Severe Acute Respiratory Syndrome (SARS) Coronavirus

To see my JBrowse Severe Acute Respiratory Syndrome Coronavirus genome annotation, SARS.

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Table of Contents:

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Severe Acute Respiratory Syndrome (SARS) Coronavirus

• NCBI Reference sequence: NC_004718.3
• Length: 29,751 nucleotides
• GC content: 40%
• Percent coding: 97%
• Topology: linear
• ssRNA
• Thirteen Genes
• Wikipedia:SARS_coronavirus
• Oxford RNA virus Database

The functional viral proteins annotated within the Severe Acute Respiratory Syndrome (SARS) Coronavirus genome:

Link to my Severe Acute Respiratory Syndrome Annotations.

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Coronaviruses

Coronaviruses are single stranded enveloped RNA viruses that have a helical geometry. Coronaviruses are the largest of RNA viruses with a genome size of up to 31kb. These viruses are grouped in the order Nidovirales. The structure common to all coronaviruses consists of spike (S), envelop (E), membrane (M), and nucleocapsid (N) proteins. There are currently three different groups of coronaviruses. Some of the notable viruses are group 2b SARS, group 1 Human coronavirus, and TGEV.

Coronavirus Phylogeny Tree based on tail spike proteins:

The phylogenetic tree was generated using ClustalW and the drawings were done using a web-based tool called Polydendron. Disclaimer: Tree was found on the web.

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Visit Fall09 Virus Phylogenetic Tree to see how coronaviruses are related to other students' viruses.

• Wikipedia:RNA_virus

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Severe Acute Respiratory Syndrome Virus Biology

SARS coronavirus belongs to a family of coronaviruses that are enveloped, positive-stranded and single stranded RNA. Its genome is about 27 to 31.5kb, which is one of the largest among RNA viruses. SARS is like other coronaviruses in that it is polycistronic and the genome expression starts with translation of two large ORFs 1a and 1b. The SARS virus has 13 known genes and 14 known proteins. It also has 14 ORFs. There are 265bp in the 5'UTR and 342bp in the 3'UTR. Conserved proteins in all of coronaviruses are encoded by the overlapping ORFs 1a and 1b and ORFs2,4,5,6,9a. Certain protein enzymes also look like they are conserved among RNA viruses; these are: PLpro, CLpro, RdRp, and 2'-O-MT. Coronaviruses usually express pp1a (the ORF1a polyprotein) and the PP1ab polyprotein with joins ORF1a and ORF1b. The polyproteins are then processed by enzymes that are encoded by ORF1a. Product proteins from the processing includes various replicative enzymes such as RNA dependent polymerase, RNA helicase, and proteinase. The replication complex in coronavirus is also responsible for the synthesis of various mRNAs downstream of ORF 1b, which are structural and accessory proteins. Two different proteins 3CLpro and PL2pro cleave the large polyproteins into 16 smaller subunits. A unique domain in SARS is SUD (SARS-CoV unique domain), which is upstream of PL2pro and has 375 amino acids. The 3'-proximal part of the genome has five unique ORFs that are not present in other group 2 coronaviruses.

The life cycle of the SARS coronavirus starts when it enters a host cell by membrane fusion. The viral nucleic acid is then replicated, and proteins are synthesized. Assembly of the nucelocapsid is done in the rough ER by putting together N protein and genomic RNA. The golgi apparatus swells to form smooth vesicles containing the nucleocapsides, which then bud from the golgi. The last step of forming the complete virus is the assembling of the envelops with the nucleocapsids. The smooth vesicles fuse with the cell membrane to release the viruses.

A representative schematic of the genus Betacoronavirus, of which Severe Acute Respiratory Syndrome virus is a species. From ViralZone.

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Severe Acute Respiratory Syndrome Virus Morphology

The morphology of the SARS coronavirus is characteristic of the coronavirus genus as a whole. These viruses have large pleomorphic spherical particles with bulbous surface projections that fomr a corona around particles. The envelop of the virus contains lipid and appears to consist of a distinct pair of electron dense shells. The internal component of the shell is a single-stranded helical ribonucleoprotein. There are also long surface projections that protrude from the lipid envelop. The size of these particles are about 80-90nm.

A general coronavirus morphology representative of all coronaviruses. From ViralZone.

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Severe Acute Respiratory Syndrome Virus Pathology

Virions first attached themselves to the surface of the host and their envelops fused with the cell membrane and the nucleocapsides enter the cell. The virus does not enter the cell via endocytosis, which is the mechanism employed by other coronaviruses. The viruses replicate their DNA inside the host cells. The SARS coronavirus infects type 2 pneumocytes, which are important for secreting pulmonary surfactants that reduce surface tension and preserves the integrity of alveolar space. SARS also infects type 1 pneumocytes, which are the primary targets early on in the infection. The spike protein also plays an important role in the pathogenesis of SARS. The S protein increases ER stress and the unfolded protein response (UPR).

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Severe Acute Respiratory Syndrome Virus Evolution

SARS is most closely related to group 2 coronaviruses, but it does not segregate into any of the other three groups of coronaviruses. The closest outgroup to the coronaviruses are the toroviruses, with which it has homology in the ORF 1b replicase and the two viron proteins of S and M. SARS was determined to be an early split off from the group 2 coronaviruses based on a set of conserved domains that it shares with group 2.

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Engineering Applications

Engineering of SARS virus has been done. In a paper published in 2006, a new transcription circuit was engineered to make recombinant SARS viruses. The recombination allowed for efficient expression of viral transcripts and proteins. The engineering of this transcription circuit reduces the RNA recombinant progeny viruses. The TRS (transcription regulatory sequences) circuit regulates efficient expression of SARS-CoV subgenomic mRNAs. The wild type TRS is ACGAAC. A doulbe mutation results in TRS-1 (ACGGAT) and a triple mutation results in TRS-2 (CCGGAT). When the remodeled TRS circuit containing viruses are genetically recombined with wild type TRS circuits, the result is a circuit reduced in production of subgenomic mRNA. The goal of modifying the SARS virus with this approach is the produce chimeric progeny that have reduced viability due to the incompatibility of the WT and engineered TRS circuits.

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Clinical/Therapeutic Considerations

Existing treatments for SARS coronavirus include the use of ribavirin, which is a nucleoside analog that has activity against numerous viruses in vitro. However, upon further study, it was shown that ribavirin did not have much activity against SARS-CoV. Another method of treatment was the use of corticosteroids, which aims to suppress cytokine-induced lung injury in phase 2 of SARS. However, this mode of therapy has the risk of complications of avascular necrosis and fungal disease. IFN-alpha was also used in combination with corticosteroids as a treatment. This drug acts to inhibit cytopathic effects of SARS-CoV, reducing viral replication. Treatment with IFN-alpha after exposure to virus yielded intermediate results. The success of clinical testing approved the treatment.

There are several other potential treatments for SARS. Glymcyrrhizin is an active component of liquorice roots. This compound can be made into a vaccine since it elicits a strong SARS-CoV specific immune response. Human monoclonal antibody (huMab) recognizes the antigen S1 protein, which recognizes the angiotensin-converting enzyme-2 receptor that leads to viral entry, in SARS-CoV. This has neutralizing activity that show huMab to be a useful inhibitor of of viral entry.

There are various proposed methods to treat and prevent SARS that are based on the discovered viral proteins, and the inhibition of their activities. A potential treatment is the inhibition of S protein cleavage by Ben-HCl. The Xa factor can cleave the S protein into S1 and S2 subunits, which is postulated to facilitate viral infection.

Another approach to tackle the virus is through vaccination. Intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of SARS-CoV spike protein can induce strong immune responses that provide protection against SARS.

Another proposed treatment is the targeting of the Mpro proteinase to prevent it from cleaving polyproteins in the SARS virus. The two large polyproteins that are translated undergo extensive proteolytic processing to produce functional proteins. The Mpro proteinase is particularly important since it cleaves the polyprotein at 11 conserved sites involving Leu-Gln sites. The 3D structure of the Mpro proteinase has been solved, which allows the possibility of inhibitor design. There are findings that a hexapeptyidyl inhibitor of the human rhinovirus 3C proteinase has a binding mode that can be used as a model for developing an inhibitor for the SARS Mpro proteinase.

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Comparison To Other Viruses

The PL2pro gene, which codes for a proteinase, is a conserved gene with group 2 Coronaviruses. The Mpro proteinase gene is highly conserved among coronaviruses. The genes that encode the Mpro proteinase is conserved among the three coronaviruses assigned, Human Coronavirus, SARS Coronavirus, and TGEV.

SARS Coronavirus is distantly related to group 2 coronaviruses, which includes the Bovine Coronavirus. It is not part of the group because it lacks genes that are present on group 2 viruses, such as PL1pro, CPD-like genes, and HE genes. A gene that is unique to SARS Coronavirus is SUD (SARS-Coronvirus unique domain). It is thought to be a proteinase.

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Multiple Sequence Alignment of Coronavirus and Rhinoviruses

This is a multiple sequence alignment of 7 coronaviruses and 3 rhinoviruses. The table below shows the sequences aligned above.

Coronaviruses and Rhinoviruses
virus	order	sequence aligned	region of significant homology
Breda	coronavirus	polyprotein 1ab	1664-1871
Rhinovirus C	picornavirus	3C protease	1-181
Rhinovirus 14	picornavirus	3C protease	1-181
Bovine Rhinovirus 2	picornavirus	3C protease	2965-3170
Human Coronavirus 229E	coronavirus	replicase polyprotein 1ab	1-204
TGEV	coronavirus	replicase polyprotein 1ab	3103-3309
Bovine	coronavirus	orf1ab polyprotein	1267-1454
SARS	coronavirus	orf1ab polyprotein	3116-3330
Bat	coronavirus	orf1ab polyprotein	1-203
infectious bronchitis virus	coronavirus	orf1ab polyprotein	1642-1848

This multiple sequence alignment shows that within the polyprotein orf1ab of various coronaviruses is a protein that shares significant homology with a protease from rhinoviruses. This protease is responsible for processing the transcribed polyprotein into more functional mature peptides by cleaving the polyprotein. Another point that this alignment illustrates is the level of conservation of this protein among coronaviruses. Determination of these homology regions in viral orders and families are important for finding drug targets for general coronaviruses. Another useful aspect of doing a sequence alignment is that a tree can be generated that tell the viewer about the evolutionary relationships between differernt viruses.

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Tree Constructed From Alignment

The phlyogram demonstrates the evolutionary relationship between these viruses. This tree was generated using the ClustalW web-based tool. This result is consistent in that rhinoviruses, coronaviruses, and breda virus (which actually belong to toroviruses, but is close to coronaviruses) are grouped separately.

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Collection of SARS PDB structures

3D crystal structures of proteins are very useful for molecular biologists since they can be used to inform the research of the location of active sites and key residues that carry out the chemistry of interest. They are also especially valuable for engineers who want to manipulate or alter the function of a particular protein. They are better informed when carrying out directed evolution or making libraries.

This is the protein structure of ORF-9b. The protein is produced from an alternate open reading frame within the nucleocapsid gene. The protein is likely to be involved in membrane attachment and associates with intracellular vesicles. The fold of the protein is a dimeric beta structure with amphipathic surfaces.

Meier C, Aricescu AR, Assenberg R, Aplin RT, Gilbert RJ, Grimes JM, Stuart DI.  The crystal structure of ORF-9b, a lipid binding protein from the SARS coronavirus.  Structure. 2006 Jul;14(7):1157-65.

This is the protein structure of Non-structural protein 3 of the replicase polyprotein 1a.

This is the protein structure of the nucleocapsid protein. The function of the protein is to form an envelop around the virus. The protein is a strong antigen and associates with various host cell motifs. This protein is of interest and a potential drug target. The interaction of this protein with the host can be more thoroughly studied since the crystal structure is available, and some means of interrupting that interaction could prove to prevent infection.

Chen CY, Chang CK, Chang YW, Sue SC, Bai HI, Riang L, Hsiao CD, Huang TH.  Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA.  J Mol Biol. 2007 May 11;368(4):1075-86. Epub 2007 Mar 2.

This is the protein structure of ORF7A accessory protein. The function of the protein has not been currently determined. The fold of the protein, however, is similar to the Ig superfamily. It is also noted that the protein is expressed and maintained intracellularly after infection.

Nelson CA, Pekosz A, Lee CA, Diamond MS, Fremont DH.  Structure and intracellular targeting of the SARS-coronavirus Orf7a accessory protein.  Structure. 2005 Jan;13(1):75-85.

This is the protein structure of the tail spike. The tail spike protein attaches to the ACE2 receptor of its host. From the study of this crystal structure, vaccine treatments can be developed based on making truncated disulfide-stabilized receptor-binding domains.

Li F, Li W, Farzan M, Harrison SC.  Structure of SARS coronavirus spike receptor-binding domain complexed with receptor.  Science. 2005 Sep 16;309(5742):1864-8.

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List of Databases and Resources for the SARS virus

Databases and Informational Sites

Coronavirus Database This site provides a convenient place that is a central place that links to genbank for various coronaviruses.

ViralZone This site is provides a lot of information about the phylogeny and unique aspects of many viruses. It is very comprehensive, and provides helpful illustrations of viral morphology and genes.

Wikipedia Wikipedia provides much of the basic knowledge about the virus and also links to many different pages. It is a great resource for getting to know the subject.

CDC This is a site from the Center for Disease Control that gives both public health information and also phylogenetic and molecular biology information on the SARS virus.

Literature References

Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, Poon LL, Guan Y, Rozanov M, Spaan WJ, Gorbalenya AE.  Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage.  J Mol Biol. 2003 Aug 29;331(5):991-1004.

Agafonov AP, Gus'kov AA, Ternovoi VA, Ryabchikova EI, Durymanov AG, Vinogradov IV, Maksimov NL, Ignat'ev GM, Nechaeva EA, Netesov SV.  Primary characterization of SARS coronavirus strain Frankfurt 1.  Dokl Biol Sci. 2004 Jan-Feb;394:58-60.

Qinfen Z, Jinming C, Xiaojun H, Huanying Z, Jicheng H, Ling F, Kunpeng L, Jingqiang Z.  The life cycle of SARS coronavirus in Vero E6 cells.  J Med Virol. 2004 Jul;73(3):332-7.

Jin DY, Zheng BJ.  Roles of spike protein in the pathogenesis of SARS coronavirus.  Hong Kong Med J. 2009 Feb;15 Suppl 2:37-40.

Yount B, Roberts RS, Lindesmith L, Baric RS.  Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: engineering a recombination-resistant genome.  Proc Natl Acad Sci U S A. 2006 Aug 15;103(33):12546-51. Epub 2006 Aug 4.

Jin DY, Zheng BJ.  Roles of spike protein in the pathogenesis of SARS coronavirus.  Hong Kong Med J. 2009 Feb;15 Suppl 2:37-40.

Anand K, Ziebuhr J, Wadhwani P, Mesters JR, Hilgenfeld R.  Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs.  Science. 2003 Jun 13;300(5626):1763-7. Epub 2003 May 13.

-- %TEACHINGWEB%.SusanChen - 07 Dec 2009