Author: Robert Lee (---.vnnyca.adelphia.net)
Date: 05-04-03 21:21
SARS Coronavirus Appears to be an FcgammaR Agent, Causing an Hyperimmune Response via a CD13 pathway - Implication for Therapeutic Interventions
Author: Robert E. Lee, M.S., M.S.W., L.C.S.W., Ph.D.
Revision Requested: 22 April 2003
Date Published: 4 May 2003
Abstract
Severe Acute Respiratory Syndrome (SARS) is an emerging disease which began to appear in the Guangdong region of China during November 2002. Understanding the infectivity of the SARS coronavirus, the putative agent of SARS disease, is imperative. This study investigated the molecular structure of the SARS coronavirus E2-spike protein, cell-surface docking proteins, and associated hyperimmune cytokines likely to be involved in this FcgammaR agent. The molecular analysis suggests that SARS coronavirus E2-spike protein is
(a) likely most closely related to the Avian Infectious Bronchitis Virus E2-spike protein rather than typical coronavirus E2-spike proteins,
(b) likely to bind to CD13 (aminopeptidase N), and trigger release of interferon-alpha, interleukin-6, and TNF-a in the hyperimmune cascade following infection (which release results in mortality in some individuals), and
(c) likely to infect MHC Class II HLA-DR, -DP, and -DQ allele displaying individuals at a higher rate and with more serious disease consequences than other individuals.
Introduction
Severe Acute Respiratory Syndrome (SARS) is an emerging disease that began to appear in the Guangdong region of China during November 2002. The disease has continued to spread through unknown vectors to the population in China and has, through international travel, spread to, in May, 2003, some 27 countries on six continents. Infecting, as of early May, 2003, some 6,000 persons worldwide with a mortality rate of approximately 6.5%, nearly 400 persons have died. Reports, e.g., a worldwide health alert, issued by the World Health Organization indicate SARS is an extremely worrisome emerging disease.
SARS is caused by a never-before-seen coronavirus called, now, the SARS Urbani coronavirus, in honor of the WHO physician killed by the disease. About April 20, 2003, the SARS Urbani coronavirus's nucleotides were sequenced by teams in Canada and the United States and published on the Internet. As the SARS disease has been described as a "mystery disease" having unknown effects on individuals and unknown vectors in its transmission, analysis of the SARS Urbani genome is important. With a mortality rate which appears to be increasing and secondary transmission of the virus documented in several countries, and a potentiality of a very serious global pandemic if the disease is not contained, it is absolutely critical that the SARS Urbani coronavirus genome be understood as completely as possible.
Understanding the infectivity of SARS Urbani coronavirus hinges on the E2 spike protein on the coronavirus's surface and human cell-surface receptors of a human cell being attacked by the SARS Urbani agent. A visual analogy of the virus E2-spike-protein and the human cell-surface-receptors is offered in considering a sea mine, laid in an ocean port to explode when in contact with a ship's hull. The spikes on the sea mine are the SARS Urbani coronavirus E2-spike proteins; the human cell-surface receptors are the hull of the ship. Determining how the virus E2-spikes interact with the human cell-surface receptors is absolutely essential in determining infectivity of the virus and may offer information that may be useful in minimizing infection and/or further understanding of the potentialities of the disease as it spreads among the world's populations.
The SARS Urbani E2-spike Protein
The accepted method of analyzing viruses is to employ a multiple sequence alignment and develop a resulting phylogeny. A multiple sequence alignment compares, side-by-side, a number of different viruses. This process is done so to detect where the compared viruses are alike and where they are different. In this way, virus components can be determined. In a phylogeny, the degree of relationship of the viruses that have been aligned is established and a picture of the relationship is the result. In this way, a multiple sequence alignment and resulting phylogeny allows the determination of what components are in viruses and how the components relate to one another. In the effort to understand the SARS Urbani coronavirus this technique was used to understand the E2-spike protein as it is the "spike on the sea mine" that will determine how the virus attaches itself to cells during infection.
E2-spike Protein Multiple Sequence Alignment
It is reasonable to compare SARS coronavirus E2-spike protein to other coronavirus's E2-spike proteins. This was done and the results indicated the SARS Urbani E2-spike proteins to be generally structurally like other coronavirus's E2-spike proteins but with notable differences as well. The multiple sequence alignment is seen in Figure 1 (click here)
Examination of the multiple sequence alignment (clust-W with standard settings) reveals the SARS coronavirus E2-spike protein is, indeed, a coronavirus-like E2-spike protein. However, the multiple sequence alignment also reveals that the SARS coronavirus-like E2-spike protein is not sufficiently similar to any other known coronavirus E2-spike protein to conclude that SARS-E2 is one of the known E2 proteins. The phylogeny provides more information concerning the SARS-E2 protein close relatives.
E2-spike Protein Phylogeny
Technical data concerning the distances in the phylogram are seen in Figure 2 (click here). The phylogram, itself, is seen in Figure 3 (click here). The phylogram reveals the closest relatives of the SARS E2-spike protein are the following E2-spike proteins:
1: S37664
V kappa Ox1=immunoglobulin V kappa-J kappa segment, V kappa Ox1=immunoglobulin V
kappa-J kappa segment {recombination site} [mice, BALB/c germ-line, Genomic, 282 nt, segment 2 of 3]
gi|250215|bbs|106807|gb|S37664.1|S37663S2[250215]
2: CAA41065
peplomeric polyprotein precursor [Avian infectious bronchitis virus]
gi|58987|emb|CAA41065.1|[58987]
3: AAK18745
spike glycoprotein, S2 subunit [Avian infectious bronchitis virus]
gi|13378217|gb|AAK18745.1|[13378217]
4: AAF08315
spike protein precursor [Avian infectious bronchitis virus]
gi|6425133|gb|AAF08315.1|[6425133]
5: CAA60684
spike protein [Avian infectious bronchitis virus]
gi|1321872|emb|CAA60684.1|[1321872]
6: AAK28146
spike glycoprotein precursor S2 subunit [Avian infectious bronchitis virus]
gi|13492241|gb|AAK28146.1|AF335555_1[13492241]
7: AAK20887
spike glycoprotein S2 subunit [Avian infectious bronchitis virus]
gi|13377887|gb|AAK20887.1|AF334685_1[13377887]The phylogram suggests that the SARS E2-spike protein and the Avian infectious bronchitis virus E2-spike protein share a common ancestor. The phylogram also suggests that SARS E2-spike protein is not Avian infectious bronchitis virus E2-spike protein. The SARS E2 and AIBV-E2 lineage appears to have shared a common ancestor with the virus that gave rise to the Murine Hepatitis Virus (as indicated in the phylogram in Figure 3). The Murine Hepatitis Virus E2-spike proteins in the lineage include:
1: AAB30950
S=viral surface peplomer {monoclonal antibody resistant} [murine hepatitis virus
MHV, Wb 1, MAR 11F/1, Peptide Mutant, 1235 aa]
gi|547041|gb|AAB30950.1||bbm|339396|bbs|147901[547041]
2: Q02385
E2 glycoprotein precursor (Spike glycoprotein) (Peplomer protein) [Contains:
Spike protein S1 (90B); Spike protein S2 (90A)]
gi|465382|sp|Q02385|VGL2_CVMJC[465382]
3: P22432
E2 glycoprotein precursor (Spike glycoprotein) (Peplomer protein) [Contains:
Spike protein S1 (90B); Spike protein S2 (90A)]
gi|138176|sp|P22432|VGL2_CVM4[138176]
4: P11225
E2 glycoprotein precursor (Spike glycoprotein) (Peplomer protein) [Contains:
Spike protein S1 (90B); Spike protein S2 (90A)]
gi|138178|sp|P11225|VGL2_CVMJH[138178]
5: AAA87062
S glycoprotein [murine hepatitis virus]
gi|561670|gb|AAA87062.1|[561670]Strangely, the SARS E2-spike protein aligns more closely to the Avian infectious bronchitis virus and the murine hepatitis virus E2-spike proteins than it does to other known human coronavirus E2-spike proteins, e.g., Human coronavirus OC-43 or Human coronavirus 229. It would be expected that the SARS E2-spike protein would be like other human coronavirus E2-spike protein but the data suggest this is not the case. In the analogy of the "ocean mines and ships" presented earlier, it would appear that the SARS E2-spike protein is not like any other mine that has attacked ships (humans) in the past. It is reasonable to conclude that the SARS E2-spike protein, though while different from any other known human coronavirus E2-spike protein, must share some commonality with these other human coronavirus's E2-spike protein else SARS would not be able to attack humans. It is therefore a reasonable investigation to discern how SARS E2-spike protein and other human coronavirus E2-spike protein are similar.
Figure 4 (click here) shows the multiple sequence alignment of SARS E2-spike proteins to other known human coronavirus E2-spike proteins; note in the alignment where the amino acids stack vertically. There are several sequences of amino acids in the human coronaviruses E2-spike proteins that match SARS E2-spike proteins. In the analogy of "mines and ships" the vertically aligned amino acids which are in-common between SARS E2-spike protein and the Human coronaviruses (OC43 and 229E) suggest how SARS E2-spike protein, though more closely resembling Avian infectious bronchitis and murine hepatitis virus than other human cornavirus's E2-spike proteins, may manage to target human cells.
There are four (4) sequences of amino acids that appear very similar between SARS E2-spike protein and the other human coronaviruses. These sequences of amino acids are:
gi|29836496|ref|NP_828851.1| QIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLT----- 921
gi|30023954|gb|AAP13567.1| QIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLT----- 921
gi|476485|pir||JQ2168 -VPFYLNVQYRINGLGVTMDVLSQNQKLIANAFNNALYAIQEGFD----- 1023
gi|306156|gb|AAA03055.1| -VPFYLNVQYRINGLGVTMDVLSQNQKLIANAFNNALYAIQEGFD----- 1023
gi|549302|sp|P36334|VGL2_CVHOC -VPFYLNVQYRINGLGVTMDVLSQNQKLIANAFNNALYAIQEGFD----- 1023
gi|138175|sp|P15423|VGL2_CVH22 -IPFSLAIQARLNYVALQTDVLQENQKILAASFNKAMTNIVDAFTGVNDA 815
gi|1869755|emb|CAA71056.1| -IPFSLAIQARLNYVALQTDVLQENQKILAASFNKAMTNIVDAFTGVNDA 718
:** : : *:* :.: :** :*** :* **:*: * :.:
gi|29836496|ref|NP_828851.1| ---------TTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDI 962
gi|30023954|gb|AAP13567.1| ---------TTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDI 962
gi|476485|pir||JQ2168 ---------ATNSALVKIQAVVNANAEALNNLLQQLSNRFGAISASLQEI 1064
gi|306156|gb|AAA03055.1| ---------ATNSALVKIQAVVNANAEALNNLLQQLSNRFGAISASLQEI 1064
gi|549302|sp|P36334|VGL2_CVHOC ---------ATNSALVKIQAVVNANAEALNNLLQQLSNRFGAISASLQEI 1064
gi|138175|sp|P15423|VGL2_CVH22 ITQTSQALQTVATALNKIQDVVNQQGNSLNHLTSQLRQNFQAISSSIQAI 865
gi|1869755|emb|CAA71056.1| ITQTSQALQTVATALNKIQDVVNQQGNSLNHLTSQLRQNFQAISSSIQAI 768
:. :** *:* *** :.::** * .** ..* ***: :: *
gi|29836496|ref|NP_828851.1| LSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMS 1012
gi|30023954|gb|AAP13567.1| LSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMS 1012
gi|476485|pir||JQ2168 LSRLDALEAEAQIDRLINGRLTALNAYVSQQLSDSTLVKFSAAQAMEKVN 1114
gi|306156|gb|AAA03055.1| LSRLDALEAEAQIDRLINGRLTALNAYVSQQLSDSTLVKFSAAQAMEKVN 1114
gi|549302|sp|P36334|VGL2_CVHOC LSRLDALEAEAQIDRLINGRLTALNAYVSQQLSDSTLVKFSAAQAMEKVN 1114
gi|138175|sp|P15423|VGL2_CVH22 YDRLDTIQADQQVDRLITGRLAALNVFVSHTLTKYTEVRASRQLAQQKVN 915
gi|1869755|emb|CAA71056.1| YDRLDTIQADQQVDRLITGRLAALNVFVSHTLTKYTEVRASRQLAQQKVN 818
.*** ::*: *:****.*** :*:.:*:: * : :: * * *:.
gi|29836496|ref|NP_828851.1| ECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPA 1062
gi|30023954|gb|AAP13567.1| ECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPA 1062
gi|476485|pir||JQ2168 ECVKSQSSRINFCGNGNHIISLVQNAPYGLYFIHFSYVPTKYVTARVSPG 1164
gi|306156|gb|AAA03055.1| ECVKSQSSRINFCGNGNHIISLVQNAPYGLYFIHFSYVPTKYVTARVSPG 1164
gi|549302|sp|P36334|VGL2_CVHOC ECVKSQSSRINFCGNGNHIISLVQNAPYGLYFIHFSYVPTKYVTARVSPG 1164
gi|138175|sp|P15423|VGL2_CVH22 ECVKSQSKRYGFCGNGTHIFSIVNAAPEGLVFLHTVLLPTQYKDVEAWSG 965
gi|1869755|emb|CAA71056.1| ECVKSQSKRYGFCGNGTHIFSIVNAAPEGLVFLHTVLLPTQYKDVEAWSG 868
*** .**.* .***:* *::*: : ** *: *:* :*:: . ..In the above sequences, where SARS is like the other human coronaviruses, it is then useful to consider how these areas of commonality in human coronavirus E2 spike protein is similar to the avian infectious bronchitis virus and other coronaviruses.
The sequence: NQKILAASFN <-- From Human coronavirus 229E E2
NQKILANAFN <-- From Feline infect. Peritonitis virus
NQQILASAFN <-- From Porcine respiratory coronavirus
NQQILASAFN <-- From Porcine transmissible gastroenteritis virus
NQKMIASSFN <-- From Rat coronavirus
NQKQIANQFN <-- From SARS coronavirus
The sequence: ALNKIQDVVN <-- From Human coronavirus 229E
ALAKVQDVVN <-- From Porcine respiratory coronavirus
ALAKVQDVVN <-- From Canine coronavirus
ALAKVQDVVN <-- From Porcine transmissible gastroenteritis virus
ALAKVQDVVN <-- From Feline coronavirus
ALGKLQDVVN <-- From SARS coronavirus
ALQQIQDVVN <-- From Avian infectious bronchitis virus
The sequence: RLDTIQADQQVDRLITGRL <-- From Human coronavirus 229E E2
LDSIQADAQVDRLITGRL <-- From Avian infectious bronchitis virus
RLEKVEADAQVDRLITGRL <-- From Feline infect. peritonitis virus
RLDKVEAEVQIDRLITGRL <-- From SARS coronavirus
The sequence: ECVKSQSKRYGFCGNG <-- From Human coronavirus 229E
ECVKSQSNRYGFCGNG <-- From Feline infect. Peritonitis virus
ECVKSQSNRYGFCGSG <-- From Avian infect. bronchitis virus
ECVKSQSHRFGFCGNG <-- From Porcine respiratory coronavirus
ECVLGQSKRVDFCGKG <-- From SARS coronavirusThe short sequences, their comparison, and their respective hosts suggest the SARS E2-spike protein is related to the avian infectious bronchitis virus and, perhaps, the feline infectious peritonitis virus, and the porcine transmissible gastroenteritis virus. Human, bird, cat, and pig. Taken all together, the large multiple sequence alignment and the very minute examination of similarities across E2-spike proteins, it is reasonable to conclude that the SARS E2-spike protein is most closely related to avian infectious bronchitis virus E2-spike protein. There are substantial models of humans being infected with bird viruses when considering influenza. There does not appear to be any evidence of out-of-the-ordinary natural evolution here in the E2-spike protein comparisons.
Therefore, it may be useful to consider the "mine spikes" in our ocean as an avian infectious bronchitis mutant E2-protein. What "ships" (i.e., cells) are targeted by this mine and what docking mechanisms exist on those cell surfaces?
Possible Cell-surface Docking Mechanisms in SARS Coronavirus Infection
Schultze, Cavanagh, and Herrler (1992) stated their "results indicate that IBV (Avian infectious bronchitis virus) attaches to receptors on erythrocytes, the crucial determinant of which is sialic acid alpha 2,3-linked to galactose. In contrast to other enveloped viruses with such a binding specificity (influenza viruses and paramyxoviruses) IBV lacks a receptor-destroying enzyme." [1] Miguel, Pharr, and Wang (2002) showed that feline cells could be infected by IBV if those cells were expressing feline aminopeptidase N [2]. Breslin et al. (2003) reported that Human Coronavirus HCoV-229E "spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN)" [3]. Bonavia et al. (2003) added "Thus, the data suggest that the domain of the spike protein between amino acids 417 and 547 is required for the binding of HCoV-229E to its hAPN receptor." [4]
Schwegmann-Wessels et al. (2002) reported "The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells." [5]. Wentworth and Holmes (2001) reported significant findings in the realization that certain differences in glycosylation between coronavirus receptors from different species are critical determinants in the species specificity of infection. They stated, "Aminopeptidase N (APN), a 150-kDa metalloprotease also called CD13, serves as a receptor for serologically related coronaviruses of humans (human coronavirus 229E [HCoV-229E]), pigs, and cats. These virus-receptor interactions can be highly species specific; for example, the human coronavirus can use human APN (hAPN) but not porcine APN (pAPN) as its cellular receptor, and porcine coronaviruses can use pAPN but not hAPN. Substitution of pAPN amino acids 283 to 290 into hAPN for the corresponding amino acids 288 to 295 introduced an N-glycosylation sequon at amino acids 291 to 293 that blocked HCoV-229E receptor activity of hAPN. Substitution of two amino acids that inserted an N-glycosylation site at amino acid 291 also resulted in a mutant hAPN that lacked receptor activity because it failed to bind HCoV-229E. Single amino acid revertants that removed this sequon at amino acids 291 to 293 but had one or five pAPN amino acid substitution(s) in this region all regained HCoV-229E binding and receptor activities. To determine if other N-linked glycosylation differences between hAPN, feline APN (fAPN), and pAPN account for receptor specificity of pig and cat coronaviruses, a mutant hAPN protein that, like fAPN and pAPN, lacked a glycosylation sequon at 818 to 820 was studied. This sequon is within the region that determines receptor activity for porcine and feline coronaviruses. Mutant hAPN lacking the sequon at amino acids 818 to 820 maintained HCoV-229E receptor activity but did not gain receptor activity for porcine or feline coronaviruses. Thus, certain differences in glycosylation between coronavirus receptors from different species are critical determinants in the species specificity of infection." [6] It is seen above that it is theoretically possible to change the species specificity of coronaviruses provided the right small nucleotide changes are made in Aminopeptidase N (APN). Tresnan and Holmes (1998) reported that "Human coronavirus HCV-229E and porcine transmissible gastroenteritis virus (TGEV), both members of coronavirus group I, use aminopeptidase N (APN) as their cellular receptors. These viruses show marked species specificity in receptor utilization as they can only use APN of their respective species to initiate virus infection." Significantly, they stated, "Thus, fAPN (feline Aminopeptidase N) acts as a common receptor for coronaviruses in group I, in marked contrast to human and porcine APN glycoproteins which serve as receptors only for human and porcine coronaviruses, respectively. These observations suggest that cats could serve as a "mixing vessel" in which simultaneous infection with several group I coronaviruses could lead to recombination of viral genomes." [7] The obvious question, given that SARS coronavirus spike-protein does not show clear relationship to human coronavirus E2-spike protein, murine hepatitis virus E2-spike protein, or porcine transmissible gastroenteritis E2-spike protein and, given that the small apparent human-animal overlapping amino-acids indicated above show both human and cat as well as pig and bird, might SARS be a recombinant virus that originated in cat?
Levis et al. (1995) indicated "Animal coronaviruses related to HCV-229E, including FIPV, CCV, and TGEV bind to cell membranes from cats, dogs, cows, pigs and humans (but not mice), while each virus infects cells from only a subset of these species. Infectious genomic HCV-229E RNA, can infect cells of all of these species. These data suggest that the species-specificity of infection for this serogroup of coronaviruses is determined at the levels of virus binding and penetration." [8] Interest in the species specificity of the coronaviruses suggests efforts to cross species barriers with coronaviruses as is evident above. Werfel et al. (1991) identify that aminopeptidase N is also known as CD13. Moreover they "demonstrate increases in the membrane expression of neutral endopeptidase (NEP, CD10, CALLA), aminopeptidase N (APN, CD13), tyrosine phosphatase (CD45/CD45Ro) and the Fc R Fc gamma-RIII (CD16) on granulocytes within minutes of treatment with human C5a." [9]. If aminopeptidase N, also known as CD13, is the target for the coronaviruses, and this is associated with FcgammaRIII, it would appear that the coronaviruses may be FcgammaR agents generally, and particularly, SARS coronavirus may be an FcgammaR agent. This possibility appears to be supported in the findings of Mizuki et al. (1992) who reported,"We recently encountered a patient with acute lymphoblastic leukemia (ALL) who showed temporal monocytosis of an unusually high cell count (5,000-30,000 monocytoid cells/microliter) five times after treatment with different chemotherapies. The leukemic cells expressed B-cell-associated antigens, CD19 and CD10, E-rosette receptor, CD2 and monocyte/myeloid antigen, CD13 simultaneously. They were peroxidase-negative. One week after the initiation of conventional chemotherapy for ALL, the leukemic blasts had disappeared. Alternatively, monocytoid cells appeared along with the recovery from nadir status. They showed several features of monocytes; they were weakly dot-positive for nonspecific esterase, reactive with CD14 and CD13 and Fc gamma-receptor-positive." [10] Kolb et al. (1998) indicated "a short stretch of 8 amino acids in the hAPN (human aminopeptidase N) protein plays a decisive role in mediating HCV 229E reception" [11]. The finding appears to suggest that a very small change in CD13 protein can have a marked effect on reception/infectivity. Lachance et al. (1998) show that hAPN (CD13) is expressed on neuronal and glial cell lines in vitro and serves as the receptor for infection by HCV-229E. This further strengthens the neurotropic potential of this human respiratory virus [12]. It would therefore appear that SARS coronavirus, which is likely to be an FcgammaR agent, keying on CD13 (aminopeptidase N), may have neurological effects.
That this small change in amino acids may have marked effects in virus infectivity is evidenced in Kolb et al. (1997) wherein they report:"Aminopeptidase N (APN) is the major cell surface receptor for group 1 coronaviruses. In this study, we have isolated and characterized a feline APN cDNA and shown that the transfection of human embryonic kidney cells with this cDNA renders them susceptible to infection with the feline coronavirus feline infectious peritonitis virus, the human coronavirus (HCV) 229E and the porcine coronavirus porcine transmissible gastroenteritis virus. By using chimeric APN genes, assembled from porcine and feline sequences, we have shown that, analogously to the human APN protein, a region within the amino-terminal part of the feline APN protein (encompassing amino acids 132-295) is essential for its HCV 229E receptor function. Furthermore, by comparing the relevant feline, human and porcine APN sequences, we were able to identify a hypervariable stretch of eight amino acids that are more closely related in the feline and human APN proteins than in the porcine APN molecule. Using PCR-directed mutagenesis, we converted this stretch of amino acids within the porcine APN molecule to the corresponding residues of the human APN molecule. These changes were sufficient to convert porcine APN into a functional receptor for HCV 229E and thus define a small number of residues that are critically important for the HCV 229E receptor function of human APN." [13] We see in the above a report of the activity of changing residues employing PCR-directed mutagenesis, to establish changes in CD13 which allows crossing the species-specificity barrier for coronaviruses. Hegyi and Kolb (1998) stated:"By using chimeric molecules assembled from porcine, human and feline APN we have analysed the determinants involved in the coronavirus receptor function of fAPN. Our results show that amino acids 670-840 of fAPN are critically involved in its FIPV and TGEV receptor function whereas amino acids 135-297 are essential for the HCV 229E receptor function. We also demonstrate that a chimeric molecule assembled from human and porcine APN is able to act as a receptor for FIPV." [14] The data above indicate it is important to examine the SARS coronavirus E2-spike protein to determine the aminopeptidase N amino acid sequence as this might reveal information concerning the species-specificity infectivity of SARS coronavirus. Hegyi and Kolb (1998) indicate this CD13 for cat is located at 670-840 in the fAPN. These amino acids are presented here:
670 ASAQKVPVTLALNNTLFLIQETEYMPWQAALSSLSYFKLMFDRSEVYGPMKRYLKKQVTP 729
730 LFNHFERVTKNWTDHPQTLMDQYSEINAVSTACSYGVPECEKLAATLFAQWKKNPQNNPI 789
790 HPNLRSTVYCNAIAQGGEEEWNFVWEQFLKAELVNEADKLRGALACSNQVW 840
The amino acid sequence from Human coronavirus 229E for aminopeptidase N, according to Hegyi and Kolb (1998), is presented here:
146 ASAHKVPVTLALNNTLFLIEERQYMPWEAALSSLSYFKLMFDRSEVYGPMKNYLKKQVTP 205
206 LFIHF-RNNTNNWREIPENLMDQYSEVNAISTACSNGVPECEEMVSGLFKQWMENPNNNP 264
265 IHPNLRSTVYCNAIAQGGEEEWDFAWEQFRNATLVNEADKLRAALACSKELW 316 A blast comparison of the two proteins shows them to be substantially similar. A multiple sequence alignment of the SARS E2-spike protein and other related coronavirus E2-spike proteins aligned with the aminopeptidase N residues above offers insight into SARS infectivity. A major difference in SARS E2-spike protein compared to all other related coronavirus E2-spike proteins as aligned by the C13 sequences above, showed the following:
Query: 2 IQESLTTTSTALGKLQDVVNQNAQALN 28
IQE T++ALGK+Q VVN NA+ALN
Sbjct: 895 IQEGFDATNSALGKIQSVVNANAEALN 921In the above, the 4 amino acid sequence SLTT in SARS coronavirus was different than all other compared coronaviruses; moreover, this difference was aligned via the aminopeptidase N sequences of many different animals. The amino acid sequence SLTT, being different from GFDA, offers insight into SARS E2-spike protein and its infectivity. A blast of the sequence IQESLTT returned the SARS E2-spike protein and a substantial number of Newcastle Disease Virus sequences. In a multiple sequence alignment of SARS E2-spike protein and Newcastle Disease Virus sequences, the following alignment, related to the infectivity of SARS, was observed:
SARS E2-spike protein and Newcastle Disease Virus alignment
gi|2792137|emb|CAA76104.1| -LGDSIRGIQESVTTSGGRRQRRFIGAIIGSVALGVATAAQITAASALIQ 86
gi|29122884|gb|AAO62651.1| ----SIRRIQESVTTSGGGKQGRLIGAIIGGVALGVATAAQITAASALIQ 46
gi|74951|pir||VGNZGB -LGDSIRRIQESVTTSGGRRQKRFIGAIIGGVALGVATAAQITAAAALIQ 143
gi|138261|sp|P06156|VGLF_NDVB -LGDSIRRIQESVTTSGGRRQKRFIGAIIGGVALGVATAAQITAAAALIQ 143
gi|6959633|gb|AAF33200.1|AF204 -LGESIGRIQESLTTSGGRRQKRFIGAIIGSVALGVATAAQITAAAALIQ 49
gi|30027620|gb|AAP13441.1| QFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQ 599
gi|30023954|gb|AAP13567.1| QFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQ 599
gi|29836496|ref|NP_828851.1| QFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQ 599
.: : :*: . . : . :*.*:: .. : : .:.* *
gi|2792137|emb|CAA76104.1| ANQN-------AASILRLKESIAATNEAVHEVTGG------------LSQ 117
gi|29122884|gb|AAO62651.1| ANQN-------AANILRLKESIAATNEAVHEVTNG------------LSQ 77
gi|74951|pir||VGNZGB AKQN-------AANILRLKESIAATNEAVHEVTDG------------LSQ 174
gi|138261|sp|P06156|VGLF_NDVB AKQN-------AANILRLKESIAATNEAVHEVTDG------------LSQ 174
gi|6959633|gb|AAF33200.1|AF204 ALQN-------AANILRIKESIAATNEAVHEVTDG------------LSQ 80
gi|30027620|gb|AAP13441.1| DVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECD 649
gi|30023954|gb|AAP13567.1| DVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECD 649
gi|29836496|ref|NP_828851.1| DVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECD 649
: *. * * :*.: *.:. * .:
gi|2792137|emb|CAA76104.1| LAVAVGKMQQFVNDQFNNTAQELDCIKITQQVGVELNLYLTELTTVFGP- 166
gi|29122884|gb|AAO62651.1| LAVAVGKM------------------------------------------ 85
gi|74951|pir||VGNZGB LAVAVGKMQQFVNDQFNKTAQESGCIRIAQQVGVELNLYLTELTTVFGP- 223
gi|138261|sp|P06156|VGLF_NDVB LAVAVGKMQQFVNDQFNKTAQELGCIRIAQQVGVELNLYLTELTTVFGP- 223
gi|6959633|gb|AAF33200.1|AF204 LAVA---------------------------------------------- 84
gi|30027620|gb|AAP13441.1| IPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTN 699
gi|30023954|gb|AAP13567.1| IPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTN 699
gi|29836496|ref|NP_828851.1| IPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTN 699
:.:. While the Newcastle Disease Virus amino acid sequences did not match the expected portions of the SARS coronavirus E2-spike protein exactly, the data appear to suggest that the infectivity of SARS may operate similarly to Newcastle Disease Virus. Pedersden et al. (1990) give evidence that Newcastle disease virus, avian infectious bronchitis virus, and infectious bursal disease virus antibodies have been discovered in humans. They state, "people having a known association with poultry showed significantly higher levels of antibodies to Newcastle disease and avian infectious bronchitis virus." [15] While Newcastle disease virus is a paramyxovirus, the SARS E2-spike protein suggests some relationship to the Newcastle disease virus. Much confusion has existed regarding whether SARS is a coronavirus or a paramyxovirus - perhaps the relationship of Newcastle disease virus to SARS E2-spike protein offers a reason for the confusion and may explain some of the activity of SARS.
The data above have suggested that SARS E2-spike protein is related to avian infectious bronchitis virus in multiple sequence alignment and phylogeny; the infectivity of SARS E2-spike protein appears to be possibly related to the Newcastle disease virus, an avian paramyxovirus. Two different lines of evidence suggest SARS E2-spike protein is related to avian viruses. There is ample evidence suggesting humans can become naturally infected with avian viruses. The evidence suggests that SARS E2-spike protein is targeting C13 (aminopeptidase N) on cell surfaces; there is evidence suggesting SARS E2-spike protein may bind to glial cells of the nervous system and may have B-cell tropism as well. There is evidence of FcgammaR activity in SARS E2-spike protein which is supported by data from Grage-Griebenow et al. (1993) where it is identified that Newcastle disease virus will stimulate interferon-alpha release from CD64- monocytes. They state:"...when CD64- and CD64+ monocytes were stimulated with Newcastle disease virus, we measured an up to 67-fold higher interferon-alpha release from CD64- than from CD64+ monocytes, indicating a higher anti-viral capacity of this subset. CD64- monocytes showed lower activity in the phagocytosis of unopsonized particles and also lower zymosan- or latex-induced chemiluminescence than CD64+ monocytes. These findings indicate that CD64- monocytes, although comprising only less than 10% of all peripheral blood monocytes, represent a monocyte subpopulation efficiently interacting in vitro with T cells and, additionally, are the major source of interferon-alpha." [16] They go on to say:
"Surface analyses revealed that the surface antigen pattern of CD64+ monocytes corresponds to the phenotype of typical unseparated monocytes. In contrast, CD64- monocytes are characterized by high expression of major histocompatibility complex (MHC) class I antigens (HLA-A, -B, -C) and MHC class II antigens (HLA-DR, -DP, -DQ), and low expression of the monocyte-specific marker CD14 which is found on nearly all CD64+ monocytes. However, 75% of the CD64- cells were found to be esterase-positive, and 85% were positive for the the CD64- cells were found to be esterase-positive, and 85% were positive for the monocyte/macrophage-specific intracellular antigen CD68. Furthermore, CD64- monocytes show significantly higher expression of CD45RA and Fc gamma receptor III (CD16) than CD64+ monocytes, but lack the natural killer cell markers CD56 and CD57. Functional studies showed that cells of the minor CD64- monocyte subset have a higher accessory cell capacity in antigen-driven T cell activation than CD64+ monocytes. CD64- monocytes pretreated with PPD (purified protein derivative of tuberculin) induced up to tentimes more interferon-gamma and also higher proliferation in responding autologous T cells than PPD-pretreated CD64+ monocytes." [17]
The above data, while reported specifically to stimulation by Newcastle disease virus, suggest that SARS coronavirus may also involve CD64 monocytes and there may be a differential effect on persons as a function of their expression of major histocompatibility comples (MHC) class I and class II antigens. Theorizing from the above, SARS coronavirus may induce strong interferon-alpha responses in persons who are CD64- which would suggest HLA-DR, -DP, and -DQ class II antigens. The above suggests that CD64+ persons would not have a profound elicitation of interferon-alpha to a virus such as SARS. It is also suggested from the above that, as CD14 is significant as well as CD16, it is likely that CD13 (aminopeptidase N) is also significant in SARS and offers further evidence of FcgammaR activity of the SARS coronavirus.
Interestingly, the HLA-DR, -DP, and -DQ, the Class II MHC, are likely to be involved in SARS disease expression and associated with interferon-alpha and TNF-alpha. Jewell et al. (1997) indicate, in a study involving interferon-alpha treatment of chronic lymphocytic leukemia, that interferon-alpha can have a substantial aversive effect on some individuals. They state:"The effect of interferon-alpha (IFN) therapy in these disorders may be to disrupt autocrine growth or survival loops. We have measured levels of circulating IL-1b, IL-6, TNF-a and soluble CD23 (sCD23) in 8 patients with Binet stage A B-cell chronic lymphocytic leukaemia (B-CLL) receiving IFN therapy, and compared these with changes in the lymphocyte count following IFN therapy. Two patients developed anti-interferon antibodies while on IFN therapy, and in both them, the changes in lymphocyte count correlated significantly with the titre of anti-interferon antibodies, as well as serum levels of IL-6, TNF-a and sCD23." [18] These data indicate that some individuals may develop anti-interferon antibodies and this will increase IL-6, TNA-a, and sCD23 while undergoing interferon-alpha therapy for leukemia. Might this same anti-interferon antibody formation, increase in IL-6, TNF-a, and sCD23 happen in individuals with SARS coronavirus infection?
Al-Humaidi (2000) indicates that IL-6, TNF-a, and sCD23 may all be elevated in Grave's disease Al-Humaidi states:"There was a marked increase in proinflammatory cytokines in Graves' disease patients: levels of IL-6 (481.5 +/- 192.3 pg/ml) and TNF-a (30.69 +/- 16.7 pg/ml) were significantly higher than those of normal controls for IL-6 (63.81 +/- 21.72 pg/ml, P<0.001) and TNF-a (8.81 +/- 1.72 pg/ml, P<0.001). Similarly the levels of sCD23 (mean 164 +/- 67.03 ng/ml) and sIL-2R (mean 2131 +/- 461.1 units/ml) were significantly higher in GD patients than in the control group (mean 31.24 +/- 11.53 ng/ml, P<0.001) and (mean 345.53 +/- 121.75 units/ml, P< 0.001) for sCD23 and sIL-2R." [19] The above information suggests a significant proinflammatory response in Graves' disease and provides further evidence of the aversive effects of elevated levels of cytokines likely occurring in SARS infection. We might expect symptoms in SARS patients to appear somewhat similar to Graves' disease. These same cytokines are seen elevated in patients with multiple sclerosis. Zaffaroni et al. (1995) reports:"IL-6 and tumor necrosis factor (TNF)-alpha synthesis was induced by PWM stimulation in all groups, but MS patients showed the most significant increase of both cytokines. Interestingly, only MS patients showed a significant increase of the soluble form of CD23 receptor (sCD23). Moreover, only sCD23 levels correlated with in vitro IgG production in MS patients. The levels of IL-6, TNF-alpha, sCD23 were greater in high responders compared to low responders in all groups. The mean value of each molecule, however, did not differ significantly among overall groups. A highly significant difference was reported for sCD23 in MS patients. We suggest that sCD23, also known as B cell growth factor, may play a role in the well-documented phenomenon of in vitro IgG hypersynthesis in MS patients, adding support to the concept of B cell up-regulation in the peripheral blood of these patients." [20]
Conclusion
This report has presented evidence concerning SARS E2-spike protein and the cell surfaces it uses for docking. The data suggest SARS E2-spike protein targets CD13 (aminopeptidase N) on cell surfaces. Data indicates SARS is highly likely to initiate an immunological cascade in some individuals, likely of HLA-DR, -DP, -DQ MHC II, which may stimulate interferon-alpha, IL-6, TNF-a, sCD23, as a result of a profound proinflammatory response. Data suggest the SARS E2-spike protein is most closely related to avian infectious bronchitis virus and further sub-analysis of SARS E2 infectivity suggests a relationship to Newcastle disease virus.
Taken all together, the above information suggests SARS coronavirus in humans may be the result of a zoonosis from birds. The data suggest a possible explanation for the coronavirus vs paramyxovirus confusion that has surrounded early SARS investigations. It is likely that SARS disease will profoundly effect HLA-DR, -DP, -DQ MHC II populations. It is likely that CD13, CD14, CD16, sCD23, and CD64 will be of interest relative to further understanding of the SARS coronavirus and its disease. Study of neurological and lymphomatous sequelae in SARS patients may be important.
We have managed to shed some light on the question of the "ocean mine's spikes" as well as on the "ship" the mine will attack. Moreover, we have seen some data suggesting the effects of detonating mines on particular types of ships. It appears there will be years of research ahead in further understanding of SARS coronavirus and its disease processes. Substantial learning about many issues in virology, molecular biology, immunology, and genetics will occur in the future as this virus and its disease are investigated.
Dr. Robert E. Lee, M.S., M.S.W., L.C.S.W., Ph.D.
May 3, 2003
References:
1. Schultze B, Cavanagh D, Herrler G: Neuraminidase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes. Virology 1992 Aug;189(2):792-4 [PubMed Abstract]
2. Miguel B, Pharr GT, Wang C: The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Brief review. Arch Virol 2002 Nov;147(11):2047-56 [PubMed Abstract]
3. Breslin JJ, Mork I, Smith MK, Vogel LK, Hemmila EM, Bonavia A, Talbot PJ, Sjostrom H, Noren O, Holmes KV: Human coronavirus 229E: receptor binding domain and neutralization by soluble receptor at 37 degrees C. J Virol 2003 Apr;77(7):4435-8 [PubMed Abstract]
4. Bonavia A, Zelus BD, Wentworth DE, Talbot PJ, Holmes KV: Identification of a receptor-binding domain of the spike glycoprotein of human coronavirus HCoV-229E. J Virol 2003 Feb;77(4):2530-8
5. Schwegmann-Wessels C, Zimmer G, Laude H, Enjuanes L, Herrler G: Binding of transmissible gastroenteritis coronavirus to cell surface sialoglycoproteins. J Virol 2002 Jun;76(12):6037-43
6. Wentworth DE, Holmes KV: Molecular determinants of species specificity in the coronavirus receptor aminopeptidase N (CD13): influence of N-linked glycosylation. J Virol 2001 Oct;75(20):9741-52
7. Tresnan DB, Holmes KV: Feline aminopeptidase N is a receptor for all group I coronaviruses. Adv Exp Med Biol 1998;440:69-75
8. Levis R, Cardellichio CB, Scanga CA, Compton SR, Holmes KV: Multiple receptor-dependent steps determine the species specificity of HCV-229E infection. Adv Exp Med Biol 1995;380:337-43
9. Werfel T, Sonntag G, Weber MH, Gotze O: Rapid increases in the membrane expression of neutral endopeptidase (CD10), aminopeptidase N (CD13), tyrosine phosphatase (CD45), and Fc gamma-RIII (CD16) upon stimulation of human peripheral leukocytes with human C5a. J Immunol 1991 Dec 1;147(11):3909-14
See also Macey MG, Jiang XP, Veys P, McCarthy D, Newland AC: Expression of functional antigens on neutrophils. Effects of preparation. J Immunol Methods 1992 Apr 27;149(1):37-42
See also: Nikolaizik WH, Simon HU, Iseli P, Blaser K, Schoni MH: Effect of 3 weeks' rehabilitation on neutrophil surface antigens and lung function in cystic fibrosis. Eur Respir J 2000 May;15(5):942-8
10. Mizuki M, Tagawa S, Nojima J, Nakamura Y, Morita T, Yumura-Yagi K, Hara J, Kawa-Ha K, Kitani T: Monocytes appearing repeatedly after chemotherapies had an identical rearrangement pattern of immunoglobulin with leukemic blasts in a patient with CD13+ acute lymphoblastic leukemia. Acta Haematol 1992;87(1-2):88-93
11. Kolb AF, Hegyi A, Maile J, Heister A, Hagemann M, Siddell SG: Molecular analysis of the coronavirus-receptor function of aminopeptidase N. Adv Exp Med Biol 1998;440:61-7
12. Lachance C, Arbour N, Cashman NR, Talbot PJ: Involvement of aminopeptidase N (CD13) in infection of human neural cells by human coronavirus 229E. J Virol 1998 Aug;72(8):6511-9
13. Kolb AF, Hegyi A, Siddell SG: Identification of residues critical for the human coronavirus 229E receptor function of human aminopeptidase N. J Gen Virol 1997 Nov;78 ( Pt 11):2795-802
14. Hegyi A, Kolb AF: Characterization of determinants involved in the feline infectious peritonitis virus receptor function of feline aminopeptidase N. J Gen Virol 1998 Jun;79 ( Pt 6):1387-91
15. Pedersden KA, Sadasiv EC, Chang PW, Yates VJ: Detection of antibody to avian viruses in human populations. Epidemiol Infect 1990 Jun;104(3):519-25
16. Grage-Griebenow E, Lorenzen D, Fetting R, Flad HD, Ernst M: Phenotypical and functional characterization of Fc gamma receptor I (CD64)-negative monocytes, a minor human monocyte subpopulation with high accessory and antiviral activity. Eur J Immunol 1993 Dec;23(12):3126-35
17. Grage-Griebenow E, Lorenzen D, Fetting R, Flad HD, Ernst M: Phenotypical and functional characterization of Fc gamma receptor I (CD64)-negative monocytes, a minor human monocyte subpopulation with high accessory and antiviral activity. Eur J Immunol 1993 Dec;23(12):3126-35
18. Jewell AP, Worman CP, Giles FJ, Goldstone AH: Serum levels of TNF, IL-6 and sCD23 correlate with changes in lymphocyte count in patients with B-cell chronic lymphocytic leukaemia receiving interferon-alpha therapy. Leuk Lymphoma 1997 Jan;24(3-4):327-33
19. Al-Humaidi MA: Serum cytokines levels in Graves' disease. Saudi Med J 2000 Jul;21(7):639-44
20. Zaffaroni M, Stampino LG, Ghezzi A, Baldini SM, Zibetti A: In vitro cytokine, sCD23 and IgG secretion in multiple sclerosis. J Neuroimmunol 1995 Aug;61(1):1-5
21: Lee RE: Further discussion of SARS Treatment hypothesis in context of FcgammaR inhibition. SARS Treatment Hypotheses. USENET misc.health.aids 2003-04-22 17:45:51 PST. Available from URL
http://groups.google.com/groups?hl=en&lr=&ie=UTF-8&selm=_mlpa.604913%24S_4.652336%40rwcrnsc53
Keywords
SARS
coronavirus
spike protein
cytokine
FcgammaR
aminopeptidase N
immunology
CD13
bioinformatics
molecular modeling
molecular docking
Acknowledgements
The writer wishes to thank Dr. Alan R. Cantwell and Dr. Brian Foley for their various input, ideas, and friendship over the last years.
Competing Interests
None declared.
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