Below, I present a path to potentially and effectively treat COVID-19 at the pre- exposure and the post-infection stages. A review of the literature indicates that, in the absence of an effective vaccine, no single medication currently has been found to effectively combat SARS-Cov-2. Here, I present a review of the life cycle of COVID-19, including binding and entry mechanisms, translation and replication of the viral genome, and viral assembly and egress, and the current treatment options for COVID-19. Because SAR-CoV and Sars-CoV-2 use multitudinal avenues for entry and egress, coupled with error-prone replication systems that provide avenues to generate quasispecies and escape mutants to monotherapies, I propose here the simultaneous and combinatorial inhibition of these myriad avenues of entry, viral replication and egress, in minimal most effective drug combinations (MMEC) to achieve effective therapeutic synergy to combat COVID19.
In December 2019, a new form of viral disease presenting with severe acute respiratory syndrome similarly to SARS was announced (1). It was quickly determined that the new SARS-like acute respiratory syndrome was indeed caused by a coronavirus, hence it was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and WHO named the disease COVID-19 (2). Analyses showed that SARS-CoV-2 is also a beta-coronavirus, with a single (+)-strand RNA genome of approximately 30 kb and sharing 79% overall identity with SARS-CoV. SARS-CoV-2 joins six previously known coronaviruses to cause disease in humans (3). The disease caused by SARS-CoV-2 is now a global pandemic with over 1.8 million persons infected worldwide to-date. Mortality is age-stratified with mortality increasing with age (4) and COVID-19 patients presenting underlying conditions are at greater risk. Presently, there are no drugs available to treat any of the human coronavirus or zoonotic coronaviruses. Thus, there is an urgent need to repurpose approved drugs to treat the pandemic.
Other reviews have focused on single agents, but since SAR-CoV and Sars-CoV-2 use myriad mechanisms for entry and egress, coupled with error-prone replication systems that effectively generate escape mutant(s) to monotherapies, we propose here the simultaneous and combinatorial inhibition of these myriad avenues of entry, viral replication and egress, via therapeutic synergy, to maximize treatment efficacy and minimize chances of generating resistant progeny. This review focuses on the minimally most effective combination for strategic inhibition of SARS-Cov-2 based on the mechanism of action of the available repurposed and investigational drugs as well as their significant side effects.
The life cycle of coronaviruses (CoVs) has been extensively studied and reviewed (5, 6). Here, I review CoV life cycle focusing on viral and cellular products, processes and events to identify and highlight suitable targets for therapeutic intervention. Of special significance to the COVID-19 pandemic is the multitudinal entry mechanisms coronaviruses use to gain entry into cells. CoVs enter cells through receptor mediated endocytosis (7, 8, 9), direct membrane fusion (10, 11), antibody mediated enhanced viral entry (ADE) (12, 13, 14, 15, 16), macropinocytosis (17, 18, 19) and/or Spike protein-mediated syncytia access (10, 11, 20). Since RNA viral populations comprise a quasispecies, consisting of mutation-generated multiple variants centered around a master sequence (21), it is conceivable that depending on viral variants or cellular target and conditions, SARS-CoV-2 may adopt one or multiple entry mechanisms to gain access into a cell. Each entry mechanism has implications for treatment.
(A) Binding, Entry and Uncoating
(i) Entry Mechanism 1: The most widely reported mechanism for entry is receptor mediated entry using angiotensin converter enzyme 2 (ACE2) for SARS- CoV and SARS-Cov-2 (5, 22, 23, 24, 25) and dipeptidyl peptidase-4 (DPP4) for MERS-CoV, the coronavirus that causes Middle East respiratory syndrome (6, 26, 27). Binding of coronaviral Spike protein to the cellular receptor ACE2 potentiates the Spike protein for cleavage by cathepsin L (9, 7, 10, 28) and subsequent activation by a cell surface transmembrane protease serine 6, TMPRSS (29, 30) for endocytosis. Following endocytosis, the virus fuses and uncoats in Niemann-Pick Compartment1-positive endolysosomes (31) after which SARS-CoV releases its positive (+) strand RNA genome into the cytosol for translation (32).
(ii) Entry mechanism 2: SARS-Cov-2 may also enter cells via direct viral fusion to cellular membranes similar to other CoVs (10, 11), a process mediated by activating cellular proteases, which include the transmembrane serine protease, TMPRSS in the case of SARS-Cov-2 (33). Upon fusion, the virus uncoats and releases its genomic cargo in the cytosol for replication (25, 34, 35, 36, 37, 38).
(iii) Entry mechanism 3: Coronaviruses also exploit antibody-mediated enhanced viral entry (ADE) to enter In ADE, exposure to non-neutralizing antibodies produced against prior non-lethal coronavirus infections provide a gateway for the chronologically ultimate Coronavirus to enter bystander cells (12, 13, 14, 15, 16). ADE is mediated by surface-expressed Fcγ molecules (13), and enables co-option of macrophages and dendritic cells to spread CoVs. ADE contributes to viral pathogenesis (39, 40). ADE mediated entry also traverses via endosomes and are blocked by compounds that raise endosomal pH (41, 42, 43).
(iv) Entry mechanism 4: Similar to other infectious pathogens including protozoa, bacteria, and other viruses, coronaviruses including SARS-CoV exploit macropinocytosis to evade and invade the host immune system (17, 18, 19, 44, 45, 46). Macropinocytosis proceeds via an endocytic pathway (18, 44, 45, 46).
(v) Entry mechanism 5: Spike-protein mediated syncytia formation is another mechanism through which CoVs can gain access to bystander cells. In this mechanism, coronavirus Spike protein, incorporated or unincorporated into virus, serves as a bridging molecule and causes infected and non-infected cells to fuse together into giant cells (47, 48, 49). The virus is thus able to penetrate hitherto naïve cells. Syncytia formation occurs independently of endocytosis.
Binding-Entry-Uncoating Treatment options: Where possible, the ideal treatment regimen to effectively curtail Coronavirus entry will simultaneously prevent and/or (i) inhibit receptor mediated endocytosis, (ii) inhibit direct viral fusion to cellular plasma membrane, (iii) inhibit antibody dependent enhanced viral entry, (iv) inhibit syncytia formation and (v) inhibit macropinocytosis. The unifying cellular machinery that links each of the preceding entry mechanisms above, except for syncytia formation, is the endosome. In each case, the endosome must be acidic for the indicated entry mechanism to be productive. Consequently, and barring syncytia-mediated entry, compounds and/or medications that raise the pH of endosomes from acidic to basic levels are apt to inhibit SARS-Cov-2 entering target cells via the entry mechanisms above.
A number of clinically approved medications that elevate endosomal pH have been proposed as treatment for COVID-19, in particular, chloroquine and hydroxychloroquine (50, 51). While shown to be effective in the reported tests,chloroquine and hydroxychloroquine induce severe allergies and arrhythmia that could conceivably complicate their effectiveness in treating COVID-19 in significant global populations (52, 53, 54, 55).
Azithromycin is a lysosomotropic antibiotic that elevates endosomal pH and inhibits virus entry (56, 57, 58, 59, 60) and therefore provide an alternative to chloroquine and hydroxychloroquine in treating COVID-19, irrespective of a population’s allergic response to the latter. Azithromycin, being also antiinflammatory and attenuator of IL-8 and GM-CSF production in primary bronchial cells, may provide additional protection from immune-pathogenesis and inflammation when used to treat COVID-19 (60).
The lysosomotropic agents referenced above may attenuate or block virus entry at the endosomal level, but viral entry into bystander cells mediated by syncytia formation can still result in virus spread, massive cytopathic effects and disease progression, since syncytia formation occurs independently of the endosomal pathway. Indeed, some CoVs including MERS-CoV, employ endosome independent and protease-inducible syncytia formation to enter cells (46, 47, 48). Additionally, even if viral assembly is blocked pharmacologically, unincorporated coronavirus Spike protein can still enhance syncytia formation (9), a process that provides an additional mechanism for CoVs in general and, SARS-Cov-2 in
particular, to enter bystander cells. This would conceivably allow viral pathogenic and immune evasion factors to still enter bystander cells and cause disease.
Spike-induced syncytia formation could be the basis for the extreme hypoxia observed during infection by SARS-CoV/SARS-Cov-2, as it leads to loss of lung orcardiac function (61, 62). Hypoxia upregulates VEGF and VEGFR expression in blood vessels and leads to fluid extravasation (1, 63, 64). Particular attention should be focused on syncytia formation, as a previously unrecognized significant contributor to coronavirus, and in particular SARS-Cov-2 infection and pathogenesis.
Together with ACE2, the cell surface transmembrane serine protease, TMPRSS, mediates SARS-Cov-2 entry into cells and potentiates spike-mediated syncytia formation. TMPRSS is inhibited by Camostat, a clinically proven protease inhibitor, leading to abrogation of syncytia formation (33).
Summary Treatment Options for SARS-Cov-2 Entry:.
From the foregoing (narrative/discussion/mechanism of COV19 entry), and attentive to the known side effects of chloroquine including allergies and cardiac arrhythmia, we propose either (i) Azithromycin/Camostat or (ii) Chloroquine/Azithromycin/Camostat combination therapies to inhibit or block all documented avenues for SARS-Cov-2 cell entry.
(B) Translation and processing of viral genome
Extensive reviews on coronavirus replication have been published (6, 65, 66, 67,8, 69) and will not be reprised here. Briefly however, following entry, uncoating and release of viral genome into the cytoplasm occurs, and viral polyproteins ORF1a and ORF1b (encoding non-structural proteins) and structural proteins are translated from the positive strand viral RNA genome with ORF1b resulting from -1 ribosomal frameshift (65, 69).Here, we will focus on 3 critical virally-encoded proteins: (i) RNA-dependent RNA polymerase (RdRp), (ii) papain-like protein (PLpro) and (iii) ExoN which are critical for the productive infection and pathogenesis of COVID-19, and can be readily targeted by repurposeable approved medications.
RNA-dependent RNA polymerase (RdRp) is the virally encoded nucleoside polymerase that incorporates nucleosides into nascent genomic and sub-genomic RNA chains (65). RdRp is conserved among Coronaviruses and absolutely required for all coronaviral replication (69, 70). The replicase-transcription machinery consists of many proteins, however, with the core comprising an integrated complex of RdRp, nsp7 and nsp8 together with nsp14 (ExoN) (71). RNA-dependent RNA polymerases are error-prone and mis-incorporate nucleosides in the order of 10-4 per nucleotide copied per round of transcription (72) resulting in viral quasispeciation (73). Thus, during replication, RNA viruses readily generate escape mutants to selective pressures, be the selective pressure vaccine, host immune response, or pharmacological (74, 75). The genetic diversity that ensues within a quasispecies contributes to pathogenesis (76, 77, 78). Viral quasispecies can increase virulence or immune evasion (79), complicate vaccine development (80, 81, 82) and result in failure of monotherapy (83).
RdRp infidelity by itself could potentially be exploited therapeutically using drugs that introduce lethal and/or chain-terminating mutations into the viral genome. However, the presence of the 3’-5’ exonuclease proof-reading activity of nsp14 (ExoN) in the RNA-synthesizing machinery assures that any therapy targeting CoV RNA synthesis must disable both RdRp and ExoN simultaneously, since the latter will excise the incorporated nucleoside(s) that introduce the lethal mutations. A clinically approved drug, namely Ribavirin (84, 85, 86) and Remdesivir (87, 88,89, 90), target RdRp. Ribavirin targets RdRp but not ExoN while Remdesivir targets and inhibits both RdRp and ExoN (86). Targeting, or not targeting ExoN,could have therapeutic significance as detailed below.
(ii) ExoN (nsp14)
Nonstructural protein 14 (ExoN) encodes bifunctional activities and is essential forviral replication (91). The N-terminal domain functions as a proofreading and DeDDh class 3’-5’ exonuclease activity that ensures genomic fidelity via prevention of lethal mutations (91, 92). The C-terminal domain of ExoN functions as a (guanine-N7)-methyltransferase (N7-MTase) for mRNA capping (93). Both enzymatic activities of ExoN are structurally and functionally integral to the viral RNA-synthesizing machinery. Coronaviruses that retain the proofreading 3’-5’ exonuclease activity are resistant to lethal mutagenesis; coronaviruses that lack the proofreading activity are susceptible to lethal mutagenesis (91). It is important to note that not suppressing both RdRp and ExoN activities may result in the emergence of even more genetically stable and robust escape progeny viruses (94, 95, 96). To be effective, therefore, a therapeutic that targets SARS-Cov-2 RNA synthesis must target both the RNA-dependent RNA polymerase (RdRp) and also the proofreading function integrated into the RNA synthesizing machinery in ExoN. As noted above, Remdesivir targets both RdRp and ExoN (89, 90, 91) and fulfills the properties needed in an effective anti-SARS-Cov-2 RNAsynthesizing therapeutic. Summary Treatment Options for SARS-Cov-2 RNA synthesis: From the foregoing, we propose Remdesivir to target both SARS-Cov-2 RNA synthesis and proof-reading functions.
SARS-CoV PLpro is a multifunctional protein embedded within the multidomain nsp3 polyprotein localized to ER membranes (97, 98). Majority of the nsp3 omains, including the catalytic domain of PLpro, is cytosolic where they partake in essential activities relevant for viral replication and/or immune evasion (99, 100). When it is acting as a cytosolic papain-like protease, PLpro catalyses cleavage and release of mature non-structural proteins nsp1, nsp2 and nsp3 from the viral polyprotein (101, 102). Polyprotein processing by PLpro is co-translational. Indeed, ORF 1a and 1ab polyproteins are not detected during infection, presumably because they are processed co- and post-translationally into intermediate and mature proteins by PLpro and the cysteine proteinase, CLpro, in nascent polyproteins (102). Upon processing of ORF1a polyprotein, PLpro, together with nsp4, participates in the assembly of virally-induced cytoplasmic double-membrane vesicles necessary for viral replication.
As a deubiquitinatase (DUB) and a deISGylatase (103, 104, 105, 106, 107, 108), PLpro processes both ‘Lys-48’- and ‘Lys-63’-linked polyubiquitin chains from cellular substrates and thereby antagonizes innate immune induction by interferon gamma. Via its effects in IFNgamma, PLpro blocks the phosphorylation, dimerization and subsequent nuclear translocation of host IRF3. PLpro also prevents host NF-kappa-B signaling (109, 110). Together, these additional activities in PLpro aid coronaviruses to evade the host innate immune responses and contribute to virulence (111, 112).
The importance of PLpro (and nsp1-4) in CoV replication and virulence was demonstrated during the successful adaptation of Murine Hepatitis Virus, a coronavirus, to Syrian baby hamster kidney (BHK) cells. The cross-species adaptation resulted in variants that gained the ability to cross species barriers to be able to efficiently grow in murine, hamster, human, and primate cells (113). Concomitant with the successful adaptation was the appearance of multiple, possibly, species-adapting mutations in pp1a, particularly in nsp3 (PLpro) and sp4 (113). A review of the literature shows that Disulfiram, an approved alcohol aversive drug, may have broad-spectrum anti-CoV PLpro activity since it has been demonstrated to inhibit MERS-CoV PLpro allosterically and SARS-CoV PLpro competitively (114, 115, 116).
Summary Treatment Option for SARS-Cov-2 PLpro:
As a multifunctional, polyprotein-processing, mutably adaptive, immune-evading inducing virulence factor, PLpro presents a significant therapeutic target. Disulfiram is proposed as the therapeutic for PLpro.
(C) Viral Assembly and Egress
Progeny CoVs assemble at the endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and bud and egress from cells in cargo vesicles (116, 117, 118, 119). Specifically, coronaviruses bud exclusively into the ERGIC (119). SARS-CoV egress depends strongly on three structural viral proteins: M, E, and N. It has been established that E localizes primarily to the ER and Golgi complex where it participates in the assembly, budding and intracellular trafficking of infectious virions (120, 121, 122). Assembly of Spike protein into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein (123). Once in the lumen of ER-Golgi intermediate compartment (ERGIC), infectious virions make their way through the host secretory pathway to be exocytosed, ultimately, from the infected cell (124).
The primacy of ERGIC in CoV assembly, budding and egress, together with the large arsenal of approved drugs that target different aspects of ERGIC function make the ER-Golgi compartment a desirable system to investigate for anti-CoV treatment. Strategically, the therapeutic intervention will inhibit a process, event or protein that is absolutely critical for the viral life cycle. Alternatively, the intervention will inhibit each critical step of the viral life-cycle that occurs within ERGIC, namely, assembly, budding and trafficking. Golgi pH, ion and redox homeostasis govern protein sorting, assembly and trafficking (125, 126). These processes that occur at acidic pHs (127, 128, 129) could be inhibited by chloroquine (130). An interrogation of a library of 640 FDA-approved drugs identified chloroquine-related quinacrine, as one of the drugs that disrupted secretory protein transport of diverse cargos (131).
That lysosomotropic agents such as chloroquine and Azithromycin would individually target both CoV entry and uncoating on the one hand and, on the other hand, CoV assembly, trafficking and egress at the ERGIC, has important implications for anti-CoV therapies: the same drugs might be used to block endosome-mediated CoV entry and endolysosomal-mediated CoV egress.
As indicated earlier, however, CoVs could also potentially exit an infected cell via another mechanism, namely, syncytia formation, wherein the spike protein, virusincorporated or not, could bridge adjacent cells to form giant syncytia and allow CoVs access to what would have been naïve bystander cells. Syncytia formation occurs when viruses are able to directly fuse at the cellular surface without the need for endocytosis, since syncytia formation is independent of endocytosis and is pH-independent (47). Thus, treatment with only lysosomotropic drugs to combat CoV entry-egress still leaves the virus with a mechanism to evade the entry/egress targeted therapy. As indicated above, Camostat, the approved pancreatitis drug, inhibits TMPRSS and blocks CoV spike-mediated syncytia formation (33). We propose Camostat would also need to be incorporated in the therapy to inhibit the syncytia bypass mechanism.
Summary Treatment Options for SARS-Cov-2 Assembly and Egress:
From theforegoing, and attentive to Chloroquine allergy, we propose either (i) hloroquine/Azithromycin/Camostat or (ii) Azithromycin/Camostat combination therapies to inhibit or block SARS-Cov-2 assembly and egress.
(D) Summary and Implications for COVID-19 Management
Since RNA viruses readily generate escape mutants to monotherapies, simultaneous administration of combination therapies to target different steps in SARS-CoV-2 lifecycle may help reduce and delay the development of viral resistance, with each drug in the cocktail raising the resistance barrier for the virus. Effective combination antiviral therapies minimize viral replication to undetectable levels and make combinational drug therapy more successful than monotherapies (132).
Multi-target synergy also achieve therapeutic selectivity, with synergistic combinations increasing the number of selective therapies (133). Mindful of reported drug interactions (134, 135), for COVID-19, we propose a combination therapy consisting of either Chloroquine or Azithromycin, with Camostatand Remdesivir as follows:
The Minimum Most Effective Combination (MMEC) of medications needed to contain SARS-Cov-2 viral entry and egress, simultaneously, would appear to be three: Chloroquine or Azithromycin separately, with Camostat combination. With consideration to the adverse reactions to chloroquine, we would recommend the following MMECs:
- For Pre-exposure prophylaxis; COVID19 contact management; Frontline medical
- For infected individuals with mild symptoms, with or without chloroquine allergy and no underlying cardiac condition.
- For infected individuals with mild symptoms with underlying cardiac conditions.
- Chloroquine may be used in combination with Camostat where chloroquine is not contraindicated.
B) Azithromycin/Camostat/ Remdesivir
1) For advanced COVID19 status – where productive infection has occurred, i.e., viral replication has commenced, critical viral proteins have been produced in vivo, shedding is ongoing and progeny virus have commenced spread in situ, we
propose combining the PEP regimen together with Remdesivir (to block further replication).
2). Introduction of Chloroquine and Disulfiram into the regimen must be
closely monitored because of the significant side-effects they evoke.
3). Multidrug tolerability and effectiveness in prophylactic or therapeutic combination will need to be established in randomized control trials to justify their use clinically.
4) Drugs in the respective classes identified herein may be investigated in combination as well.
5). The proposals might be extended to other Coronavirus outbreaks, including SARS and MERs.
Figure 1: SAR-CoV-2 and Coronavirus Life Cycle and Strategic Grids for Therapeutic Synergy Intervention
1) SAR-CoV-2 or CoV virus life cycle is illustrated in Grid A-1 to A-7. Stages in SAR-CoV-2/CoV lifecycle for therapeutic targeting are indicated by X: I) Binding, entry and uncoating (Grid A-1 to A-3); II) Replication (Grid A-4 to A-5); and III) Assembly, trafficking and egress (Grid A-6 to A-7).
2A) Entry mechanisms used by SAR-CoV-2/CoV are illustrated in Grid B-1 to B-3. I) SAR-CoV-2/CoV use five different mechanisms to enter a cell. Four of the entry mechanisms are endocytosis-dependent: i) ACE2 receptor; ii) Membrane fusion; iii) Antibody-dependent Entry (ADE); and iv) Macropynocytosis (Grid B-2). II) Syncytia is an endocytosis-independent mechanism and is mediated by the CoV spike protein and cell surface TMPRSS serine protease (Grid B-3).
2B) COVID19 or CoV’s viral replication and assemby are illustrated in Grid B-4 to B-7. I) Once COVID19 or CoVs achieve productive entry and uncoating, viral replication ensues with viral RNA synthesis and proofreading (Grid B-4) and polyprotein processing (Grid B-5). II) Virus assembly, trafficking and budding proceed in the ER-Golgi intermediate compartment (ERGIC) (Grid B6) for egress at the plasma membrane or through syncytia formation (Grid B-7).
3) Clinically approved drugs recommended for SAR-CoV-2 therapeutic synergy intervention and their mechanisms are listed in Grid C-1 to C-7. I) Drugs against Cellular Targets. i) Endocytosis-dependent entry proceeds via endosomes and can be blocked by lysosomotropic drugs such as Chloroquine or Azithromycin which act by elevating endosomal pH (Grid C-2). The same lysosomotropic drugs Chloroquine or Azithromycin block assembly, trafficking and processing at the ERGIC by raising endo-vesicular pH (Grid C-6). ii) Spike-mediated syncytia formation is dependent on cell surface protease TMPRSS and can be blocked by Camostat via inhibition of TMPRSS activity (Grid C-3). Camostat also blocks viral egress mediated by spike-mediated TMPRSS-dependent syncytia (Grid C-7). II) Drugs against Viral Proteins. Viral encoded RdRp mediates SARS-CoV2/CoV RNA synthesis and its fidelity is provided by ExoN. Remdesivir inhibits the activities of both RdRp and ExoN (Grid C-4). CoV PLpro partakes in viral polyprotein processing and can be inhibited allosterically or competitively by Disulfiram (Grid C-5).
(E) Other Matters
1. Multitudinal entry assures that blocking any one entry mechanism, such as ACE2-mediated entry alone, may reduce infection rate but ultimately be ineffective since the other entry mechanisms could provide bypass mechanisms for SARS-Cov-2 to enter cells.
2. Multitudinal entry also assures that re-infection of an individual can occur
even after initial viral clearance since multiple entry mechanisms accentuate reinfection and superinfection.
3. Antibody-mediated entry has significant implications, namely:
a). Previous exposure to non-lethal, immune-cleared CoVs could predispose an individual to more extensive COVID-19 virulence: ADE exposes Fcgamma-bearing dendritic cells and macrophages to infection and/or simultaneously co-opts them to spread the virus further afield to other tissues (heart, liver, kidneys, intestines and eyes) which will ultimately be attacked by SARS-Cov-2 even if the lungs somehow escaped cytopathy.
b). The combination of ADE with dual egress, particularly syncytia formation, assures rapid devastation of primary SARS-Cov-2 target organs, particularly the lungs, or whichever organs they may be.
c) A patient’s previous history of exposure to nonlethal CoV infections may provide an insight into the severity of disease progression and may help explain age-stratification of COVID-19 mortality. The same could help explain “outliers” of age-stratified COVID-19 mortality.
d). A test that distinguishes exposure to other CoVs from exposure to SARS-CoV-2 may be a predictor of COVID-19 disease severity and possible outcome.
4. The error prone nature of RdRp, even in the presence of functional ExoN, assures there will be polyspeciation with mutations that could confer resistance to any single COVID-19 (mono)therapy. We propose that combinatorial therapy, therefore, as the best treatment option for COVID-19.
- Huang C, Wang Y, Li X, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020 Feb 15;395(10223):497-506. DOI:https://doi.org/10.1016/S0140-6736(20)30. Epub 2020 Jan 24.
- World Health Organization (WHO). Naming the coronavirus disease (COVID-19) and the virus that causes it. Archived from the original on 28 February 2020. Retrieved 28 February 2020 https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/naming-the-coronavirus-disease-(covid-2019)-and-the-virus-that-causes-it
- Zhu N, Zhang D, Wang W, et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med 2020; 382:727-733
- Mahase E, Covid-19: death rate is 0.66% and increases with age, study estimates. BMJ2020; 369 doi: https://doi.org/10.1136/bmj.m1327(Published 01 April 2020)
- de Wit E, van Doremalen N, Falzarano D, et al. SARS and MERS: recent insights into emerging coronaviruses. Nat Rev Microbiol. 2016 Aug;14(8):523-34. doi: 10.1038/nrmicro.2016.81. Epub 2016 Jun 27.
- Fehr AR and Perlman S. Coronaviruses: An Overview of Their Replication and Pathogenesis. Methods Mol Biol. 2015;1282:1-23. doi: 10.1007/978-1-4939-2438-7_1.
- Li W, Moore MJ, Vasilieva N et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature426, 450–454 (2003). DOI: 10.1038/nature02145.
- Burkard C, Verheije MH, Wicht O et al. Coronavirus Cell Entry Occurs through the Endo-Lysosomal Pathway in a Proteolysis-Dependent Manner. PLOS Pathogens 11(2): e1004709. https://doi.org/10.1371/journal.ppat.1004709
- Belouzard S, Millet JK, Licitra BN et al Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein. Viruses.2012 Jun;4(6):1011-33. doi: 10.3390/v4061011. Epub 2012 Jun 20
- Heald-Sargent T, Gallagher T. Ready, Set, Fuse! The Coronavirus Spike Protein and Acquisition of Fusion Competence. Viruses. 2012 Apr; 4(4): 557–580. Published online 2012 Apr 12. doi: 10.3390/v4040557
- Alsaadi EAJ and Jones IM. Membrane binding proteins of coronaviruses. Future Virol. 2019 Apr; 14(4): 275–286. Published online 2019 Apr 29. doi: 10.2217/fvl-2018-0144
- Wan Y, Shang J, Sun S, et al. Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry. J Virol.2020 Feb 14;94(5). pii: e02015-19. doi: 10.1128/JVI.02015-19.
- Jaume M, Yip MS, Cheung CY. et al Anti-Severe Acute Respiratory Syndrome Coronavirus Spike Antibodies Trigger Infection of Human Immune Cells via a pH- and Cysteine Protease-Independent FcgammaR Pathway. J Virol. 2011 Oct; 85(20): 10582–10597. doi: 10.1128/JVI.00671-11
- Lum F-M, Couderc T, Chia B-S. et al, Antibody-mediated enhancement aggravates chikungunya virus infection and disease severity. Sci Rep8, 1860 (2018). https://doi.org/10.1038/s41598-018-20305-4 .
- Yip M-S, Leung NHL, Cheung CY. et al Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus. Virol J 11, 82 (2014). https://doi.org/10.1186/1743-422X-11-82
- Wang SF, Tseng SP, Yen CH et al Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins. Biochem Biophys Res Commun. 2014 Aug 22;451(2):208-14. doi: 10.1016/j.bbrc.2014.07.090. Epub 2014 Jul 26.
- Freeman MC, Peek CT, Becker MM et al Coronaviruses Induce Entry-Independent, Continuous Macropinocytosis. mBio. 2014 Aug 5;5(4):e01340-14. doi: 10.1128/mBio.01340-14.
- 18. Mercer J, Helenius A. Virus entry by macropinocytosis. Nat Cell Biol 11, 510–520 (2009). DOI: 1038/ncb0509-510.
- Falcone S, Cocucci E, Podini P. et al Macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events. Journal of Cell Science2006 119: 4758-4769; https://jcs.biologists.org/content/119/22/4758
- Chan JFW, Lau SKP, To KKW, et al Middle East Respiratory Syndrome Coronavirus: Another Zoonotic Betacoronavirus Causing SARS-Like Disease. Clinical Microbiology Reviews Mar 2015, 28 (2) 465-522; DOI: 10.1128/CMR.00102-14.
- Biebricher CK, Eigen M. What is a quasispecies? Curr Top Microbiol Immunol. 2006;299:1–31. DOI: 10.1007/3-540-26397-7_1.
- Song W, Gui M, Wang X. et al Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLoS Pathog. Published: August 13, 2018 https://doi.org/10.1371/journal.ppat.1007236
- Zhang H, Penninger JM, Li Y. et al.Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target. Intensive Care Med 46, 586–590 (2020). https://doi.org/10.1007/s00134-020-05985-9
- Jia HP, Look DC, Shi L. et al ACE2 Receptor Expression and Severe Acute Respiratory Syndrome Coronavirus Infection Depend on Differentiation of Human Airway Epithelia. Journal of VirologyNov 2005, 79 (23) 14614-14621; DOI:10.1128/JVI.79.23.14614-14621.2005.
- Ou X, Liu Y, Lei X. et al.Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat Commun11, 1620 (2020). https://doi.org/10.1038/s41467-020-15562-9.
- Yang Y, Du L, Liu C. et al Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus. Proc Natl Acad Sci U S A. 2014 Aug 26; 111(34): 12516–12521. Published online 2014 Aug 11. doi: 10.1073/pnas.1405889111
- Walls AC, Xiong X, Park YJ. et al Unexpected receptor functional mimicry elucidates activation of coronavirus fusion. Cell2019 Feb 21; 176(5): 1026–1039.e15. Published online 2019 Jan 31. doi: 10.1016/j.cell.2018.12.028.
- Simmons G, Zmora P, Gierer S, et al Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research. Antiviral Res. 2013 Dec;100(3):605-14. doi: 10.1016/j.antiviral.2013.09.028. Epub 2013 Oct 8
- Bosch BJ, Bartelink W, Rottier PJ. Cathepsin L functionally cleaves the severe acute respiratory syndrome coronavirus class I fusion protein upstream of rather than adjacent to the fusion peptide. J Virol.2008 Sep;82(17):8887-90. doi: 10.1128/JVI.00415-08. Epub 2008 Jun 18.
- Millet JK, Whittaker GR. Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein. Proc Natl Acad Sci USA. 2014 Oct 21;111(42):15214-9. doi: 10.1073/pnas.1407087111. Epub 2014 Oct 6.
- Mingo RM, Simmons JA, Shoemaker CJ, et al Ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to NPC1+ endolysosomes is a rate-defining step. J Virol. 2015 Mar;89(5):2931-43. doi: 10.1128/JVI.03398-14. Epub 2014 Dec 31.
- Yamauchi Y, Greber UF. Principles of Virus Uncoating: Cues and the Snooker Ball. Traffic.2016 Jun;17(6):569-92. doi: 10.1111/tra.12387. Epub 2016 Mar 31.
- Hoffman M, Kleine-Webber H, Schroeder S. et al SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 1–10 April 16, 2020. https://doi.org/10.1016/j.cell.2020.02.052
- Millet JK, Whitaker GR. Host cell proteases: critical determinants of coronavirus tropism and pathogenesis. Virus Res.2015 Apr 16; 202:120-34. doi: 10.1016/j.virusres.2014.11.021. Epub 2014 Nov 22.
- Belouzard S, Chu VC, Whittaker GR. Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites. PNASApril 7, 2009 106 (14) 5871-5876; https://doi.org/10.1073/pnas.0809524106
- Iwata-Yoshikawa N, Okamura T, Shoimizu Y., et al TMPRSS2 Contributes to Virus Spread and Immunopathology in the Airways of Murine Models after Coronavirus Infection. J Virol. 2019 Mar 15; 93(6): e01815-18. Published online 2019 Mar 5. Prepublished online 2019 Jan 9. doi: 10.1128/JVI.01815-18
- 37. Earnest JT, Hantak MP, Li K. et al The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases. PLoS Pathog. 2017 Jul 31;13(7):e1006546. doi: 1371/journal.ppat.1006546. eCollection 2017 Jul.
- Simmons G, Gosalia DN, Rennekamp AJ. et al Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. PNASAugust 16, 2005 102 (33) 11876-11881; https://doi.org/10.1073/pnas.0505577102
- Acevedo OA, Diaz FE, Beals TE. et al Contribution of Fcγ Receptor-Mediated Immunity to the Pathogenesis Caused by the Human Respiratory Syncytial Virus. Front Cell Infect Microbiol. 2019 Mar 29; 9:75. https://doi.org/10.3389/fcimb.2019.00075. eCollection 2019.
- González AE, Lay MK, Jara EL. et al Aberrant T cell immunity triggered by human Respiratory Syncytial Virus and human Metapneumovirus infection. Virulence 2017 Aug 18;8(6):685-704. doi: 10.1080/21505594.2016.1265725. Epub 2016 Dec 2.
- Furuyama W, Marzi A, Carmody AB et al Fcγ-receptor IIa-mediated Src Signaling Pathway Is Essential for the Antibody-Dependent Enhancement of Ebola Virus Infection. PLoS Pathog. 2016 Dec 30;12(12):e1006139. doi: 10.1371/journal.ppat.1006139. eCollection 2016 Dec.
- Carro AC, Piccini LE, Damonte EB. Blockade of dengue virus entry into myeloid cells by endocytic inhibitors in the presence or absence of antibodies PLoS Negl Trop Dis. 2018 Aug; 12(8): e0006685. Published online 2018 Aug 9. doi: 10.1371/journal.pntd.0006685
- He X, Sun X, Wang J. et al Antibody-enhanced, Fc gamma receptor-mediated endocytosis of Clostridium difficile toxin A. Infect Immun. 2009 Jun; 77(6): 2294–2303. Published online 2009 Mar 23. doi: 10.1128/IAI.01577-08
- 44. Lim JP, Gleeson PA (2011). Macropinocytosis: an endocytic pathway for internalising large gulps. Immunol Cell Biol 2011 Nov;89(8):836-43. doi: 1038/icb.2011.20. Epub 2011 Mar 22
- Mercer J, Helenius A. Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells. Science 2008 Apr 25;320(5875):531-5. doi: 10.1126/science.1155164.
- Lin HP, Singla B, Ghoshal P. et al Identification of novel macropinocytosis inhibitors using a rational screen of Food and Drug Administration‐approved drugs. Br J Pharmacol. 2018 Sep; 175(18):3640-3655. doi: 10.1111/bph.14429. Epub 2018 Aug 1.
- Qian Z, Dominguez SR, Holmes KV. Role of the Spike Glycoprotein of Human Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Virus Entry and Syncytia Formation. PLoS ONE 8 (10): e76469. doi: 10.1371/journal.pone.0076469.
- Shirato K, Kawase M, Matsuyama S. Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2. J Virol. 87: 12552–12561. Published online November 1, 2013. https://doi.org/10.1128/JVI.01890-13
- Mesel-Lemoine M, Millet J, Vidalain P-O. et al A Human Coronavirus Responsible for the Common Cold Massively Kills Dendritic Cells but Not Monocytes. J Virol. 2012 Jul; 86(14): 7577–7587. doi: 10.1128/JVI.00269-12
- Gao J, Tian Z, Yang X. Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies. BioScience Trends 2020 Mar 16; 14(1):72-73. doi: 10.5582/bst.2020.01047. Epub 2020 Feb 19.
- Colson P, Rolain JM, Lagier JC, et al Chloroquine and hydroxychloroquine as available weapons to fight COVID-19. Int. J. Antimicrobial Agents 2020 Mar 4:105932. doi: 10.1016/j.ijantimicag.2020.105932.
- Bussaratid V, Walsh D, Wilairatana P, et al Frequency of pruritus in plasmodium vivax malaria patients treated with chloroquine in Thailand. Trop Doct. 2000 Oct;30(4):211-4. DOI: 10.1177/004947550003000410
- Osifo NG. Chloroquine Pharmacokinetics in tissue of pyrogen treated rats and implications for chloroquine related pruritus. Res Commun Chem Pathol Pharmacol. 1980, 30:419–430.
- George AO. (1996) Chloroquine pruritus: a possible fixed cutaneous reaction. Int J Dermatol. 1996 May;35(5):323-4. DOI: 10.1111/j.1365-4362.1996.tb03631.x
- Chatre C, Roubille F, Vernhet H. et al. Cardiac Complications Attributed to Chloroquine and Hydroxychloroquine: A Systematic Review of the Literature. Drug Saf. 2018 Oct;41(10):919-931. doi: 10.1007/s40264-018-0689-4.
- Tyteca D, Van Der Smissen P, Mettlen M. et al Azithromycin, a Lysosomotropic Antibiotic, Has Distinct Effects on Fluid-Phase and Receptor-Mediated Endocytosis, but Does Not Impair Phagocytosis in J774 Macrophages. Exp Cell Res. 2002 Nov 15;281(1):86-100. DOI: 10.1006/excr.2002.5613
- Tyteca D, Van Der Smissen P, Van Bambeke F. Azithromycin, a lysosomotropic antibiotic, impairs fluid-phase pinocytosis in cultured fibroblasts. European Journal of Cell Biology 80, 466 – 478 (2001, July) https://doi.org/10.1078/0171-9335-00180
- Tyteca D, Schanck A, Dufrêne YF et al The Macrolide Antibiotic Azithromycin Interacts with Lipids and Affects Membrane Organization and Fluidity: Studies on Langmuir-Blodgett Monolayers, Liposomes and J774 Macrophages., J. Membr. Biol.2003 Apr 1;192(3):203-15. DOI: 10.1007/s00232-002-1076-7
- Tran DH, Sugamata R, Hirose T. et al.Azithromycin, a 15-membered macrolide antibiotic, inhibits influenza A(H1N1)pdm09 virus infection by interfering with virus internalization process. J Antibiot72, 759–768 (2019). https://doi.org/10.1038/s41429-019-0204-x
- Melchjorsen J. Learning from the Messengers: Innate Sensing of Viruses and Cytokine Regulation of Immunity—Clues for Treatments and Vaccines. Viruses. 2013 Jan 31;5(2):470-527. doi: 10.3390/v5020470.
- Zhang J, Zhou L, Yang Y et al Therapeutic and triage strategies for 2019 novel coronavirus disease in fever clinics. Lancet 8, Issue 3, PE11-E12, March 01, 2020. DOI:https://doi.org/10.1016/S2213-2600(20)30071-0.
- Nieto-Torres JL, DeDiego ML, Verdiá-Báguena C. et al Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis. PLoS Pathog 10(5): e1004077. https://doi.org/10.1371/journal.ppat.1004077.
- Vinores SA, Wang Y, Vinores MA. et al Blood-retinal barrier breakdown in experimental coronavirus retinopathy: association with viral antigen, inflammation, and VEGF in sensitive and resistant strains. J. Neuroimmunol. 2001 Oct 1; 119(2): 175–182. Published online 2001 Sep 27. doi: 10.1016/S0165-5728(01)00374-5
- Vinores SA, Xiao W, Zimmerma R. et al Upregulation of vascular endothelial growth factor (VEGF) in the retinas of transgenic mice overexpressing interleukin-1ß (IL-1ß) in the lens and mice undergoing retinal degeneration. Histol. Histopathol. 18(3):797-810 · August 2003. DOI: 10.14670/HH-18.797
- Sola I, Almazán F, Zúñiga S. et al Continuous and Discontinuous RNA Synthesis in Coronaviruses. Annu Rev Virol. 2015 Nov;2(1):265-88. doi: 10.1146/annurev-virology-100114-055218.
- Fung TS, Liu DX. Human Coronavirus: Host-Pathogen Interaction. Annu. Rev. Microbiol. 2019 Sep 8;73:529-557. doi: 10.1146/annurev-micro-020518-115759. Epub 2019 Jun 21.
- Jin Y, Yang H, Ji W. et al Virology, Epidemiology, Pathogenesis, and Control of COVID-19. Viruses 2020 Mar 27;12(4). pii: E372. doi: 10.3390/v12040372.
- Xu J, Zhao S, Teng T. et al Systematic Comparison of Two Animal-to-Human Transmitted Human Coronaviruses: SARS-CoV-2 and SARS-CoV. Viruses 2020 Feb 22;12(2). pii: E244. doi: 10.3390/v12020244.
- Koonin EV, Dolja VV. Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem. Mol. Biol. 1993;28(5):375-430. DOI: 10.3109/10409239309078440
- Ziebuhr J. The Coronavirus Replicase. Curr Top Microbiol Immunol. 2005; 287:57-94. DOI:10.1007/3-540-26765-4_3.
- Ganeshpurkar A, Gutti G. Singh SK. RNA-Dependent RNA Polymerases and Their Emerging Roles in Antiviral Therapy in Gupta SP (Ed.) Viral Polymerases, Pages 1-42, Academic Press, Published Date: 1st November 2018: DOI: 10.3390/v7082829.
- Mandary MB, Masomian M, Poh CL. Impact of RNA Virus Evolution on Quasispecies Formation and Virulence. Int J Mol Sci. 2019 Sep; 20(18): 4657. doi: 10.3390/ijms20184657.
- Presloid JB, Novella IS. RNA Viruses and RNAi: Quasispecies Implications for Viral Escape. Viruses 2015 Jun; 7(6): 3226–3240. doi: 10.3390/v7062768
- Gerrish PJ, Garcia-Lerma JG. Mutation rate and the efficacy of antimicrobial drug treatment. Lancet Infect Dis. 2003;3:28–32. https://doi.org/10.1016/S1473-3099(03)00485-7.
- Vignuzzi M, Stone JK, Arnold JJ, Cameron CE, Andino R. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature. 2006;439:344–348.DOI: 10.1038/nature04388
- Vignuzzi M, Wendt E, Andino R. Engineering attenuated virus vaccines by controlling replication fidelity. Nat Med. 2008;14:154–161. DOI: 10.1038/nm1726
- Yao H, Lu X, Chen Q et al Patient-derived mutations impact pathogenicity of SARS-CoV-2. medRxiv preprint posted April 23, 2020. doi: https://doi.org/10.1101/2020.04.14.20060160
- Lu X, Rowe L.A, Frace M et al. Spike gene deletion quasispecies in serum of patient with acute MERS-CoV infection. J. Med. Virol. 2017, 89, 542–545. DOI: 10.1002/jmv.24652.
- Baric RS, Fu K, Chen W, Yount B. High recombination and mutation rates in mouse hepatitis virus suggest that coronaviruses may be potentially important emerging viruses. Adv Exp Med Biol. 1995;380:571–576. DOI: 10.1007/978-1-4615-1899-0_91.
- Baric RS, Fu K, Schaad MC, Stohlman SA. Establishing a genetic recombination map for murine coronavirus strain A59 complementation groups. Virology. 1990;177:646–656. DOI: 10.1016/0042-6822(90)90530-5.
- Makino S, Keck JG, Stohlman SA, Lai MM. High-frequency RNA recombination of murine coronaviruses.J Virol.1986 57:729–737.
- Domingo, E.; Sheldon, J.; Perales, C. Viral quasispecies evolution. Microbiol. Mol. Biol. Rev. 2012, 76, 159–216. DOI: 10.1128/MMBR.05023-11.
- Cameron CE, Castro C. The mechanism of action of ribavirin: lethal mutagenesis of RNA virus genomes mediated by the viral RNA-dependent RNA polymerase. Curr Opin Infect Dis. 2001 Dec; 14(6):757-64. DOI: 10.1097/00001432-200112000-00015.
- Crotty S, Maag D, Arnold JJ. et al. The broad-spectrum antiviral ribonucleoside Ribavirin is an RNA virus mutagen. Nat Med. 2000 Dec;6(12):1375-9. DOI: 10.1038/82191
- Te HS, Randall G, Jensen DM. Mechanism of action of Ribavirin in the treatment of chronic hepatitis C. Gastroenterol Hepatol. (N.Y) 2007 March 3 (3):218 –225.
- de Wit E, Feldmann F, Cronin J. et al Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection. PNASMarch 24, 2020 117 (12) 6771-6776; first published February 13, 2020 https://doi.org/10.1073/pnas.1922083117.
- Agostini ML, Andres EL, Sims AC et al. Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease. mBio 9:e00221-18. https://doi.org/10.1128/mBio.00221-18
- Wang M, Cao R, Zhang L. et al Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res30, 269–271 (2020). https://doi.org/10.1038/s41422-020-0282-0
- Sheahan TP, Sims AC, Leist SR et al Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV. Nat Commun11, 222 (2020). https://doi.org/10.1038/s41467-019-13940-6
- Minskaia E, Hertzig T, Gorbalenya AE. et al. Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis. PNAS 2006 103 (13) 5108-5113; https://doi.org/10.1073/pnas.0508200103
- Smith EC, Blanc H. Vignuzzi M. et al Coronaviruses Lacking Exoribonuclease Activity Are Susceptible to Lethal Mutagenesis: Evidence for Proofreading and Potential Therapeutics PLoS Pathog 9(8): e1003565. doi: 10.1371/journal.ppat.1003565
- Ma Y, Wu L, Shaw N. et al Structural basis and functional analysis of the SARS coronavirus nsp14–nsp10 complex. PNASJuly 28, 2015 112 (30) 9436-9441; first published July 9, 2015 https://doi.org/10.1073/pnas.1508686112
- Pfeiffer JK, Kirkegaard K. A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity. Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7289-94. Epub 2003 May 16. DOI: 10.1073/pnas.1232294100.
- Khatun A, Shabir N, Yoon K-J. et al Effects of ribavirin on the replication and genetic stability of porcine reproductive and respiratory syndrome virus. BMC Vet Res. 2015; 11: 21. Published online 2015 Feb 7. doi: 10.1186/s12917-015-0330-z
- Denison MR, Graham RL, Donaldson EF. et al Coronaviruses: An RNA proofreading machine regulates replication fidelity and diversity. RNA Biology 2011 Mar-Apr;8(2):270-9. Epub 2011 Mar 1. DOI: 10.4161/rna.8.2.15013.
- Lei J, Kusov Y, Hilgenfeld R. Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein. Antiviral Res. 2018 Jan;149:58-74. DOI:
- Báez-Santos YM, St John SE, Mesecar AD. The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds. Antiviral Res. 2015 Mar;115:21-38. DOI:10.1016/j.antiviral.2014.12.015. Epub 2014 Dec 29
- Hagemeijer MC, Verheije MH, Ulasli M. et al Dynamics of coronavirus replication-transcription complexes. J. Virol. 2010, 84:2134–2149. doi:10.1128/JVI.01716-09
- Oostra M, Hagemeijer MC, van Gent M. et al Topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning. J. Virol. 2008 Dec; 82(24):12392-405. doi: 10.1128/JVI.01219-08. Epub 2008 Oct 8
- Han YS, Chang GG, Juo CG. et al Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, puriﬁc ation, characterization, and inhibition. Biochemistry 2005 44, 30, 10349–10359. https://doi.org/10.1021/bi0504761.
- Harcourt BH, Jukneliene D, Kanjanahaluethai A. et al Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity. J. Virol. Dec. 2004, p. 13600–13612. DOI: 10.1128/JVI.78.24.13600-13612.2004.
- Bekes M, Rut W, Kasperkiewicz P. et al SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme. Biochem J. 2015 Jun 1; 468(2): 215–226. doi: 10.1042/BJ20141170
- Wang D, Fang L, Li P et al The Leader Proteinase of Foot-and-Mouth Disease Virus Negatively Regulates the Type I Interferon Pathway by Acting as a Viral Deubiquitinase. J. Virology Mar 2011, 85 (8) 3758-3766; DOI:10.1128/JVI.02589-10.
- Matthews K, Schäfer A, Pham A. et al. The SARS coronavirus papain like protease can inhibit IRF3 at a post activation step that requires deubiquitination activity. Virol J.11, 209 (2014). DOI https://doi.org/10.1186/s12985-014-0209-9
- Mielech AM, Kilianski A, Baez-Santos YM. et al MERS-CoV papain-like protease has deISGylating and deubiquitinating activities. Virology 2014 Feb;450-451:64-70. doi: 10.1016/j.virol.2013.11.040. Epub 2013 Dec 22.
- Bailey-Elkin BA, Knaap RCM, Kikkert M. et al Structure and Function of Viral Deubiquitinating Enzymes. J Mol Biol. 2017 Nov 10;429(22):3441-3470. doi: 10.1016/j.jmb.2017.06.010. Epub 2017 Jun 16.
- Ma-Lauer Y, Carbajo-Lozoya, Hein MY. et al p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1. PNASAugust 30, 2016 113 (35) E5192-E5201; first published August 12, 2016. https://doi.org/10.1073/pnas.1603435113
- Lindner HA, Lytvyn V, Qi H et al. Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease. Arch Biochem Biophys. 2007 Oct 1;466(1):8-14. Epub 2007 Jul 14. DOI:10.1016/j.abb.2007.07.006
- Frieman M, Ratia K, Johnston RE, et al Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease Ubiquitin-Like Domain and Catalytic Domain Regulate Antagonism of IRF3 and NF-B Signaling. J. Virol. July 2009, p. 6689–6705. doi: 10.1128/JVI.02220-08.
- Li SW, Wang CY, Jou YJ et al SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway. Sci Rep. 2016 May 13; 6:25754. doi: 10.1038/srep25754
- Chen X, Yang X, Zheng Y. et al SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex. Protein Cell. 2014 May; 5(5):369-81. doi: 10.1007/s13238-014-0026-3. Epub 2014 Mar 14.
- Baric RS, Sullivan E, Hensley L, et al Persistent Infection Promotes Cross-Species Transmissibility of Mouse Hepatitis Virus. J. Virol. Jan. 1999, 73, 638–649.
- Lin MH, Moses DC, Hsieh CH et al Disulfiram can inhibit MERS and SARS coronavirus papain-like proteases via different modes. Antiviral Res. 2018 Feb;150:155-163. DOI 10.1016/j.antiviral.2017.12.015. Epub 2017 Dec 28.
- Venkatagopalan P, Daskalova SM, Lopez LA, et al Coronavirus envelope (E) protein remains at the site of assembly. Virology 2015 Apr; 478: 75–85. Published online 2015 Feb 27. doi: 10.1016/j.virol.2015.02.005.
- Krijnse-Locker J, Ericsson M, Rottier PJ et al Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step. J. Cell Biol. 1994 Jan; 124(1-2):55-70. DOI:10.1083/jcb.124.1.55.
- Tooze J, Tooze SA. Infection of AtT20 murine pituitary tumour cells by mouse hepatitis virus strain A59: virus budding is restricted to the Golgi region. Eur. J. Cell Biol.1985 May;37:203-12.
- Klumperman J, Locker JK, Meijer A et al Coronavirus M Proteins Accumulate in the Golgi Complex beyond the Site of Virion Budding. J. Virol. 1994 Oct;68(10):6523-34.
- Siu YL, Teoh KT, Lo J. et al The M, E, and N Structural Proteins of the Severe Acute Respiratory Syndrome Coronavirus Are Required for Efficient Assembly, Trafficking, and Release of Virus-Like Particles. J. Virol. Nov. 2008, 82:11318–11330. doi:10.1128/JVI.01052-08
- Westerbeck JW, Machamer CE A Coronavirus E Protein Is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway J Virol. 2015 Sep 15; 89(18): 9313–9323. doi: 10.1128/JVI.01237-15
- Godeke GJ, de Haan CAM, Rossen JWA. et al Assembly of Spikes into Coronavirus Particles Is Mediated by the Carboxy-Terminal Domain of the Spike Protein. J. Virol Feb. 2000, 74:1566-1571. DOI: 10.1128/jvi.74.3.1566-1571.2000.
- Ruch TR, Machamer CE The Coronavirus E Protein: Assembly and Beyond. Viruses 2012 Mar; 4(3): 363–382. doi: 10.3390/v4030363
- Paroutis P, Touret N, Grinstein S. The pH of the secretory pathway: measurement, determinants, and regulation. Physiology 19, 207–215. doi: 10.1152/physiol.00005.2004.
- Kellokumpu S. Golgi pH, Ion and Redox Homeostasis: How Much Do They Really Matter? Front. Cell Dev. Biol., 11 June 2019 https://doi.org/10.3389/fcell.2019.00093
- Appenzeller-Herzog C, Roche AC, Nufer O. et al pH-induced conversion of the transport lectin ERGIC-53 triggers glycoprotein release. J. Biol. Chem. 279, No. 13, Issue of March 26, pp. 12943–12950, 2004. doi: 10.1074/jbc.M313245200
- Appenzeller-Herzog C, Hauri HP. The ER-Golgi intermediate compartment (ERGIC): in search of its identity and function. J. Cell. Sci. 2006 119: 2173-2183; doi: 10.1242/jcs.03019
- Cancino J, Capalbo A, Di Campli A. et al Control systems of membrane transport at the interface between the endoplasmic reticulum and the Golgi. Dev. Cell 2014 Aug 11;30(3):280-94. doi: 10.1016/j.devcel.2014.06.018.
- Palokangas H, Ying M, Vaananen K. et al Retrograde transport from the pre-Golgi intermediate compartment and the Golgi complex is affected by the vacuolar H+-ATPase inhibitor Bafilomycin A1. Mol. Biol. Cell 1998 Dec; 9(12): 3561–3578. doi 10.1091/mbc.9.12.3561
- Hassinen A, Pujol FM, Kokkonen N et al. Functional organization of Golgi N- and O-glycosylation pathways involves pH-dependent complex formation that is impaired in cancer cells. J. Biol. Chem. 2011 286, 38329–38340. doi: 10.1074/jbc.M111.277681
- Thorens B, Vassalli P. Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion. Nature321, 618–620 (1986). https://doi.org/10.1038/321618a0
- Menendez-Arias L. Molecular basis of human immunodeficiency virus type 1 drug resistance: Overview and recent developments. Antiviral Res. 2013;98:93–120. Doi: 10.1016/j.antiviral.2013.01.007)
- Lehár J, Krueger AS, Avery W et al Synergistic drug combinations tend to improve therapeutically relevant selectivity. Nat. Biotechnol. 2009 Jul;27(7):659-66. doi: 10.1038/nbt.1549. Epub 2009 Jul 5. doi 10.1038/nbt.1549
- Zithromax (azithromycin tablets). Package Insert: New York, NY: Pfizer labs; 2013.
- Antabuse (disulfiram tablet). Package Insert. Tulsa, Ok: Physicians Total Care, Inc.; 2012.
ACKNOWLEDGMENTS: We express sincere thanks to Dr. Charlene F. Barroga for discussions and, to her and Dr. Tony Hunter (Salk Institute) for critical review of the manuscript.
CONFLICT OF INTEREST DECLARATION: The author declares no conflict of interest.
ACCREDITATION STATUS: Thrivus Institute for Biomedical Science and Technology is undergoing program review by the National Accreditation Board of Ghana
NONMEDICAL USE STATEMENT: The therapeutic agents identified herein, and combinations thereof, are for research purposes only. They are not prescriptions and are not to be taken as self-medication for the treatment or cure for any disease or sickness. Contact your health-care provider for medical advice and care.