pH-Dependent Entry of Severe Acute Respiratory Syndrome Coronavirus Is Mediated by the Spike Glycoprotein and Enhanced by Dendritic Cell Transfer through DC-SIGN
The severe acute respiratory syndrome coronavirus (SARS-CoV) synthesizes several putative viral envelope proteins, including the spike (S), membrane (M), and small envelope (E) glycoproteins. Although these proteins likely are essential for viral replication, their specific roles in SARS-CoV entry have not been defined. In this report, we show that the SARS-CoV S glycoprotein mediates viral entry through pH-dependent endocytosis. Further, we define its cellular tropism and demonstrate that virus transmission occurs through cell-mediated transfer by dendritic cells. The S glycoprotein was used successfully to pseudotype replication-defective retroviral and lentiviral vectors that readily infected Vero cells as well as primary pulmonary and renal epithelial cells from human, nonhuman primate, and, to a lesser extent, feline species. The tropism of this reporter virus was similar to that of wild-type, replication-competent SARS-CoV, and binding of purified S to susceptible target cells was demonstrated by flow cytometry. Although myeloid dendritic cells were able to interact with S and to bind virus, these cells could not be infected by SARS-CoV. However, these cells were able to transfer the virus to susceptible target cells through a synapse-like structure. Both cell-mediated infection and direct infection were inhibited by anti-S antisera, indicating that strategies directed toward this gene product are likely to confer a therapeutic benefit for antiviral drugs or the development of a SARS vaccine.
The severe acute respiratory syndrome coronavirus (SARS-CoV) is the likely cause of an acute infectious respiratory disorder identified in highly lethal outbreaks during the past year (10, 18, 21, 32, 40). Infection is characterized by acute flu-like symptoms that progress to a severe febrile respiratory illness with significant mortality. Coronaviruses, comprising a genus of the Coronaviridae family, are enveloped positive-strand RNA viruses. In general, coronaviruses cause respiratory and enteric diseases in humans and domestic animals (15, 20). Two previously known human coronaviruses caused only mild upper respiratory infections (15, 20). In contrast, a highly pathogenic, severe respiratory disease is caused by the SARS-CoV, especially in the elderly (44). Coronaviruses can be divided into three serologically distinct groups (15). Phylogenetically, SARS-CoV is not closely related to any of the three groups (26), though it is most similar to the group II coronaviruses (33, 36).
Although the organization of the SARS-CoV genome is related to that of animal coronaviruses, its genetic sequence is unique, and the structure and function of its gene products are not known. At least 14 open reading frames (ORFs) can be identified in its genome (26, 34, 36). Among these, the replicase/transcriptase genes are located in the 5′ portion of the genome. At its 3′ end, the four major structural proteins (S, M, N, and E) are made through different subgenomic RNAs. Based on comparison to animal coronaviruses, three structural gene products are predicted to be present on the viral envelope: the spike (S), membrane (M), and small envelope (E) proteins (20, 26, 34). The structure of the SARS-CoV envelope differs in some respects from that of other enveloped viruses, such as retroviruses and lentiviruses, many of which contain one viral envelope protein.
Envelope or spike proteins from enveloped viruses have been used to pseudotype retroviral and lentiviral vectors for functional and gene transfer studies (29, 35, 43, 45); however, whether coronavirus glycoproteins could pseudotype these viruses was unknown. Here we report that replication-defective retroviral (Moloney murine leukemia virus) and lentiviral (human immunodeficiency virus type 1 [HIV-1]) vectors can be pseudotyped with the SARS-CoV S protein, and the properties of S related to entry have been defined. Using these pseudoviruses, we were able to determine the relative contributions of SARS-CoV envelope proteins to viral entry and fusion and to examine the roles of these different viral envelope gene products with respect to entry, cell specificity, and potential inhibition of viral replication.
In contrast, influenza and Ebola viruses are prototypes for viruses that utilize a pH-dependent endocytotic pathway (43). To determine the pathway utilized by the SARS-CoV, the pH dependence of the SARS-CoV S-pseudotyped lentiviral vector was analyzed. Addition of ammonium chloride, which prevents acidification of the endosome, caused a dose-dependent reduction in viral entry (Fig. (Fig.1B,1B, left) at concentrations similar to those described for other pH-dependent viral glycoproteins (3, 11, 43). This effect was also observed with another inhibitor of endosomal acidification, bafilomycin, also in a dose-dependent fashion (Fig. (Fig.1B,1B, right).
Previous research from Meridian Institute Article :
Possible Relevance to SARS
The World Health Organization has concluded that SARS is produced by a new virulent strain of coronavirus. Specific research on the possible pH dependency of the SARS virus has not yet been done. It is well known that coronavirus infectivity is exquisitely sensitive to pH. For example, the MHV-A59 strain of coronavirus is quite stable at pH 6.0 (acidic) but becomes rapidly and irreversibly inactivated by brief treatment at pH 8.0 (alkaline). Human coronavirus strain 229E is maximally infective at pH 6.0. Infection of cells by murine coronavirus A59 at pH 6.0 (acidic) rather than pH 7.0 (neutral) yields a tenfold increase in the infectivity of the virus.
ChooseLife : If the strain of coronavirus responsible for SARS shares the pH characteristics of these other coronaviruses that are pH-dependent, this could be a valuable clue to effective prevention and treatment strategies for this potential epidemic. Perhaps keeping a balanced or slightly alkaline pH environment for the body’s tissues can provide viral protection or enhanced healing for SARS and common viral agents that cause respiratory infections.
Inter-related to this, is research on MUC5B, which has shown that those of lower pH, are much more prone to having inhibited mucous membrane formation:
“Moreover, we demonstrate that the conformation of these highly entangled linear polymers is sensitive to calcium concentration and changes in pH. In the presence of calcium (Ca2+, 10 mM) at pH 5.0, MUC5B adopted a compact conformation which was lost either upon removal of calcium with EGTA, or by increasing the pH to 7.4. These results suggest a pathway of mucin collapse to enable intracellular packaging and mechanisms driving mucin expansion following secretion. They also point to the importance of the tight control of calcium and pH during different stages of mucin biosynthesis and secretion, and in the generation of correct mucus barrier properties. “
ChooseLife Related Thoughts :
The above shows that there are multiple potential protective methodologies in play, some people may feel a glass of cold water with 1/2 teaspoon of Sodium Bicarbonate every two hours on the first day may be effective (outlined at the bottom of this page), this is one method I would consider myself (Arm and Hammer or Bobs Mill being Aluminium free). Also small Sips of highly Alkaline Milk of Magnesia, every hour, may coat the upper respiratory regions fairly well and rapidly bring up the pH, out of the greater danger zones of lower pH < 6.5 (this is my go to for my kids with sniffles or worse), I would likely do this myself for this situation.
Personally I am going to use this outbreak as a good time to bring my own (and childrens) pH up, using methods as above, plus make some Moreless Alkalising Mineral Mixture, which both Alkalises and significantly raises the Calcium levels in the body but in a complexed form (pre-bonded to Molasses or Honey) which does not hamper the Mucous membrane process outlined above, which as shown above in the scientfic literature is exactly what our bodies need to be ready to either repel, or minimise the effects of such threats.
Moreless Alkalising Mix :
1918 Flu Prevention : Baking Soda:
“The proven value of Bicarbonate of Soda as a therapeutic agent (from a letter to the Church and Dwight Company):
In 1918 and 1919 while fighting the Flu with the U.S. Public Health Service it was brought to my attention that rarely any one who had been thoroughly alkalinized with bicarbonate of soda contracted the disease, and those who did contract it, if alkalinized early, would invariably have mild attacks. I have since that time treated all cases of Cold, Influenza and LaGripe by first giving generous doses of Bicarbonate of Soda, and in many, many instances within 36 hours the symptoms would have entirely abated.
Further, within my own household, before Women’s Clubs and Parent-Teachers’ Association, I have advocated the use of soda as a preventative for ‘Colds’, with the result that now many reports are coming in stating that those who took ‘Soda’ were not affected, while nearly everyone around them had the ‘Flu’.
…An occasional three-day course of the Bicarbonate of Soda increases the alkalinity of the blood, assists elimination and increases the resisting power of the body to all Infectious Diseases…
Whenever taking a bicarbonate solution internally, the soda should be dissolved in cold water. In the event of a threatened attack we recommend the following treatment: During the first day take six doses of half a teaspoon of Bicarbonate of Soda in a glass of cool water, at about two hour intervals.”
General essay about pH and Toxicity:
This should not be construed as Advice, simply reflections of my own thoughts.
Be Well, Rich