Why does pH make a really big difference?
Changes in the pH of a cell, like your endosomes, can encourage or discourage viral fusion.
In this video below, you can hear the guy talks about how hydroxychloroquine changes the pH of your endosomes, which is a necessary step for viral fusion to take place.
"Chloroquine is alkaline, and it can enter into cells like the lysosomes and the organelles, and they can enter into endosomes as well, to actually increase the pH of those organelles, decreasing those organelle function."
UPDATE 12-17-21: As with many videos with compelling evidence that explain Hydroxychloroquine's efficacy, it seems that this one has been made private. It's a shame, as it was very informative, and I could see from a search of the embed code using this part of the address: S6kPUFseTWQ. I could see that many others had referenced it as well. You can see those references, in search results I will paste further down the page. I believe the video was called "Mechanism & Overview of Antiviral Effects" according to the references on this page (the last reference at the bottom). https://www.slideshare.net/MdIrfanUddin2/anti-viral-properties-of-hydroxychloroquinepharmacology
- 9. 9 Treatment:- For treatment of covid-19 there are no specific medication. But for some particular reasons, we can use hydroxychloroquine. Bibliography:- 1.Katzung, Bertram G. 2012. Basic & Clinical Pharmacology. 12th. New york : Lange Medical Publications, 2012. p. 861. 2.Medicine, JJ. 2020. Chloroquine,Hydroxychloroquine & SARS-coV-2(COVID- 19):Mechanism & Overview of Antiviral Effects. you tube. [Online] april 11, 2020. https://www.youtube.com/watch?v=S6kPUFseTWQ.
You should definitely have a look at this article. I highlighted the parts about pH that you may want to see.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461643/
Targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases
Abstract
Emerging viruses such as HIV, dengue, influenza A, SARS coronavirus, Ebola, and other viruses pose a significant threat to human health. Majority of these viruses are responsible for the outbreaks of pathogenic lethal infections. To date, there are no effective therapeutic strategies available for the prophylaxis and treatment of these infections. Chloroquine analogs have been used for decades as the primary and most successful drugs against malaria. Concomitant with the emergence of chloroquine‐resistant Plasmodium strains and a subsequent decrease in the use as antimalarial drugs, other applications of the analogs have been investigated. Since the analogs have interesting biochemical properties, these drugs are found to be effective against a wide variety of viral infections. As antiviral action, the analogs have been shown to inhibit acidification of endosome during the events of replication and infection. Moreover, immunomodulatory effects of analogs have been beneficial to patients with severe inflammatory complications of several viral diseases. Interestingly, one of the successful targeting strategies is the inhibition of HIV replication by the analogs in vitro which are being tested in several clinical trials. This review focuses on the potentialities of chloroquine analogs for the treatment of endosomal low pH dependent emerging viral diseases.
Introduction
Emerging and re‐emerging pathogens such as Ebola and Marburg viruses; dengue and hepatitis C viruses; severe acute respiratory syndrome (SARS) and Middle‐East respiratory syndrome (MERS), coronaviruses; human and avian influenza viruses; Chikungunya virus (CHIKV); human immunodeficiency virus (HIV) and other viruses represent huge challenges to human and veterinary medicine. Researchers, physicians, and healthcare professionals work together to evaluate their pandemic potentials and plan mitigating strategies. For entry into hosts, viruses bind to surface molecules on the plasma membrane of susceptible cells ‐ such as macrophages, monocytes, dendritic cells, endothelial cells, and hepatocytes and lead to them being internalized into vesicles which traffic through the endosomal/lysosomal pathways (Kissing et al. 2014; Shivanna et al. 2014; Bekerman and Einav 2015; Ekins et al. 2015; Kraft et al. 2015; Long et al. 2015). In order to infect susceptible cells, the viruses require endosomal/lysosomal acidification and the acidic pH dependent endosomal proteases cleave the viral glycoprotein segments to cross the replication events (Chandran et al. 2005; Marzi et al. 2012). Without endosomal acidification and cleavage processes, the viral replication and infection are abrogated (Martinson et al. 2014; Shivanna et al. 2014). Therefore, targeting the endosomal/lysosomal acidification and their acidic pH dependent proteases by the therapeutic agents will be highly effective in combating the present world viral epidemic.
Inhibition of viral infection with the increase pH by chloroquine analogs ((Al‐Bari 2015). Steps: 1. Endosome formation; 2. Fusion; 3. posttranslational modification; 4. uncoating virus and CQ, chloroquine.
Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner
Introduction
To achieve successful infection enveloped viruses need to fuse with a host cell membrane to deliver the viral genome into the host cell. Some viruses, such as herpes simplex virus, Sendai virus, and human immunodeficiency virus, appear to be capable of direct fusion at the plasma membrane after initial attachment [1]–[5]. However, the majority of enveloped viruses use endocytosis for uptake and transport prior to fusion. Since endocytic cargo may eventually end up in the destructive environment of the lysosome, environmental cues are crucial to trigger viral fusion at the right stage of trafficking. These triggers, which may include a decrease in pH, changes in redox environment, and proteolytic activity [6]–[8], induce conformational changes in the viral fusion proteins leading to the merger of viral and host membranes. Two well-studied viruses; influenza A virus (IAV) and vesicular stomatitis virus (VSV), are known to undergo fusion upon exposure to low pH [9]–[12]. Other enveloped viruses, such as respiratory syncytial virus (RSV) and Ebola virus, require proteolytic processing of their viral fusion proteins in the endosomal system for fusion to occur [13]–[16].
Coronaviruses (CoVs) are enveloped, plus-strand RNA viruses belonging to the family Coronaviridae in the order Nidovirales. They are capable of infecting a wide variety of mammalian and avian species. In most cases they cause respiratory and/or intestinal tract disease. Human coronaviruses (HCoVs) are known as major causes of the common cold (e.g. HCoV-229E and HCoV-OC43). However, the emergence of new HCoVs of zoonotic origin has shown the potential of CoVs to cause life-threatening disease in humans as was demonstrated during the 2002/2003 SARS-CoV epidemics and more recently for MERS-CoV in the Middle East [17], [18]. The well-studied mouse hepatitis virus (MHV) is often used as a safe model to study CoV infections.
All CoV virions contain a canonical set of four structural proteins. The viral genomic RNA is encapsidated by the nucleocapsid protein (N) to form the helical nucleocapsid, which is surrounded by the lipoprotein envelope, containing membrane glycoprotein (M), the small envelope protein (E), as well as the spike glycoprotein (S) (reviewed in [19]). Trimers of the CoV S protein, a type I membrane protein belonging to the class I fusion proteins, form the peplomers that protrude from the virion surface [20]. The S protein can be divided into two functional subunits. The amino-terminal S1 subunit contains the receptor-binding domain; while the carboxy-terminal S2 subunit contains domains required for fusion, including the fusion peptide (FP), heptad repeat domains (HR) HR1 and HR2, and the transmembrane (TM) domain.
Various entry routes have been described as being used by different CoVs for infection of cells. Clathrin-dependent as well as clathrin- and caveolae-independent entry pathways have been reported for SARS-CoV [21], [22]. Also feline infectious peritonitis virus (FIPV) was suggested to enter via a clathrin- and caveolae-independent endocytic route [23], [24]. For the HCoV-229E a caveolae-dependent endocytic uptake has been suggested [25]. Although the ability of MHV S proteins to cause cell-cell fusion at a neutral pH was initially interpreted as an indication for fusion of virions at the cell surface, more recent studies indicate the requirement for clathrin-mediated endocytosis for entry of MHV [26]–[29]. However, while some studies report that MHV strain A59 is sensitive to lysosomotropic agents that affect endocytosis [26], this is not the case according to others [27].
Proteolytic cleavage of the CoV S proteins appears to be important for the induction of cell-cell fusion and/or virus entry into host cells. Different cleavage sites have been identified for different CoVs, the importance of which seems to differ for cell-cell and virus-cell fusion. Some CoV S proteins, including that of MHV strain A59, are cleaved at the S1/S2 boundary by furin(-like) proteases during transport of the newly assembled virions through the secretory pathway of the producer cell [30]–[33]. Inhibition of this S protein cleavage was shown to inhibit cell-cell fusion, but not to affect entry of MHV strain A59 into host cells [30], [34], [35]. MHV strain 2 contains an S protein that is not cleaved at the S1/S2 boundary. Interestingly, although MHV strains 2 and A59 were both reported to enter via clathrin-mediated endocytosis, entry of MHV 2 but not of MHV A59, was blocked by inhibitors of low-pH activated cathepsin proteases [27], [36]. Inhibitors of cathepsin proteases have also been shown to inhibit entry of SARS-CoV and feline CoVs [23], [37], [38], while treatment of cell-bound virus particles with different proteases was shown to enhance virus entry and/or cell-cell fusion [27], [34], [39]–[45]. For SARS-CoV and infectious bronchitis virus (IBV), it appears that a proteolytic cleavage of the S protein at a more downstream position than the S1/S2 boundary upon receptor binding is of importance for cell entry [40], [43], [46]–[49].
In the present study we performed a detailed investigation of the entry of different CoVs. Using siRNA gene silencing, we found that the prototypic coronavirus MHV strain A59 (further referred to as MHV) requires proteins known to be important for late endosomal maturation and endosome-lysosome fusion for efficient infection of cells. By using recombinant MHVs expressing reporter genes as well as by applying a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV virions to lysosomal compartments and processing of the S protein by lysosomal proteases was required for productive entry to occur. Our results indicate that a cleavage site in the S protein of CoVs immediately upstream of the FP determines the site of fusion. In agreement herewith FIPV, which requires processing by lysosomal proteases, was also shown to depend on trafficking to lysosomes. In contrast, MERS-CoV, which contains a minimal furin-cleavage site consensus sequence in the S protein immediately upstream of the FP, was negatively affected by inhibition of furin, but not of lysosomal proteases.
Mechanism of action of hydroxychloroquine as an antirheumatic drug
If you click on the play button on the youtube video about Influenza (an enveloped virus, like a coronavirus) below, you can go straight to the part where they talk about pH. It changes the hemaglutinnin structure.
hemagglutinin or haemagglutinin (British English; both /ˌhɛməˈɡluːtɪnɪn/) are glycoproteins which cause red blood cells (RBCs) to agglutinate or clump together
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3140544/
Perforin: an important player in immune response
Perforin is a glycoprotein responsible for pore formation in cell membranes of target cells. Perforin is able to polymerize and form a channel in target cell membrane. Many research groups focus on the role of perforin in various diseases, immune response to bacterial and viral infections, immune surveillance and immunopathology. In addition, perforin is involved in the pathogenesis of autoimmune diseases and allogeneic transplant rejection. Natural killer (NK) cells and CD8-positive T-cells are the main source of perforin. However, CD4-positive T-cells are also able to express a low amount of perforin, when classic cytotoxicity is ineffective or disturbed.
Calcium ions might be also responsible for inhibition of perforin polymerization and are able to block transmembrane channels. Such phenomenon is observed in low pH and increased concentration of calcium ions. Other factors inhibiting perforin functions include the activity of protein S. Protein S is a glycoprotein inhibiting lytic activity of complement components (membrane attacking complex). Due to the structural homology between perforin and C9 complement component, protein S is able to inhibit perforin functions. Protein S competitively binds to the perforin binding site on the target cell and therefore inhibits pore formation [11, 12].
Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells
Nature Immunology 12, 770–777(2011)
Abstract
How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.
Perforin pores in the endosomal membrane trigger release of endocytosed granzyme B to the cytosol of target cells
PFN inhibits early endosome acidification
Gzms need to be released into the target cell cytosol to trigger apoptosis23, 24. We hypothesized that Gzms are released when PFN forms endosomal membrane pores. However, early endosomes normally rapidly acidify through the actions of the vacuolar ATPase35, and PFN pore formation is severely compromised at pH<6.5 (36 and data not shown). We therefore predicted that PFN might interfere with endosomal acidification. We first assessed whether PFN-mediated delivery of GzmB and apoptosis induction requires endosomal acidification by treating target cells with Bafilomycin A1, an inhibitor of the vacuolar-type H+-ATPase37, 38 (Fig. 2a) or with ammonium chloride, a weak base that increases endosomal pH by unidirectional diffusion into endosomes39 (Fig. 2b). PFN and GzmB-mediated apoptosis was not altered by pre-treating HeLa cells with these agents that interfere with endosomal acidification. Similarly, pre-incubation of target cells with Bafilomycin A1 did not affect NK cell-mediated killing (Fig. 2c). Moreover, Bafilomycin A1 pre-treatment did not lead to more PFN-induced necrosis (Supplementary Fig. 4a). Therefore, PFN delivery of GzmB does not require endosomal acidification.
https://www.nature.com/articles/ncb0206-107
V-ATPase: a potential pH sensor
Nature Cell Biology 8, 107–109(2006)
An interaction between V-ATPase, a multi-subunit complex responsible for endosome acidification, and ARNO, the GDP/GTP exchange factor for ARF1 and ARF6, indicates that V-ATPase is the long-sought pH-sensor that regulates trafficking in the endocytic pathway.
https://www.sciencedirect.com/science/article/abs/pii/S0161589010004700?via%3Dihub
Human perforin permeabilizing activity, but not binding to lipid membranes, is affected by pH
Abstract
The various steps that perforin (PFN), a critical mediator of innate immune response, undertakes to form a transmembrane pore remains poorly understood. We have used surface plasmon resonance (SPR) to dissect mechanism of pore formation. The membrane association of PFN was calcium dependent irrespective of pH. However, PFN does not permeabilize large or giant unilamellar vesicles (GUV) at pH 5.5 even though the monomers bind to the membranes in the presence of calcium. It was possible to activate adsorbed PFN and to induce membrane permeabilization by simply raising pH to a physiological level (pH 7.4). These results were independently confirmed on GUV and Jurkat cells. The conformational state of PFN at either pH was further assessed with monoclonal antibodies Pf-80 and Pf-344. Pf-344 maps to a linear epitope within region 373–388 of epidermal growth factor (EGF)-like domain while the Pf-80 appears to recognize a conformational epitope. Pf-344 interacts with the EGF-like domain after PFN monomers undergo pore formation, the site recognized by Pf-80 is only accessible at acidic but not neutral pH. Thus, the Pf-80 mAb likely interacts with a region of the monomer that participates in oligomerization prior to insertion of the monomer into the lipid bilayer and thus may have therapeutic utility against PFN-mediated immunopathology.
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