Role of Endolysosomes in Severe Acute Respiratory Syndrome Coronavirus-2 Infection and Coronavirus Disease 2019 Pathogenesis: Implications for Potential Treatments

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an enveloped, single-stranded RNA virus. Humans infected with SARS-CoV-2 develop a disease known as coronavirus disease 2019 (COVID-19) with symptoms and consequences including acute respiratory distress syndrome (ARDS), cardiovascular disorders, and death. SARS-CoV-2 appears to infect cells by first binding viral spike proteins with host protein angiotensin-converting enzyme 2 (ACE2) receptors; the virus is endocytosed following priming by transmembrane protease serine 2 (TMPRSS2). The process of virus entry into endosomes and its release from endolysosomes are key features of enveloped viruses. Thus, it is important to focus attention on the role of endolysosomes in SARS-CoV-2 infection. Indeed, coronaviruses are now known to hijack endocytic machinery to enter cells such that they can deliver their genome at replication sites without initiating host detection and immunological responses. Hence, endolysosomes might be good targets for developing therapeutic strategies against coronaviruses. Here, we focus attention on the involvement of endolysosomes in SARS-CoV-2 infection and COVID-19 pathogenesis. Further, we explore endolysosome-based therapeutic strategies to restrict SARS-CoV-2 infection and COVID-19 pathogenesis.


INTRODUCTION
Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) that causes the pandemic disease known as coronavirus disease 2019 (COVID-19) (Contini et al., 2020;Gudbjartsson et al., 2020) is an enveloped virus that contains a large single-stranded RNA genome Huang et al., 2020;Lu et al., 2020;Ren et al., 2020). SARS-CoV-2 belongs to the same beta-coronavirus family as does SARS-CoV that caused the SARS outbreak in China in 2002 (Cherry, 2004) and Middle East respiratory syndrome coronavirus (MERS-CoV) that caused the MERS outbreak in Saudi Arabia in 2012 (Zaki et al., 2012;Li and Du, 2019). Similar to other enveloped coronaviruses, SARS-CoV-2 enters host cells by endocytosis and uses host cell machinery for replication. Spiked glycoproteins on the outer surface of coronaviruses are recognized by and bind to cell surface receptors such as angiotensin-converting enzyme 2 (ACE2) (Huang et al., 2006;Hoffmann et al., 2020b;Shang et al., 2020b) as well as possibly other co-receptors (Raj et al., 2013). Following binding, receptorbound virus is endocytosed whereupon the viral genome is delivered into the cytoplasm; endocytosis mechanisms are pHdependent and -independent (Dimitrov, 2004;White and Whittaker, 2016). Viruses that co-opt pH-independent mechanisms, an example of which is HIV-1, fuse with cell surface membranes and use endocytic pathways to achieve infection (White and Whittaker, 2016). Viruses that enter cells by pH-dependent mechanisms fuse with endosome membranes and use host factors associated with endosomes to enable viral entry into cells (Yang et al., 2004;White and Whittaker, 2016).
Coronaviruses use endolysosome-associated cathepsin B and L proteases under acidic conditions and are considered to be late penetrating viruses (late-entry kinetic mechanism) (Follis et al., 2006;Bosch et al., 2008;Millet and Whittaker, 2014;Coutard et al., 2020;Hoffmann et al., 2020a;Hoffmann et al., 2020b;Pranesh et al., 2020). Following entry, coronaviruses are released into the cytosol from endolysosomes or are targeted for degradation in lysosomes. In addition, some coronaviruses including SARS-CoV-2 can escape endolysosomes and replicate in autophagosome-like structures in the cytosol (Maier and Britton, 2012;Gassen et al., 2019;Gassen et al., 2020). Accordingly, it is important to focus attention on the role of endolysosomes in early stages of interactions between the virus and host cells as well as COVID-19 pathogenesis.

THE ACIDIC NATURE OF ENDOLYSOSOMES
Endosomes are formed from plasma membrane invaginations; a process known as endocytosis. These acidic organelles are categorized further as early, late and recycling endosomes; all with different compositions and hydrogen ion (H + ) content (Luzio et al., 2007;Huotari and Helenius, 2011;Gautreau et al., 2014). Rab4 and Rab5 are important components of early endosomes and function optimally at a pH range of 5.5-6.0. Early endosomes participate in signaling between the extracellular and intracellular environments (Pálfy et al., 2012;Villaseñor et al., 2016); they can recycle to plasma membranes thereby returning endocytosed constituents back to the cell surface (McCaffrey et al., 2001;Grant and Donaldson, 2009;Hsu and Prekeris, 2010). Alternatively, early endosomes can mature and transform into late endosomes (Bright et al., 2005;Luzio et al., 2007); these are differentiated from early endosomes by the expression of Rab7 and have an optimal pH range of 5.0-5.5 (Vanlandingham and Ceresa, 2009;Guerra and Bucci, 2016). Late endosomes can also recycle to plasma membranes (Guerra and Bucci, 2016), can produce multi-vesicular bodies from which extracellular vesicles (exosomes) originate, or can fuse with lysosomes (Piper and Luzio, 2001;Traub, 2010). The fusion of late endosomes with lysosomes generates endolysosomes under more acidic conditions ranging from pH 4.5-5.0 ( Figure 1) (Mullock et al., 1998;Luzio et al., 2007;Luzio et al., 2010). The tight range of H + concentrations in these organelles controls enzymatic activities as well as fusions between autophagolysosomes and lysosomes, and lysosomes and endosomes; pH also affects autophagy and other important cellular processes (Luzio et al., 2007;Luzio et al., 2010;Nakamura and Yoshimori, 2017). Vacuolar-ATPase (v-ATPase) activity largely regulates the acidic nature of endolysosomes and does so by controlling the flux of cations and anions via hydrolysis of free ATP that drives protons against their electrochemical gradient into the lumen of endolysosomes (Mindell, 2012;Halcrow et al., 2019a;Khan et al., 2019a).
Endolysosomes are involved in a wide range of cellular processes including membrane trafficking, catabolism of extracellular and intracellular components, immune responses and antigen presentation, cell secretions, and cell life and death (Eskelinen and Saftig, 2009;Munz, 2012;Repnik et al., 2013;Bright et al., 2016;Truschel et al., 2018;Khan et al., 2019a;Afghah et al., 2020). These acidic organelles have also been implicated in various pathological conditions; structural and functional changes have been reported in various neurodegenerative disorders as well as in cancer (Repnik et al., 2013;Bright et al., 2016;Davis, 2018;Halcrow et al., 2019a; FIGURE 1 | The endolysosome pathway: Extracellular signaling molecules upon binding to cell surface receptors can be engulfed by endocytosis. These endocytosed vesicles can mature and differentiate into early endosomes (pH 5.5-6.0), late endosomes (pH 5.5-5.0), lysosomes (pH 5.0-4.5), and endolysosomes (a fusion process of lysosomes and late endosomes). Various marker substances can differentiate early from late endosomes including Rab4 (early endosomes), and Rab5 and Rab7 (late endosomes). Both early and late endosomes regulate recycling processes that return constituent molecules back to plasma membranes. Late endosomes can produce multi-vesicular bodies, which can fuse with lysosomes or can be released from cells in the form of extracellular vesicles (exosomes). Lysosomes regulate the degradation of extracellular materials in endolysosomes produced by fusions with late endosomes. Lysosomes can also fuse with autophagosomes to form autolysosomes; sites where extracellular and intracellular components are degraded. EL, endolysosomes; ER, endoplasmic reticulum; EE, early endosomes; LE, late-endosomes; MVBs, multi-vesicular bodies; AP, autophagosomes; Rab, ras-related protein 4, 5 and 7). 2019a). Because endolysosome pH regulates structural and functional features of endolysosomes, the involvement of v-ATPase in disease pathogenesis has received much attention and the v-ATPase complex has been targeted for therapeutic reasons. Indeed, inhibitors of v-ATPase and other strategies to keep endolysosomes from de-acidifying has shown benefit against diverse pathological conditions including different types of cancer (Whitton et al., 2018;Halcrow et al., 2019a;Halcrow et al., 2019b), neurological complications (Colacurcio and Nixon, 2016), and infectious diseases (Luzio et al., 2007).
TPCs are present in two forms; TPC1 and TPC2. TPC1s are mainly localized on early endosomes while TPC2s are mainly found on late endosomes/lysosomes (Brailoiu et al., 2009;Pitt et al., 2010;Zakon, 2012). Both subtypes of TPCs can help orchestrate interactions between endolysosomes and such viruses as Ebola (Sakurai et al., 2015), MERS-CoV (Gunaratne et al., 2018), and SARS-CoV-2 (Ou et al., 2020); TPCs regulate the trafficking of virus to late-endosomes/lysosomes following entry into cells. Not surprisingly then, TPC inhibitors can block entry of SARS-CoV-2 into cells and restrict the release of viral RNA into the cytosol ( Figure 2) (Ou et al., 2020). TPCs are also involved in chloroquine-mediated endolysosome leakage and facilitated the release of HIV-1 Tat protein from endolysosomes thus enabling activation of HIV-1 LTR transactivation in the nucleus . Therefore, TPCs appear to promote virus entry and facilitate the release and transport of viral RNA to replication sites by inducing endolysosome permeability and depolarization. NPC1 appears to also play a role in virus entry and infectivity. SARS-CoV enters into early endosomes, traffics to NPC1-positive late endosomes and lysosomes, and accesses highly active cathepsin L protease that triggers fusion mechanisms ( Figure 2) (Shah et al., 2010;Zheng et al., 2018). MERS-CoV, Ebola, and SARS-CoV-2 use similar mechanisms to enter into host cells (Mingo et al., 2015;Zhou et al., 2016;Ballout et al., 2020).
The process of autophagy degrades invading viruses, enhances antigen processing and presentation, and induces adaptive immune responses (Lee and Kim, 2007;Delgado et al., 2009;Richetta and Faure, 2013;Choi et al., 2018a). For example, tolllike receptors are pattern recognition receptors that sense viral RNA and DNA in endolysosomes, induce type I-interferon responses, and following induction of autophagy antiviral immune responses are decreased and invading viruses are degraded (Lee and Kim, 2007;Dalpke and Helm, 2012;Choi et al., 2018a). Autophagy has antiviral effects independent of the degradation process; interferon-γ can suppress replication of norovirus (Hwang et al., 2012;Baldridge et al., 2016;Biering et al., 2017). Additionally, viruses can modulate, escape, and inhibit autophagy at multiple steps to survive and replicate in host cells (Pattingre et al., 2005;Kyei et al., 2009;Chaumorcel et al., 2012).

ENDOLYSOSOME-BASED THERAPEUTIC STRATEGIES TO INHIBIT SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS-2 INFECTION
Because endolysosomes influence coronavirus infections, these organelles might be targeted against SARS-CoV-2 infection and COVID-19 pathogenesis. Given the urgency of need and the tremendous costs involved in developing new drugs, a good approach to therapeutic drug development is the repurposing of drugs known to accumulate in and affect the function of endolysosomes. The diprotic weak base drugs chloroquine (CQ) and hydroxychloroquine (HCQ), that de-acidify endolysosomes, have shown effectiveness in controlling SARS-CoV-2 infection in in vitro studies, however the effectiveness of CQ/HCQ against COVID-19 has not been established for COVID-19 patients Wang et al., 2020;Yao et al., 2020). Endolysosome de-acidification can restrict replication of SARS-CoV-2 because acidic conditions are necessary for SARS-CoV-2 to enter into and be released from host cells. In the context of SARS-CoV-2 infection, CQ and HCQ have been used in combination with azithromycin (Andreani et al., 2020;Carlucci et al., 2020); a weak base antibiotic known to accumulate in endolysosomes (Kong FIGURE 2 | Endolysosome-mediated therapeutic strategies against SARS-CoV-2: SARS-CoV-2 enters cells following interactions between viral spike proteins and cell surface ACE2 receptors. Once endocytosed, spike proteins in endosomes are primed in late endosomes/lysosomes by cathepsin enzymes (B/L); this enhances virus entry. Post-fusion, virus is either released from or degraded in endolysosomes. SARS-CoV-2 once released from endolysosomes, enters the cytosol where it produces a replication complex to generate viral genomic and sub-genomic RNA. Following replication, viral structural proteins get inserted into the ER and move to the ERGIS (endoplasmic reticulum-Golgi intermediate compartment) secretory pathway for virus assembly. Following assembly, virions are transported to vesicles and released from cells by exocytosis. Thus, various stages are targetable for intervention. The first target might be fusion between spike proteins and host ACE2 receptors. A second target might be de-acidification of endolysosomes and blocking the priming of spike proteins by deactivating serine proteases. A third target might be clathrinmediated endocytosis. Fourth, TPC and NPC1 inhibitors could effectively inhibit the virus infection by de-acidifying endolysosomes and blocking the trafficking of cholesterol. Endolysosome acidification may also be a therapeutic target because of its capacity to block the escape of viral RNA to the cytosol and enhance the degradation of the virus in lysosomes. Shown in the figure are multiple compounds and drugs capable of targeting each of these important steps in the virus cycle. SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; TMPRSS2, transmembrane protease serine 2; ACE2, angiotensin-converting enzyme 2; EL, endolysosome; EE, early endosome; LE, late-endosome; TPC1/2, two-pore channel 1 and 2; NPC1, Niemann-Pick disease type C1; viral RNAs, viral ribonucleic acids; DMVs, doublemembrane vesicles; APLS, autophagosome-like structures; ER, endoplasmic reticulum.

EFFECTS OF ENDOLYSOSOME PH ON CORONAVIRUS INFECTION
The coronavirus spike protein is activated under acidic conditions by the endolysosome proteases TMPRSS2 and cathepsins B, L; conditions that promote fusion with host cell membranes and entrance into cells (Hoffmann et al., 2020b). Consistent with this, de-acidification of endolysosomes by CQ, bafilomycinA1, and ammonium chloride have all been shown to deactivate TMPRSS2 and cathepsin B, L as well as suppress coronavirus infection (Figure 2) (Simmons et al., 2004;Vincent et al., 2005;Wang et al., 2008;Shirato et al., 2013;Al-Bari, 2017;Gao et al., 2020;Hoffmann et al., 2020b). Although mentioned earlier, it is important to consider more specifically the involvement of endolysosome-resident ion channels and proteins that regulate endolysosome pH including TPCs, NPC1, and v-ATPase.

CONCLUSION
The high fatality rate of COVID-19 especially among people with pre-existing co-morbities and rapidly increasing case numbers of SARS-CoV-2 infections has created a huge global need for effective therapeutic interventions against COVID-19. Because of the urgent need for therapeutics, re-purposing already approved pharmaceuticals might be the quickest available strategy. SARS-CoV-2 enters into endolysosomes where it can escape detection by immune surveillance and from there can traffic to the cytosol where it can propagate. Endolysosomes generally and endolysosome pH more specifically may represent important targets against SARS-CoV-2 replication and COVID-19 pathogenicity, and several compounds and drugs are available that may be repurposed for immediate testing. Reviewed above were several potential targets to block SARS-CoV-2 infection including endocytosis following binding of the spike protein with its receptor (ACE2), RNA replication and transcription, translation and proteolytic processing of viral proteins, virion assembly, and release from infected cells (Guy et al., 2020;Poduri et al., 2020); all targets involving the endolysosome Frontiers in Pharmacology | www.frontiersin.org October 2020 | Volume 11 | Article 595888 system. In considering approaches against SARS-CoV-2 infection and COVID-19 pathogenesis, the involvement of endolysosomes should be considered.