Yet the virus has also become known for affecting other parts of the body in ways not as well understood, sometimes with longer-term consequences, such as heart arrhythmia, fatigue and “brain fog.”
Researchers at University of California San Diego School of Medicine are using stem cell-derived organoids – small balls of human cells that look and act like mini-organs in a laboratory dish—to study how the virus interacts with various organ systems and to develop therapies to block infection.
“We’re finding that SARS-CoV-2 doesn’t infect the entire body in the same way,” said Tariq Rana, Ph.D., professor and chief of the Division of Genetics in the Department of Pediatrics at UC San Diego School of Medicine and Moores Cancer Center. “In different cell types, the virus triggers the expression of different genes, and we see different outcomes.”
Rana’s team published their findings February 11, 2021 in Stem Cell Reports.
Like many organs, the team’s lung and brain organoids produce the molecules ACE2 and TMPRSS2, which sit like doorknobs on the outer surfaces of cells. SARS-CoV-2 grabs these doorknobs with its spike protein as a means to enter cells and establish infection.
The fluorescent label allowed them to quantify the binding of the virus’ spike protein to ACE2 receptors in human lung and brain organoids, and evaluate the cells’ responses.
The team was surprised to see approximately 10-fold more ACE2 and TMPRSS2 receptors and correspondingly much higher viral infection in lung organoids, as compared to brain organoids. Treatment with viral spike protein or TMPRSS2 inhibitors reduced infection levels in both organoids.
“We saw dots of fluorescence in the brain organoids, but it was the lung organoids that really lit up,” Rana said.
Besides differences in infectivity levels, the lung and brain organoids also differed in their responses to the virus. SARS-CoV-2-infected lung organoids pumped out molecules intended to summon help from the immune system – interferons, cytokines and chemokines.
Infected brain organoids, on the other hand, upped their production of other molecules, such as TLR3, a member of the toll-like receptor family that plays a fundamental role in pathogen recognition and activation of innate immunity
Rana explained that, while it might seem at first like the brain organoid reaction is just another form of immune response, those molecules can also aid in programmed cell death. Rana’s team previously saw a similar brain cell response to Zika virus, an infection known to stunt neonatal brain development.
Of course, organoids aren’t exact replicas of human organs. They lack blood vessels and immune cells, for example. But they provide an important tool for studying diseases and testing potential therapies.
According to Rana, organoids mimic the real-world human condition more accurately than cell lines or animal models that have been engineered to over-express human ACE2 and TMPRSS2.
“In animals over-expressing ACE2 receptors, you see everything light up with infection, even the brain, so everyone thinks this is the real situation,” Rana said. “But we found that’s likely not the case.”
In addition to their work with the pseudovirus, the team validated their findings by applying live, infectious SARS-CoV-2 to lung and brain organoids in a Biosafety Level-3 laboratory – a facility specially designed and certified to safely study high-risk microbes.
Now Rana and collaborators are developing SARS-CoV-2 inhibitors and testing how well they work in organoid models derived from people of a variety of racial and ethnic backgrounds that represent California’s diverse population.
They were recently awarded new funding from the California Institute for Regenerative Medicine to support the work.
Pathophysiology of COVID-19
Coronaviruses (CoVs) are non-segmented nucleocapsid-protein enveloped + SS-RNA viruses that are known to cause infection in mammals, including humans and other host species (Pal et al. 2020). Genomic studies have revealed that the CoVs has the most abundant RNA genome of around 30–30 kb in length with a 5′-capping site and 3′poly-A tail region that promotes host genome transcription and translation.
Based on the genomic structure and target host, this virus is generally categorized into four groups, including alpha, beta, gamma, and delta. Among these forms, only alpha and beta forms of viruses known to infect mammals and typically cause upper respiratory infections (Rabi et al. 2020). Seven human coronaviruses (HCoVs) have been identified to date, such as 229E, OC43, HKU1, NL63, SARS-CoV, MERS-CoV, and SARS-CoV-2 was identified as belonging to the beta coronavirus family.
In the host genome, the pathogenic in vivo appearance of SARS-CoVs falls in different stages. The binding of the virus to the host receptors, entry of the virus into the host cells by membrane fusion or endocytosis, the release of virus particles within the host cell followed by replication and biosynthesis of viral proteins, and budding/release of viral particles are sequential steps.
According to the virologists, the pathophysiology and virulence mechanism of human SARS-CoV and SARS-CoV-2 are possibly similar (82% identical) to the work of non-structural proteins (nsps) and structural proteins (sps) (Chan et al. 2020a). To date, the coronavirus genome has recorded six open reading frames (ORFs), these ORFs can serve as a template for the biosynthesis of subgenomic mRNAs. Among these, ORF1a and ORF1b encode 16 nsps that are typically crucial for virus transcription and replication.
Other ORFs encode four major sps: spike (S), membrane (M), envelope (E) and nucleocapsid (N), and other vital proteins (Snijder et al. 2003; Luk et al. 2019). Spike a glycoprotein surface consists of two structural components S1 and S2. Among them, S1 binds to the angiotensin-converting enzyme 2 (ACE2) receptor that highly expressed in the host genome’s lung epithelial cells.
S2 also contains a transmembrane domain, cytoplasmic domain, and fusion protein that facilitates virus fusion in the envelope and host cell membranes to enable viral fusion. Scientists are, therefore looking S2 as a promising target for antiviral drug therapy (Kirchdoerfer et al. 2016; Xu et al. 2020a). Zhou et al. (2020) recently reported that the novel SARS-CoV-2 could also identify ACE-2 receptors in human cells.
Unlike other coronaviruses, the SARS-CoV-2 has a unique furin cleavage site at the S1/S2 boundary (“RPPA” sequence) that makes this virus pathogenic. Other cell surface proteases such as transmembrane protease serine 2 (TMPRSS2) and cathepsin L also facilitate the cleavage of the complex S1/S2-ACE2 and help to activate viral spike proteins for entry into the host genome (Ou et al. 2020; Hoffmann et al. 2020).
In pathogenesis, the envelope proteins play an essential role by facilitating the assembly and release of coronavirus. The N-terminal N-protein binds to the single positive RNA genome and plays a crucial role in the replication and transcription cycle. Currently, two groups of antiviral drugs, such as theophylline and pyrimidone, may inhibit the binding of viral RNA to N-terminal protein (Sarma et al. 2020).
When the virus reaches the body, the host’s immune system it activates a sequence of inflammatory events triggered by antigen-presenting cells (APCs). APCs add foreign antigen to cells containing CD4+-T-helper (TH1) and release interleukin-12 to activate TH1.
Besides, the activated TH1 cells trigger CD8+-T-killer cells which recognise and eliminate infected host cells. TH1 cells also activate B-cells to synthesize viral-specific antibody that is unique to viruses. The infection spreads in the mucosa of the nasal and larynx, then targets lung epithelial cells, which express ACE2 receptors in abundance (Chen et al. 2020a; Bennardo et al. 2020). Activated WBCs activate positive cytokines, including IL-6. However, the IL-6 level elevates in the extreme state of the disease, which raises the aggressiveness of coronavirus.
Molecular mechanism of COVID-19
Coronaviruses are ubiquitous pathogens that cause respiratory or gastrointestinal infections. A recent phylogenetic analysis found that 2015-SARS coronavirus sequence and human 2019-SARS-CoV-2 emerged in the congregation of a bat. In addition, the Bayesian Quick Unconstrained AppRoximation (FUBAR) analysis revealed genomic mutations in the viral S glycoprotein and nucleocapsid protein (Benvenuto et al. 2020).
Compared to the ability of infection with coronaviruses, SARS-CoV-2 has a higher binding affinity to human ACE2 than SARS-CoV, which may explain the frequency of SARS-CoV-2 than SARS-CoV (He et al. 2020). Diverse work has shown that this link between SARS-coronavirus and ACE2 enables a smooth entry into human cells (Struck et al. 2012).
Unlike non-ACE2 expressing cells in the ACE2-expressing cells, the novel SARS-CoV-2 uses ACE2 after binding to infected host cells as an entry receptor. Chemokine ligand 2 (CCL2) is the small chemical molecules that attract immune cells including lymphocytes, basophils, and monocytes.
This also penetrated the various inflammatory reactions and in the S-ACE2 signaling (Hoffmann et al. 2020), and serves as a critical cytokine. Cheung et al. confirmed the increased expression of chemotactic protein CCL2/monocyte 1 in patients with SARS-CoV and hence CCL2 can be a promising therapeutic marker (Cheung et al. 2005).
Heurich et al. found that infected lung epithelial cells facilitate casein kinase II (CK II) which induces phosphorylation of the ACE2 receptor at the Ser-787 site, whereby SARS-CoV recognises and binds to the ACE2 receptor leading to a structural change of the ACE2 receptor.
It also involved the establishment of ACE2 induced downstream signal transduction pathways, including ERK1/2 and AP-1(Heurich et al. 2014). Simmons and colleagues reported that the SARS-CoV accomplishes its entry into host cells via membrane fusion between the virus and lipid bilayer (Simmons et al. 2004).
Another finding from Belouzard et al. revealed that the occurrence of a crucial proteolytic cleavage on the S protein of SARS-CoV at S2 position mediates the membrane fusion involving host and CoV pathogen (Belouzard et al. 2009). Besides CoV, the MERS-CoV required furin activation for membrane fusion (Millet and Whittaker 2014).
In addition to membrane fusion, clathrin-dependent and independent endocytosis has been reported to be mediated by the SARS-CoV entry (Kuba et al. 2010). Once the virus enters the host, eventually, the viral RNA releases into the cytoplasm.
Further, it translates itself into two polyproteins and structural proteins followed by replication of the viral genome (Perlman and Netland 2009). Later, the newly formed envelope glycoproteins enter the cell organelles naming the endoplasmic reticulum (ER) or Golgi complex.
The nucleocapsid contains the mixing of genomic RNA and capsid protein. Then the virus buds are sprout into the ER-Golgi intermediate compartment. Lastly, the vesicles containing the virus particles blend with the plasma membrane to discharge the virus (de Wit et al. 2016). Details related to this is graphically presented in Figs. 1 and and22.
Structure of SARS-CoV 2
(Modified from Shereen et al. 2020)
Life cycle and mechanism of infection of SARS-CoV 2 in host cells. SARS-CoV 2 begins its life cycle when spike protein (S) binds to the cellular receptor ACE2. After receptor binding, the conformation change in the S protein facilitates viral envelope fusion with the cell membrane through the endosomal pathway. Then SARS-CoV 2 releases RNA. Genome RNA gets translated into viral replicase polyproteins (pp1a and 1ab) and further cleaved into small products by viral proteinases. The polymerase generates a series of subgenomic mRNAs by discontinuous transcription and finally translated into appropriate viral proteins. Viral proteins and genome RNA are subsequently assembled into virions in the ER and Golgi body and then transported via vesicles and released out of the cell. ACE2 Angiotensin-converting enzyme 2, ER Endoplasmic reticulum, ERGIC ER–Golgi intermediate compartment
Additionally, it has been reported that the SARS-CoV is involved in the antigen-dependent presentation of MHC I molecules, but MHC II also contributes to its presentation (Liu et al. 2010). Want et al., conducted a polymorphism-based study and found that the human leukocyte antigen (HLA) polymorphisms such as HLA-B4601, HLA-B0703, HLA-DR B1 * 1202, and HLA Cw0801 are associated with the susceptibility of SARS-CoV (Keicho et al. 2009).
In comparison, they found that the HLA-DR0301, HLA-Cw1502, and HLA-A0201 play a vital role in SARS infection and functioning as protective alleles (Wang et al. 2011). It has also been observed that mannose-binding lectin (MBL) gene polymorphisms are associated with antigen presentation and thus linked to the risk of infection with SARS-CoV (Tu et al. 2015).
Research has indicated that acute respiratory distress syndrome (ARDS) is the leading cause of death in COVID-19 and one of the main routes for the cytokine storm associated with ARDS. Nevertheless, lethal systemic inflammatory response leading to elevated levels of pro-inflammatory cytokines such as IFN-α, IFN-α, TNF-α, TGFβ, IL-1β, IL-6, IL-12, IL-18, IL-33 and chemokines such as CCL2, CCL3, CCL5, CXCL8, CXCL9, CXCL10 (Williams and Chambers 2014; Channappanavar and Perlman 2017).
Xu et al. recently reported that the peripheral blood of SARS-CoV-2 patients displayed a substantial reduction in immune defence cells such as CD4+ and CD8+ T cells. In contrast, high concentrations of HLA-DR (CD4 3.47%) and CD38 (CD8 39.4%) were also found in double-positive fractions within the same patients (Xu et al. 2020b).
SARS-CoV viruses are adequate to employ many methods to prevent the survival of the immune system in host cells. Snijder et al. reported that SARS-CoV and MERS-CoV could provoke the assemblage of membrane vesicles that require for Porcine reproductive and respiratory syndrome (PRRS) and avoiding the host detection of their dsRNA (Snijder et al. 2006). These findings are valuable for the effective treatment of COVID-19.
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832422/
More information: Shashi Kant Tiwari et al. Revealing tissue-specific SARS-CoV-2 infection and host responses using human stem cell-derived lung and cerebral organoids, Stem Cell Reports (2021). DOI: 10.1016/j.stemcr.2021.02.005
