Researchers have identified a set of human genes that fight SARS-CoV-2 infection

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Scientists at Sanford Burnham Prebys have identified a set of human genes that fight SARS-CoV-2 infection, the virus that causes COVID-19. Knowing which genes help control viral infection can greatly assist researchers’ understanding of factors that affect disease severity and also suggest possible therapeutic options. The genes in question are related to interferons, the body’s frontline virus fighters.

The study was published in the journal Molecular Cell.

“We wanted to gain a better understanding of the cellular response to SARS-CoV-2, including what drives a strong or weak response to infection,” says Sumit K. Chanda, Ph.D., professor and director of the Immunity and Pathogenesis Program at Sanford Burnham Prebys and lead author of the study.

“We’ve gained new insights into how the virus exploits the human cells it invades, but we are still searching for its Achille’s heel so that we can develop optimal antivirals.”

Soon after the start of the pandemic, clinicians found that a weak interferon response to SARS-CoV-2 infection resulted in some of the more severe cases of COVID-19. This knowledge led Chanda and his collaborators to search for the human genes that are triggered by interferons, known as interferon-stimulated genes (ISGs), which act to limit SARS-CoV-2 infection.

Based on knowledge gleaned from SARS-CoV-1, the virus that caused a deadly, but relatively brief, outbreak of disease from 2002 to 2004, and knowing that it was similar to SARS-CoV-2, the investigators were able to develop laboratory experiments to identify the ISGs that control viral replication in COVID-19.

“We found that 65 ISGs controlled SARS-CoV-2 infection, including some that inhibited the virus’ ability to enter cells, some that suppressed manufacture of the RNA that is the virus’s lifeblood, and a cluster of genes that inhibited assembly of the virus,” says Chanda.

“What was also of great interest was the fact that some of the ISGs exhibited control across unrelated viruses, such as seasonal flu, West Nile and HIV, which leads to AIDS.”

We identified eight ISGs that inhibited both SARS-CoV-1 and CoV-2 replication in the subcellular compartment responsible for protein packaging, suggesting this vulnerable site could be exploited to clear viral infection,” says Laura Martin-Sancho, Ph.D., a senior postdoctoral associate in the Chanda lab and first author of this study.

“This is important information, but we still need to learn more about the biology of the virus and investigate if genetic variability within these ISGs correlates with COVID-19 severity.”

As a next step, the researchers will look at the biology of SARS-CoV-2 variants that continue to evolve and threaten vaccine efficacy. Martin-Sancho notes that they have already started gathering variants for laboratory investigation,

“It’s vitally important that we don’t take our foot off the pedal of basic research efforts now that vaccines are helping control the pandemic,” concludes Chanda. “We’ve come so far so fast because of investment in fundamental research at Sanford Burnham Prebys and elsewhere, and our continued efforts will be especially important when, not if, another viral outbreak occurs.”


Role of Epigenetics in SARS-CoV-2 Infection, Immune-Pathogenesis, and Comorbidities
Epigenetic Regulation of Innate Host Immunity and Viral Pathogenesis

Epigenetic mechanisms clearly appear to be a vital part of SARS-CoV-2, which can make inroads into the host through these mechanisms resulting in the severity of illness and mortality that has been so frequently seen during the COVID-19 pandemic (Mehta et al., 2020).

We know for many decades that invariably, all viruses use epigenetic mechanisms, especially CpG methylation, to induce enterocytosis and syncytium development- a critical feature of coronaviruses in general (Mehta et al., 2020; Xia et al., 2020). The underlying molecular and epigenetic mechanisms that regulate the pathogenesis of coronaviruses are complex and dependent on the host-virus interactions guiding viral entry, replication, and immuno-pathogenesis.

SARS-CoV-2 is no different from other coronaviruses, as it also has an intrinsic ability to tamper with the host innate immune system. Although with subtle differences between DNA and RNA viruses, invariably, all viruses make use of host epigenetic reprogramming, which assists them in evading the host immune responses (Mehta et al., 2020).

To successfully survive and replicate through their life cycle in the host, viral pathogens have an arsenal of a variety of epigenetic strategies they use in subverting the host’s immune system. These strategies include pathogen-directed modification of host proteins and chromatin by virus-specific gene products or viral proteins, modulation of activators and repressors in innate immunity that can attenuate pattern recognition receptor (PRR) sensing and signaling pathways.

In response, the host immunity also counters pathogen-induced changes to their epigenomes to maintain effective control of anti-viral immunity. Thus, viruses have evolved diverse strategies to tamper with the host epigenetic machinery by targeting DNA methylation and reprogramming the host DNA methylome for its own benefit (Zhang and Cao, 2019).

While mutations can change the genetic code, thereby directly affecting the genetic material, epigenetic regulation, in contrast, bridges genotype and phenotype, therefore these modifications result in changes in the chromatin structure or modification of nucleic acid without any alteration in the genetic code. It implies the reversibility, flexibility, and quick responsiveness of epigenetic alterations to rapid changes in the environment and other exposures.

Here, the environment and the genome serve as powerful interfaces for any epigenetic modification (Goldberg et al., 2007). Rapid progress has been made in many areas of medicine, which include cancer biology, infectious diseases, and immunity, and it is thought that some viral pathogens that modify chromatin and other epigenetic machinery, are excellent candidates for drug targets (Esteller, 2008; Obata et al., 2015).

The research has primarily focused on molecular mechanisms of RNA viruses that tamper with the components that regulate host innate immunity, which forms the anti-viral defense arsenal of the host. It has been suggested that the RNA viruses, especially that replicate in the cell cytoplasm, have evolved a sophisticated mechanism that is designed to not only to exert influence on the host epigenome but also regulate it, thereby taking charge of subverting the anti-viral defense of the host at its own terms to promote its own replication and successful establishment in the host (Bird, 2007; Goldberg et al., 2007).

In support of this, it has been previously shown that both DNA and RNA viruses have evolved this function to antagonize the regulatory machinery of the host epigenome to facilitate viral replication and spread (Busslinger and Tarakhovsky, 2014).

Epigenetic mechanisms can switch genes on or off and determine which proteins need to be transcribed at any given time, thereby an essential role in regulating normal cellular processes. Different kinds of cells and organs have a different set of genes that turn on and off as per functional needs. Epigenetic silencing is one way to turn genes off, which contributes to differential expression, implying that such genomic changes that result in no sequence alteration in the host can be reversed in certain situations.

Within these different cells that perform diverse functions, three systems act in tandem to silence genes, and this involves RNA-mediated silencing, DNA methylation, and histone modifications (Egger et al., 2004; Schäfer and Baric, 2017). These three systems, which are the functional regulators that modulate gene expression, work in tandem in a highly flexible and seamless manner.

Epigenetic modification of cellular genomes occurs in a highly structured and specific manner and is carefully orchestrated, particularly DNA methylation and other specific histone modifications that assure precise and reliable transmission of gene expression to the progeny cells (Schäfer and Baric, 2017). Upon infection and also during chronic infection, there is a disruption of cellular epigenetic balance (Schäfer and Baric, 2017), and it is this balance that defines the pathogenesis, in part, during infection, in addition to various disease processes in cancer, neurodegenerative and metabolic disorders.

There is an intense research activity concerning SARS-CoV-2 on how this and the family of coronaviruses affect the host gene expression regulation. Although the complex epigenetic interactions of coronaviruses and epigenetic processes with host cells have been researched since the discovery of SARS virus in 2002 (Froude and Hughes, 2020), the current investigations are focussed on how the histone methylation/chromatin remodeling, DNA transcription, cellular packaging of DNA and non-coding RNAs epigenetically regulate gene expression (Schäfer and Baric, 2017).

These processes are critical and vital for the virus as it relies on the host cell to replicate its genetic material and continue its progeny. There are four main aspects of epigenetic regulation (DNA methylation and oxidation, histone modifications/chromatin remodeling, and non-coding RNAs (miRNA), which are involved in shaping the innate immune response during any viral infection.

Therefore, a clear understanding of these aspects can guide us in treating viral infections and various other diseases that afflict humans. High throughput genomic technologies are already allowing a comprehensive visualization and investigation of such epigenetic modifications with high resolution. With data integration being a reality, the possibilities are endless for a holistic view of these processes.

DNA Methylation, Histone Modification, and Chromatin Remodeling
DNA methylation is a chemical process that adds a methyl group to DNA and is involved in transcriptional silencing. It is highly orderly and takes place post-DNA replication across all mammalian cells. It occurs explicitly at the 5′ position of the cytosine ring within the CpG islands, where a methyl group is added to create 5-methylcytosine (5 mC).

This reaction is mediated by the DNA methyltransferases (DNMTs), which preferentially targets unmethylated CpG islands to achieve DNA methylation, and the insertion of methyl groups alters the structural appearance of DNA (Egger et al., 2004; Jones, 2012).

Through this epigenetic alteration, as stated earlier, viruses can switch on and switch off genes at multiple host gene locations. Such chemical modifications in DNA methylation and histone modification collectively subjugate the production of antigen presentation molecules vital in mounting anti-viral response during infection, as shown for both MERS-CoV and H5N1 viruses (Menachery et al., 2018).

The two critical antigen-presenting cells of the innate immune system are the dendritic cells and macrophages, and they the primary sensors of “danger” signals, and recognize structurally conserved viral proteins via the Toll-like receptors (TLRs- e.g., TLR 3, 4, 7, and 8), which are dominant PRRs recognized by pathogens and are expressed on the surfaces of these immune cells. Defects in TLR function of these innate immune cells have been shown to increase the severity of pneumonia in mice, especially in the context of aging and low-grade chronic inflammation which develops with aging (Inflammaging), and this could be the difference between the aging immune system and disease severity seen during COVID-19 pandemic in the elderly (Zhang and Cao, 2019).

Moreover, upon activation, both cell-and stimulus-specific signals are mounted to initiate temporal and spatial responses mediated via both cell-to-cell contact or secretion of interferon (IFN) and tumor necrosis factor (TNF), implying the intrinsic ability of their epigenome to change in real-time following the sensing of danger signal and mounting a vigorous anti-viral host response. This is the way epigenome primes the immunological memory for managing the clear and present danger, and for subsequent future insults.

Thus, to overcome anti-viral restriction by the host, almost all known viruses downregulate the interferon production by immune cells fighting the infection. Interestingly, the subversion of interferons in the host is mainly achieved via the induction of de novo methylation of the IFN-γ promoter leading to epigenetic silencing of the interferon secreting genes (ISGs) to block host’s anti-viral arsenal (Zhang and Cao, 2019), but the mechanisms independent of epigenetic silencing directly through viral pathogenic mechanisms have also been described for other DNA and RNA viruses to play a role in the silencing of interferon secreting genes (Haller and Weber, 2007).

Lu et al. (2020) have shown the value of a dynamic post-transcriptional RNA modification epigenetic change, known as N6-methyladenosine modification or Adenosine methylation (also known as m6A methylation), in modifying the viral activity and reinstating the host’s immune system to fight the virus in a mouse model (Lu et al., 2020). N6-Methyladenosine or m6A accounts for over 80% of all RNA methylation, influencing structure, splicing, localization, translation, stability, turnover, and biology of RNA (Lu et al., 2020). As the m6A exhibits both pro- and anti-viral activities, depending on the virus species and host cell type, its value in disease and treatments is essential.

The m6A and m6M affect the viability of specific RNA viruses by modulating viral replication, viral cap structures, innate sensing, and innate immune response pathways (Gonzales-van Horn and Sarnow, 2017). The primary interaction between virus and host during viral infection is affected by m6A, and multiple m6A-modified viral RNAs have been defined, which alter the epi-transcriptome of m6A in the host following viral infection.

Viral life cycle right from viral gene expression, replication, and production of progeny virions are all modulated by m6A modifications (Kuppers et al., 2019). It has become evident that m6A methylation makes the virus able to hide from the immune system by masking and mimicking the host RNA to evade immune recognition as being non-self RNA- thereby assuring virus persistence goes undetected in the host. Thus, targeting this viral strategy could pay dividends in anti-viral control.

The SARS-CoV-2 RNA genome has more than 50 potential m6A sites based on the presence of sequence-specific motifs for m6A modification by the RNA methylase complex METTL3/14, including GGACU(T), GGACA, and GGACC. As a result, >0.64% of all adenosines, or 0.18% of all bases, in SARS-CoV-2 RNA could acquire m6A (Kuppers et al., 2019; Lu et al., 2020).

Gain or loss of m6A can result in significant functional changes to RNA viruses, at the level of entry, fusion, replication, transmission, host immune evasion, and pathogenesis. Importantly, the m6A epi-transcriptome of host cells, which plays a vital role in resisting viral infection, can also undergo alterations following viral infection (Zaccara et al., 2019; Kim et al., 2020). It is also important to emphasize that as the members of the coronaviruses, including SARS-CoV-2, can encode their own methyltransferases for self-methylating adenosine residues, promoting immune evasion (Zhang and Cao, 2019). Overall, studies on m6A modification in the virus and host can unveil factors that affect SARS-CoV-2 infection and will lead to the identification of novel targets for treatment, and possibly vaccines for COVID-19.

Specific SARS-CoV-2 Proteins Interfere With Significant Epigenetic Processes of the Host Involving Innate Immunity and Immuno-Pathogenesis

It is now apparent that all three recently emerged coronaviruses- the SARS-CoV, MERS-CoV, and SARS-CoV-2, have the intrinsic ability to delay pathogen recognition and subjugate, antagonize interferon-stimulated genes (ISGs) effector function. It is thus crucial to understand the role of a variety of SARS-CoV-2-encoded proteins that can effectively and epigenetically modulate host innate immune signaling (Schäfer and Baric, 2017). There are several known viral proteins that associate with viral pathogenesis and are controlled epigenetically (see Figure 3 for an interactome of epigenes that SARS-CoV-2 encodes and its interaction with the host). Below are some of the critical examples of epi-genes in the context of SARS-CoV-2 that have provided some insights into host-virus interactions.

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Figure 3
SARS-CoV-2 proteins and gene ontologies of their interacting host genes. Size of gene ontology circle is proportional to the number of genes in the ontology, while thickness of the lines linking SARS-CoV-2 proteins and gene ontologies represents number of interacting genes in the ontology. SARS-CoV-2 proteins interacting with significantly higher number of host genes than expected are marked by asterisks, with representation: *P < 0.05, **P < 0.01, ***P < 0.001.

ORF3b
The SARS-CoV-2, uniquely encodes for a shorter protein-ORF3b (open-reading frame 3b) (Gordon et al., 2020), which has been recently described in hampering innate immune reaction in vitro by limiting the induction of the type I interferon response, which is the most crucial aspect of viral pathogenesis. This protein can block host defenses early in the infection cycle by shutting off the host’s interferon secretion through an epigenetic mechanism before the T and B cells come into play. SARS-CoV-2 ORF3b is considerably shorter than its SARS-CoV ortholog, encoding a protein just 22 amino acids long due to premature stop codon, compared to the 154 amino acids of SARS-CoV ORF3b.

In an in vitro experiment, a premature stop codon was introduced into the SARS-CoV ORF3b coding sequence, the resulting 135-amino acid protein was better at suppressing type I interferon than the wildtype SARS-CoV protein was a difference that could be possibly attributed to higher virulence of SARS-CoV-2. Still, these data are preliminary, and further investigation using human samples is needed, because a 56-amino acid variant of SARS-CoV-2-ORF3b circulated in Ecuador showed no evidence of severity, except in some cases (Konno et al., 2020). Moreover, when compared to the related SARS-CoV-1, IFN antagonism could also be attributed to ORF3b, ORF6, and the nucleocapsid (N) gene products (Frieman et al., 2010).

In another recent study (Blanco-Melo et al., 2020), this immune response was also shown to be defined by low levels of type I and III interferons juxtaposed to elevated chemokines coupled with high expression of interleukin (IL)-6 suggesting that reduced innate antiviral defenses, coupled with vigorous inflammatory cytokine production, are the most likely drivers of viral pathogenesis. In this context, it is notable that the common respiratory viruses, including influenza A virus (IAV), encode a variety of antagonists to the IFN-I and -III response (García-Sastre, 2017) in comparison to SARS-CoV-2.

Viroporins
Further, novel coronaviruses (nCoVs) are also known to encode 3 ion-channel proteins, also called Viroporins, such as protein E, ORF3a, and ORF8a. These are small, hydrophobic proteins with a multifunctional ability that aids in modifying cellular membranes and renders permeability with both features assisting the release of virions from infected cells (Carrasco, 1995). They also have additional roles in homeostasis-mediated by protein-protein interactions with host cellular proteins, cellular metabolism, and in enhancing viral growth rates in the host (Gonzalez and Carrasco, 2003). They are particularly common among RNA viruses implicated in causing human diseases, which include HIV, HCV, influenza A virus, poliovirus, respiratory syncytial virus, SARS-CoV, and CoV-2 (Gonzalez and Carrasco, 2003; Nieva et al., 2012; Nieto-Torres et al., 2015; Chen et al., 2019).

Viroporins, via mechanisms that involve intracellular lysosomal disruption and ion-redistribution, lead to the activation of the innate immune signaling receptor NLRP3-(NOD-, LRR-, and pyrin domain-containing 3) inflammasome. This, in turn, triggers the production of inflammatory cytokines [interleukin 1β (IL-1β), IL-6, and TNF] resulting in tissue inflammation, tissue damage, and severe respiratory illness, as seen in COVID-19 pandemic. NLRP3 inflammasome in macrophages can be activated by several viruses and viral proteins, including SARS-CoV-2 (Chen et al., 2019).

The inflammasome is a vital arm of innate immunity that detects and senses a variety of endogenous or exogenous, sterile or infectious stimuli encountered within the cell, thereby inducing cellular responses and effector mechanisms to ward off pathogens or threat.

Emerging evidence shows epigenetic factors, including DNA methylation and histone acetylation, regulate NLRP3 mRNA expression (Wei et al., 2016), suggesting that NLRP3 inflammasome could be an attractive drug target for SARS-CoV-2 (Shah, 2020). It is worth emphasizing that the transmembrane E protein is another epigenetic player in SARS-CoV-2, which through binding affinity with the BRD2 and BRD4 epigenetic, allows interaction with acetylated histones to regulate gene expression (Lai et al., 2020b). These interactions play a vital role in influencing the inflammatory response and determining the severity of disease in SARS-CoV-2 patients.

Nsp5
It is the central protease of SARS-CoV-2, and has been shown to interact with one of the epigenetic regulators- the histone deacetylase 2 (HDAC2). This interaction affects inflammatory and interferon responses mediated by HDAC2 (Schäfer and Baric, 2017). In agreement with these findings, Gordon et al. (2020) through a detailed interactome of SARS-CoV-2 and host have also identified high-confidence interaction between Nsp5 and the epigenetic regulator histone deacetylase 2 (HDAC2), and predicted a cleavage site between the HDAC domain and the nuclear localization sequence, suggesting that Nsp5 may inhibit HDAC2 transport into the nucleus potentially impacting the function of HDAC2 in mediating inflammation and interferon response (Chamberlain and Shipston, 2015; Gordon et al., 2020). They also identified an interaction of Nsp5 (C145A) with tRNA methyltransferase 1 (TRMT1), and predict that TRMT1, which can remove its zinc finger and nuclear localization through cleavage by Nsp5, forcing the TRMT1 to localize exclusively in mitochondria.

reference link :https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019793/


More information: Laura Martin-Sancho et al. Functional Landscape of SARS-CoV-2 Cellular Restriction, Molecular Cell (2021). DOI: 10.1016/j.molcel.2021.04.008

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