question. If the viruses that cause up to a third of common cold cases in the human population are not undergoing significant mutation over time, how is it that people can be re-infected with them every couple of years?
Part of the answer to this likely lies in the quality and longevity of antibody response mounted against these viruses. A study published 30 years ago in which volunteers were infected with common cold coronaviruses found that antibodies declined soon after infection. More worrying is that after a year people could be reinfected when challenged with the same virus. One possibility is that the low pathogenicity of these viruses (most cases of the common cold are relatively mild) results in a half-hearted immune response that is sufficient to clear the virus but fails to generate lasting protection. Evidence that this might be a common problem with coronaviruses comes from a study performed in healthcare workers who recovered from MERS. In this study, authors found that the longevity of the antibody response against MERS was far more variable among individuals who had had mild symptoms than those having severe illness. Since so many cases of SARS-Cov2 infection are thought to be asymptomatic or entailing mild-severity, it is a possibility that a large proportion of infected people lack robust or long-lasting antibody response against the virus.
“Two critical questions remain unanswered: Are antibodies generated against SARS-Cov2 effective at combating the infection? and if so, is the immunization long-lasting?
In a recent pre-print looking at serology of 175 patients who experienced mild symptoms, almost 50% of people had medium or low antibody levels, and 10 patients showed no neutralising antibodies at all. The ability of the antibodies to block infection were assessed through an in vitro neutralisation assay. However, the extent to which this assay correlates with protection in vivo remains unclear. An important aspect that has not been possible to assess yet is how long-lasting this antibody response will be. Hence, whether exposure to SARS-Cov2 will lead to long-term protection is uncertain. Studies with patients infected with SARS-Cov1 (which is closely related to SARS-Cov2) during the outbreak in 2003, indicated that antibody levels declined after 2 years. The risk that natural infection might not provide sufficient long-term protection is an important justification for focusing resources on the development of an effective vaccine and antiviral drugs. By training our immune system with the ‘right’ antigen and following a robust immunization regimen, it might be possible to induce long-term and dependable immunity across the population. Whilst the antibody test will be important in informing policy decisions and epidemiological models in the short term, evidence about the efficacy of natural immunity will critical to identify a long-term solution for this pandemic.
Written by Louise Iselin, DTP rotation student in the Castello lab