HMR/D
Viral Hijacking
Viral Hijacking of Human Cellular Metabolic Machinery leads to HMR/D
The virulent outcome of a SARS-CoV-2 infection depends on viral hijack of cellular metabolic machinery through sequential steps of viral attachment, invasion, RNA replication and propagation. The initial step of COVID-19 pathogenesis involves that the viral spike (S)-protein interacts and anchors to susceptible host tissue through hijacking of specific CSRs (i.e., ACE2 and NRP1). Subsequent cellular invasion (internalization) of the pathogen takes place as the viral S-protein/host receptor complex is primed by furin cleavage at two sites: S1/S2 and S2'. This proteolytic cleavage induces conformational changes that favor S-protein recognition by host cell membrane proteases (i.e., TMPRSS2, CTSL). The cleavage of S2' triggers fusion between viral envelope and cell membrane to facilitate SARS-CoV-2 entry into the host cell. Structure-functional properties of the above FIVE specific viral-hijacked host cellular factors, their ultimate pathophysiological consequence(s) contributes to the onset of viral-induced HMR/D in acute COVID-19 and in chronic virus-free PASC disease state.
The pathophysiology of COVID-19 and PASC is described right from the initiation of SARS-CoV-2 infection by primordial hijacking of host cellular metabolic machinery, subsequent progression of viral-induced HMR/D in a susceptible host, inflicting the patient through a severe tri-phasic symptomatic clinical onset of COVID-19 (lasting 3 to 4 weeks), and ultimate transition of a survivor into PASC or long-COVID, a virus-free state, lingering with earlier and/or new onset clinical manifestations from pre-acquired HMR/D (lasting for weeks to months).
Viral Hijack of Human Angiotensin-Converting Enzyme-2 (hACE2)
SARS-CoV-2 binds to the human angiotensin-converting enzyme-2 (hACE2) as a potential CSR for binding to specific cellular tissue sites in the body. Human cells that express ACE2 are at potential targets for SARS-CoV-2 infection; however, other cellular factors such as human proteases that prime the viral S-protein are also critical for next sequential steps of the viral infection process. ACE2 exists either in free soluble form (sACE2) or in bound form immobilized on cell membranes (mACE2) of intestinal, renal, testicular, gall bladder, pulmonary, and cardiovascular epithelia. Both soluble and membrane bound ACE2 proteins are critical for regulation of blood pressure in the body.
ACE2 is an essential counter-regulatory carboxypeptidase of the hormonal RAAS, a vital regulator of blood volume, systemic vascular resistance; thereby, the cardio-vascular and circulatory homeostasis. ACE2 is expressed in most human tissues and cell types as a type I integral membrane protein solubilized by the action of a disintegrin and metallopeptidase (ADAM)-17. Therefore, hijacking hACE2 may shift RAAS homeostasis and compromise cardiovascular function.
Consequences of ACE2 hijack: The viral hijack of hACE2 could compromise patient’s health for extended periods of time, especially among COVID-19 and PASC with comorbidities (i.e., CVD, T2DM, brain and kidney dysfunctions). Human ACE2 gene is strongly associated with diabetes; therefore, any loss of hACE2 decreases the insulin secretion and impairs the glucose tolerance. This partly explains the higher morbidity and mortality rates among COVID-19 patients with preexisting diabetes. Human ACE2 expression is high in tubular epithelia (from kidney), and the viral hijack of this enzyme could alter sodium transport, affect blood volume/ pressure and lead to AKI. Viral hijack of hACE2 in the BBB axis could impair autonomic nervous system (ANS) and dysregulate blood pressure and respiration. Viral hijack of hACE2 in the brain stem may increase sympathetic nerve drive, alter baroreflex, and exacerbate hypertension. Loss of hACE2 in the vasculature may lead to endothelial dysfunction, inflammation and aggravate atherosclerosis and diabetes. A loss of pulmonary hACE2 may cause hypertension, respiratory distress, and fibrosis post-viral infection. Thus, SARS-CoV-2- mediated cell surface reduction of hACE2 receptors could trigger widespread inflammatory sequelae observed in COVID-19, which may linger through PASC for extended period.
Viral Hijack of Human Neuropilin (hNRP)-1
Neuropilin-1 (NRP1), is another prominent CSR that facilitates entry of SARS-CoV-2 into the CNS through olfactory epithelium in the nasal cavity. Also, NRP1 (but not ACE2) serves as the principal CSR to mediate SARS-CoV-2 infection of astrocytes in the brain tissue. Viral infection of astrocytes resembles reactive astrogliosis with elevated type-I IFN production, increased inflammation, and down-regulation of transporters for water, ions, choline, and neurotransmitters. These events lead to dysfunction and death of uninfected bystander neurons that could inflict severe symptoms of neuro-COVID including anosmia, ageusia, headache, delirium, acute psychosis, seizures, and stroke. NRP1 avidly binds to furin-cleaved S1 fragment of viral S-protein and potentiates cellular entry of SARS-CoV-2. NRP1 is abundantly expressed in the respiratory and olfactory epithelia, with highest localization in endothelial and epithelial cells
NRPs are single-pass transmembrane, non-tyrosine kinase surface glycoproteins, expressed by endothelial, immune, and vascular smooth muscle cells and are regulators of numerous signaling pathways within the vasculature. NRP1 plays a key role in VEGF-dependent angiogenesis (i.e., new blood vessel formation). NRP2 is important for migration, antigen presentation, phagocytosis and cell-cell contact in the immune system. Both NRPs play a multifunctional role in several physiological pathways including nervous and vascular development, as well as in immunity and tumorigenesis.
Consequences of NRP1 hijack: The SARS-CoV-2 S-protein could hijack NRP1 signaling and directly affect the VEGF-alpha-mediated pain. This may rise the possibility that pain, an early symptom of COVID-19, could be diminished by SARS-CoV- 2 S-protein interaction with NRP1. Such ‘silencing’ of pain through subversion of VEGF-A/NRP-1 signaling could be an underlying factor for disease transmission through SARS-CoV-2 infected asymptomatic or minimally symptomatic individuals. As a key player in VEGF-induced vascular permeability and angiogenesis, the viral hijack of NRP1 could impede capillary formation, tissue repair and organ function in the body. Furthermore, loss of NRP1 could compromise integrity of vascular endothelium, a selective barrier that regulates macromolecular exchange between the blood and tissues. The ensuing vascular hyper-permeability of plasma molecules and leukocytes may lead to acute tissue edema and inflammation. SARS-CoV-2 infection of astrocytes via NRP1 hijack could disrupt normal neuron function, induce neuronal cell death leading to abnormal clinical manifestations in the central nervous system (CNS). NRP1 deficiency in visceral smooth muscle cells could negatively impact GI contractility and motility. Also, the viral hijack-mediated suppression of epithelial NRP1 could weaken the gut barrier function. The NRP-1-mediated SARS‐CoV‐2 entry into bone marrow‐derived macrophages (BMM) could impede osteoclast differentiation and affect calcium/phosphorus metabolism in COVID‐19 patients. Accordingly, viral hijacking of NRP1 could exert chronic disorders of bone metabolism such as osteoporosis or osteopetrosis in PASC patients.
Viral Hijack of Human Transmembrane Protease, Serine 2 (TMPRSS2)
Human transmembrane protease, serine 2 (TMPRSS2) has been identified as a key host cell factor that determines the route of viral entry for SARS-CoV-2 infection and the pathogenic spectrum of COVID-19. Specifically, TMPRSS2 processes the SARS-CoV-2 S-protein and enables the viral entry into host cells within <10 min in a pH-independent manner. In TMPRSS2-defecient cellular tissue sites, SARS-CoV-2 is endocytosed into lysosomes, and an alternative route of viral entry into cytosol is achieved in about 40-60 min of post-infection via acid-activated CTSL protease. TMPRSS2 cleaves the viral S protein at multiple sites, including the canonical S1/S2 cleavage site. TMPRSS2 expression is high in the human prostate gland under androgenic hormone regulation. As an apical surface serine protease, TMPRSS2 regulates epithelial sodium homeostasis in the prostate gland and plays a vital role in male reproduction. In normal prostate, TMPRSS2 is involved with proteolytic cascades to activate prostate-specific antigen (PSA) in the seminal fluid (like fibrinolytic blood coagulation). TMPRSS2 expression is high in ciliated cells and type I alveolar epithelia (AT1), which is known to upregulate with ageing. This may explain the relative protection of infants and children from severe respiratory illnesses. Recently, both TMPRSS2 and ACE2 were detected in human corneal epithelia, which suggests that ocular surface is a potential route of cellular entry for SARS-CoV-2. TMPRSS2 plays a vital role in several biological functions such as digestion, salt-water balance, iron metabolism, tissue remodeling, blood coagulation, auditory nerve development, and fertility. It is also required for many pathobiological pathways that involve inflammatory responses, tumor cell invasion, apoptosis, and pain. TMPRSS2 modulates dendritic cells and regulates cytokine release, a major clinical manifestation in COVID-19 pathology.
Consequences of TMPRSS2 hijack: As a membrane-anchored enzyme of the host cellular machinery, TMPRSS2 activates precursor molecules in pericellular milieu to establish metabolic homoeostasis. Viral hijacking of this protease could dysregulate lipid metabolism, adipose tissue phenotype, and thermogenesis via direct growth factor activation or indirect hormonal mechanisms. TMPRSS2 expression is high in human prostate gland and this enzyme regulates sperm function in the seminal prostasome. Increasing evidence suggests that COVID-19 could inflict detrimental effects on spermatogenesis and hormonal regulation in male patients. Abnormal serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) levels were also reported, which suggests a dysfunctional hypothalamic-pituitary-gonadal (HPG) axis in COVID-19 patients. These male reproductive health issues may aggravate and continue to persist among PASC patients. Dysregulation of TMPRSS2 expression and/or catalytic activity may cause both tumor formation and metastasis, contributing to the etiology of several cancer types, especially prostate cancer. TMPRSS2 is reportedly associated with tumor cell expression, different complex(es) formation, and pathways, as well as transcriptional mis-regulation in prostate cancer among COVID-19 and PASC patients.
Viral Hijack of Human Furin
SARS-CoV-2 contains a unique insertion of AAs (PRRAR685↓) at the S1/S2 boundary of its S-protein, which is clearly absent in SARS-CoV and other related hCoVs. Interestingly, this PRRAR insertion generates a ‘furin cleavage site’ on S-protein at the S1/S2 multi-basic region, considered as a potential ‘gain of function’ for the viral pathogen. Furin-mediated priming of viral S protein at S1/S2 (PRRAR685↓) [the underlined basic AAs refer to critical residues needed for the furin recognition] is a key determinant in the pathogenesis of COVID-19.
Furin is a member of the proprotein convertase (PC) family of enzymes, known to process latent precursor proteins into their biologically active state. Humans encode nine members of PCSK family (from 1–9), where the PCSK3 represents ‘furin’. Most viral envelope glycoproteins, like bacterial exotoxins, also require proteolytic cleavage to mediate entry into host cells. Accordingly, viral pathogens hijack cellular endo-proteases, such as furin/PCSK3 that prime polybasic cleavage sites and provide a critical access for tissue tropism and viral spread in an infected host.
Furin/PCSK3 cleaves basic AA motifs; therefore, also termed as PACE (Paired basic AA Cleaving Enzyme). It cleaves diverse types of protein precursors in the secretory pathway at downstream of basic-AA target sequence (canonically, R-X(R/K)-R). Furin most likely cleaves and activates more than 150 mammalian, viral and bacterial substrates. These include viral envelope glycoproteins and bacterial toxins, as well as cellular factors that promote tumorigenesis. Substrates for furin cleavage possess a specific 20-residue recognition sequence motif, which include: pro-parathyroid hormone (PTH), transforming growth factor (TGF)-β1 precursor, pro-albumin, membrane type-1 matrix metalloproteinase (MMP), β-subunit of pro-nerve growth factor, and von Willebrand factor. Several bacterial and viral pathogens exploit human furin enzymes for proteolytic activation of their own virulent factors during the infectious process. In bacterial pathogens, furin-activated toxins may promote tissue invasion, increase transmission rates, or suppress cellular immune responses.
Consequences of furin hijack: Furin is essential for cardiovascular function; therefore, viral hijack of this proprotein convertase enzyme could alter lipid metabolism, affect blood pressure regulation and vascular remodeling in COVID-19 patients. Furin also regulates the transcription factor NKX2-5, which is essential for both normal heart development and cardio-vascular function. Viral hijack of furin may increase the susceptibility of tissue epithelia for ferroptosis-like cell injury. Ferroptosis is a type of cell death linked to altered iron metabolism, glutathione (GSH) depletion, GSH peroxidase 4 (GPX4) inactivation, and increased OxS, which is a prominent clinical feature of COVID-19. Furin is an integral part of specialized cellular machinery that regulates membrane type-1 matrix metalloproteinases (MT1-MMPs) and its deterrence by SARS-CoV-2 could compromise proteolytic events on host cell surface and affect nutrient transport for cellular metabolism. Loss of furin due to viral pre-utilization may result in increased growth, invasiveness and cytokine production in the bone-joint synovium and aggravate rheumatoid arthritis. Dysregulation of furin may lead to neurodegenerative and neuropsychiatric disorders known to persist during PASC. Co-existence of furin with ACE2/RAAS in the female and male reproductive systems pose a potential risk on human fertility in COVID-19 patients. Viral hijacking of host cellular factors in the female reproductive tract may disrupt ovarian function and thereby the oocyte quality. Furin also regulates placenta-specific-1-mediated oocyte meiosis and fertilization. Furin plays a major role in oocyte development beyond the early secondary follicle stage and any virus-mediated loss of its proteolytic activity could lead to follicular dysplasia and female infertility.
Viral Hijack of Human Cathepsin L (hCTSL)
In COVID-19 pathogenesis, the cleavage and priming of S-protein is critical for viral entry into host cells. SARS-CoV-2 uses different routes of host cell entry: i) membrane fusion (with cells that express both ACE2 + serine proteases i.e., TMPRSS2 and furin) and/or ii) receptor-mediated endocytosis (to target cells that express only ACE2 + cysteine proteases i.e., CTSL). Interestingly, CTSL alone could activate membrane fusion of viral S-protein and facilitate host cellular entry of the virus. Therefore, viral hijack of CTSL provides an alternative entry mechanism (via endo/lysosomal route) for SARS-CoV-2 invasion of host cells that lack TMPRSS2 enzyme. The activated/ primed S protein then mediates fusion of viral envelope with host cell membrane and releases the SARS-CoV genome into the cytoplasm for subsequent viral expression/ replication. The CTSL expression is up-regulated during chronic inflammation and is involved in the degradation of extracellular matrix, an important process for SARS-CoV-2 to enter host cells. The circulating level of CTSL is elevated after SARS-CoV-2 infection and correlates with disease course/ severity. The SARS-CoV-2 Omicron variant, which dominated the pandemic, prefers the endo/ lysosomal cysteine protease CSTL over TMPRSS2 for host cell entry.
Cathepsin L (CTSL) a member of the lysosomal cysteine protease family, containing lysosomal targeting motifs with maximal catalytic activity at acidic pH (3.0–6.5) in the presence of thiol (-SH) compounds. The enzyme activity and stability of CTSL at physiological pH strictly depends on ionic strength of the milieu. The endopeptidase activity of CTSL generates active enzymes, receptors, transcription factors, and biologically active peptides by limited proteolysis. Limited endosomal proteolytic activity of CTSL is critical for diverse cellular processes such as normal lysosome mediated protein turnover, antigen/ proprotein processing, regulation of signaling molecules, extracellular matrix remodeling, and apoptosis. CTSL plays a vital role in functional development of immune system, in skeletal physiology including bone collagen degradation/ resorption and thyroid hormone release. Human cysteine proteases are involved in pathogenesis of several diseases including rheumatoid arthritis, osteoporosis, tumor metastasis, renal diseases, diabetes, periodontal diseases, and viral infections.
Consequences of CTSL hijack: In the cytosol and nuclei, CTSL is critical for several biological pathways, including cell division. CTSL regulates oocyte maturation and early embryonic divisions. Any interference with CTSL activity could impair female competence for embryonic development. The viral hijack of CTSL may reduce female competence for embryonic development (also a major cause of infertility) and may account for early miscarriages during COVID-19 pandemic. Furthermore, several lysosomal enzymes are involved in female ovulation, especially CTSL, in ovarian follicle growth and maturation. The CTSL-mediated activation of progesterone receptors in granulosa degrades extracellular matrix in the follicular tissue during female ovulation. Viral hijack of CTSL could severely compromise female reproductive health.
In secretory vesicles, CTSL generates active neuropeptides including enkephalin, β-endorphin, and dynorphin, as well as proopiomelanocortin (POMC)-derived peptide hormones adreno-corticotropin hormone (ACTH), and melanocyte stimulating hormone (MSH), are essential for cell-cell communication in the nervous and endocrine systems. CTSL also converts proenkephalin into the active enkephalin, an opioid peptide neurotransmitter that mediates pain relief. Inhibition of CTSL alleviates microglia-mediated neuroinflammatory responses from caspase-8 and NF-κB pathways. A majority of COVID-19 and PASC patients show neurological symptoms, including headache, impairment of memory, seizures, and encephalopathy, as well as anatomical abnormalities, such as changes in brain morphology. The viral hijack of CTSL could be a contributing factor for these cognitive dysfunctions in SARS-CoV-2 infection.
In addition to cardiac injury (i.e., myocardial infarction, fulminant myocarditis, venous thromboembolism, arrhythmias, and cardiomyopathies), the vasculature is severely affected in COVID-19 and PASC, directly by the SARS-CoV-2 hijack of host factors, and indirectly from the systemic inflammatory cytokine storm. Obesity, diabetes, and other related metabolic syndrome pose a higher risk of severe COVID-19 infection with poor prognosis. CTSL is known to degrade fibronectin, insulin receptor (IR), and insulin-like growth factor-1 receptor (IGF-1R), essential molecules for adipogenesis and glucose metabolism. Inhibition of CTSL results in reduced body weight, low serum insulin levels, and increased glucose tolerance. New onset T2DM, arterial hypertension and dyslipidemia are possible sequelae of COVID-19.