STRUCTURAL AND MOLECULAR ORGANIZATION WITH THEIR FUNCTIONAL STATUS OF CORONA VIRUSES: A REVIEW

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STRUCTURAL AND MOLECULAR ORGANIZATION WITH THEIR FUNCTIONAL STATUS OF CORONA VIRUSES: A REVIEW Balwant Singh*1, Shivangi Tripathi2, Sakshi Tripathi3 *1Research

2Junior

3Post

Scholar, Department of Botany, Dr. Ram Manohar Lohiya Avadh University Ayodhya, Uttar Pradesh, India.

Research Fellow, Department of Microbiology, King Georg Medical University Lucknow, Uttar Pradesh, India.

Graduate Student, Department of Zoology, B. P. P. G. College Narayanpur Maskanwa, Gonda, Uttar Pradesh, India.

ABSTRACT The corona virus is a most thundering and expressive virus that are pathogenic from since 1965. Till date three major pandemic human corona viruses are evolved time to time at different locations. Corona virus causes pathogenesis, not only humans as well as animals also that was known earlier. Structurally corona viruses are spherical with different complex morphological structural proteins. Corona viruses share +ssRNA as genetic material that represent it highly specific and advanced. This article express about the structural and their functional status that would be helpful for academic as well as researchers.

KEYWORDS: Corona Virus, SARS, MERS, Covid-19, Structural Organization.

I.

INTRODUCTION

The first Human Corona virus (HCoV) was cultivated in human ciliated embryonic tracheal cells in 1965.(1) Prior to present Severe Acute Respiratory Syndrome Corona Virus-2 (SARS CoV-2), six corona viruses were known to cause disease in humans including Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS).(2) Resent evolved corona virus (SARS CoV-2) reported from Wuhan City, Huanan Seafood Wholesale Market in Hubai Province of China, associated with severe cause of pneumonia and respiratory illness, since 08 December 2019. (3-5) SARS was first evolved severe corona virus of human in November 2002 from Guangdong Province of China that was first pandemic of 21st Century,(6-7) whereas MERS was first identified in January 2012 from Bisha area of Saudi Arabia with their associated diseases.(8-9) The evolution of these human corona viruses, bat might be original host with some intermediate hosts like Palm Civets for SARS CoV, Camel for MERS CoV and may be Pangolins, Snake, Cat for SARS CoV-2.(10-14) SARS CoV, MERS CoV and SARS CoV-2 are cause severe respiratory disease to human respectively SARS, MERS and Covid-19, whereas remaining four other corona viruses (HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1) cause mild upper respiratory disease such as common cold.(2) There are all severe human corona viruses; SARS CoV-2 has more transmissibility with low mortality rate than prior known SAR CoV and MERS CoV.(10)(15-16) Corona viruses belong to family Coronaviridae in under Nidovirales order. The family Coronaviridae has two sub-family Coronavirinae and Torovirinae, whereas Coronavirinae subdivided into four genera, Alpha CoV, Beta CoV, Delta CoV and Gamma CoV. Alpha CoV (HCoV-229E, HCoV-NL63) and Beta CoV (HCoV-OC43, HCoV-NL63, SARS CoV, MERS CoV and SARSCoV-2) include Human Corona Viruses.(2)(10)(17)

II.

STRUCTURE

HCoV are spherical shaped with a nucleocapsid of helical symmetry. (18-19) They are enveloped viruses and about 100-120 nm in diameter.(16) Exteriorly virions surrounded by long halo crowns protein structure consist of spike glycoprotein (S-Protein).(11) Some other shorter spike proteins are also line the outside of virion that consists of hemagglutinin-esterase glycoprotein (HE-Protein).(10) The matrix glycoproteins (MProtein) are present in both internal core structure and the envelope which is made by lipid bilayer. The envelop protein (E-Protein) also form a part of the viral envelope and it is present in much smaller quantities than other viral envelope proteins.(2)(10)(18)(20) Interiorly virion is a positive sense single stranded ribonucleic acid (+ssRNA) genome that ranges from 26-32 Kilo bases (Kb) in length, the largest www.irjmets.com

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of all known RNA viral genomes.(12) Separately genome of SARS CoV-2 IS 29.9 Kb while earlier corona virus’s genome of SARS CoV and MERS CoV is 27.9 Kb and 30.1 Kb respectively. (2)(10) The genome (RNA) is associated with nucleocapsid phosphoprotein (N-Protein) that allows it to form a long, flexible, helical nucleocapsid. This helical nucleocapsid is enclosed within a 65 nm in diameter, spherical, possibly icosahedral Internal Core Structure (Core).(10) This virus core is encapsidated by a lipoprotein envelope that is formed during virus budding from intracellular membranes.(21)

Fig-1: Diagrammatic Structure of Corona Virus

III.

STRUCTURAL PROTEINS

S-Protein S-Protein is encircling club-shaped and about 9-12 nm that responsible for the solar corona like appearance of the virus and hence the viruses are named corona virus. S-Protein is the major protein present in the viral membrane forming the typical spike structure found on all corona virions. (2)(11)(18) Mature proteins form oligomers in the form of homotrimers and are known to be glycosylated. (22) Post translational glycosylation occurs on at least four different sites of the spike protein. (23) Once fully processed the protein exists as a 180 kDa single protein species with the homotrimers species of approximately 500 kDa.(21) Although some S-Protein oligomers can be found on the infected cell surface where it may mediate cell-cell fusion most newly synthesized S-Protein accumulates in the Golgi Bodies (GB) of infected cell where it participates in virus particle assembly.(24) S-Protein seems to be cleaved into S1 and S2 subunits in infected cells. (25) The S1 subunit contains the receptor binding domain which causes it to be the main determinate of viral tropism. (2) Angiotensin Converting Enzyme-2 (ACE2 for SARS CoV and SARS CoV-2)(26) and Dipeptidyl Peptidase-4 (DPP4 for MERS CoV) have been identified to be two receptors for the corona viruses. (21) S1 subunit responsible to www.irjmets.com

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the determination for virus-host range cellular tropism with the key function as receptor binding domain (RBD). S2 subunit contains internal hydrophobic sequences which are thought to be responsible for the membrane fusion activity by two tandem domains, Heptad Repeats-1 (HR1) and HR2. Expression of the SProtein alone has been shown to induce membrane fusion on cells that are susceptible to Coronavirus infection.(2)(10)(21) HE-Protein Another protein found on some corona viruses is the hemagglutinin esterase glycoprotein (HEProtein).(27) HE-Protein exists as a homodimer of 65-70 kDa protein that form short spikes on surface of corona virus virions.(28-29) This protein complex is incorporated into the envelope of budding virus particles after being transported to the GB and does not incorporated into the virion envelope is expressed on cellular surfaces.(29-30) Some studies hypothesized that the HE-Protein is dispensable for viral replication since the presence or absence is highly inconsistent the corona viruses. In addition the HE gene is frequently mutated of completely deleted during serial virus passing in cell culture. (31) HEProtein also has an acetyl esterase activity that cleaves acetyl group from 9-O-acetylated neuraminic acid which reverses or prevents the hem-agglutination and hem-adsorption activity induced by S and HEProtein.(30) This acetyl esterase activity along with the inhibition of corona viruses infectivity through neutralization of HE-Protein with monoclonal antibodies, points to a role for HE-Protein in virus entry or virus release from infected cells.(32) E-Protein The small envelope protein (E-Protein) is typically 9-12 kDa protein that is a component of the virion envelope.(33-34) In comparison to other structural proteins, the E-Protein is much less abundant relative to M, N and S-Proteins.(33)(35) E-Proteins share a general structure with a short hydrophilic region on the amino terminus, followed by a large hydrophobic region, preceding a large hydrophilic carboxyl terminus.(35) Topologically E-Protein is an integral membrane protein presenting its carboxyl terminus on the cytoplasmic face of the endoplasmic reticulum (ER) or GB which corresponds to the interior of the assembled virion.(36) E-Protein localized primarily in the per-nuclear space of infected cells, although it also has been detected on the cell surface.(33-34) The main function of E-Protein is the formation of corona virus envelope.(21) For several corona virus, expression of M and E-Protein alone has been shown to be sufficient for the formation of virion like particles which are exported from the cell except the sever corona viruses.(36-37) In these, expression of M and N-Protein is alone sufficient for capsid formation.(38) Another function of E-Protein is its ability to induce apoptosis in the infected cells. (39) It also suggested that E-Protein appears to have a unique structural feature in that it forms a highly unusually short, palindromic transmembrane helical hairpin around a previously unidentified pseudo-center of symmetry.(40) It is through the action of this hairpin, by way of deforming the lipid bilayer and increasing membrane curvature, that there finally may be a molecular explanation of the E-Protein play vital role in virus budding.(21)(40) M-Protein The membrane glycoprotein (M-Protein) the most abundant envelope component is a triple membrane spanning protein with a short amino terminus on the virion exterior surface, alpha helical, and a large carboxyl terminal domain inside the virion envelope. (41-42) The protein is inserted into the ER membrane through the action of a signal sequence after being synthesized on membrane bound polysomes. (43) After being synthesized, the protein undergoes post-translational modification in the form of glycosylation.(21) Through the use of a virus like particle system it has been demonstrated that neither glycosylation of the M-Protein nor its interaction with the S-Protein is necessary for virus assembly.(44-45) Mature matrix protein accumulates in the GB and is not transported to the plasma-membrane.(46-47) The nature of the membrane targeting sequence that causes this accumulation in the GB varies for each corona virus. (42) One of the main function of M-Protein is to direct the incorporation of the S-Protein and the N-Protein into the budding virion particle.(48) It also determine that the expression of M and E-Protein alone is not

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sufficient for capsid formation, however the expression of M and N-Protein alone is able to produce virus capsid.(38) N-Protein Corona virus nucleocapsid protein (N-Protein) is 50-60 kDa highly basic protein which interacts with the viral genome in order to form viral nucleocapsid.(49-50) Translations of the protein occur on free polysomes and are rapidly phosphorylated on serine residues in the cytosol, the extent and physiological relevance of phosphorylation however remains unclear.(51) The synthesized protein consists of three highly conserved domains that are separated by spacer regions of variable length.(52) The most prominent feature of N-Protein is its ability to bind RNA. Several studies have pointed out the RNA binding region of the N-Protein to the second of the three conserved domain found in the N-Protein.(53-54) The complexes formed between N-Protein and viral genome (RNA) of corona virus is more easily disrupted at high salt concentration and offer little protection against RNAse. (55) The second type of interaction is N-Protein interaction with itself. Disulfide linked multimeric forms of the N-Protein have been shown to exist, where as carboxyl terminal conserved domain functions as a dimerization domain.(56-57) The third type of interaction is the N-Protein interaction with M-Protein which leads to the formation of virus particles. (58) It is also hypothesized that it may function in viral RNA synthesis, transcription translation and virus budding.(21) Specifically it has been shown that the N-Protein participates in MRN synthesis and also found that RNA synthesis was inhibited by greater than 90% when antibodies to N-Protein were introduced into an in-vitro synthesizing system.(59) This inhibition implies a critical role for the N-Protein in transcription and replication of the viral RNA.(21) Some N-Protein may be complexed with cellular membranes where it functions in budding of the virus.(60) N-Protein also may selectively activate the activator protein-1 (AP-1) signal transduction pathway except in SARS CoV.(61)

IV.

NON-STRUCTURAL PROTEIN

Replicase Protein Two replicase polyproteins, termed PP1a and PP1ab, are produced from two open reading frames (ORF1a and ORF1b) contained within the corona virus genome.(27)(49) PP1a is an polyproteins of about 486 kDa that has been predicted to include a papain-like protease (PLpro), two putative membrane proteins (MP1 = nsp4 and MP2 = nsp6), a picorna virus 3C-Like protease (3CLpro), and several other products of unknown function.(62) PP1ab is about 790 kDa and generated by ribosomal frame-shifting so that both ORF1a and ORF1b are included in the translation. It is predicted that ORF1b contains a helicase domain (nsp13) as well as the predicted core RNA polymerase (nsp12), exo-nuclease (nsp14), endo-ribonuclease (nsp15) and methyl-transferase (nsp16) activities.(63) A total number of 16 protein products (non structural proteins = nsp1-nsp16) produced from the processed pp1a and pp1ab polyproteins, are predicted to assemble into a membrane associated viral replication complex.(63-64) This processing of the polyproteins has been hypothesized to be coordinated by two virus-encoded proteinases, the picorna-virus 3C-like proteinase (3CL pro) and a papin-like proteinase (PLP).(63) The precursor proteins and mature processed replicase proteins likely mediated the progression from replication complex formation, followed by sub-genomic mRAN transcription and finally genome replication. Essential functions carried out by the replication complex are transcription of genome length negative and positive stranded RNAs. Replication complex activity has been shown to take place at double membrane vesicles in the host cell cytoplasm.(64) Corona Virus Specific Proteins In addition to the four main structural proteins (S, E, M and N-Protein) and the viral encoded RNA dependent RNA polymerase, there are nine other potential ORFs found in the corona virus genome that vary from 39-274 amino acids in length.(49-50)(64) The functions of these additional ORFs are largely unknown and further characterization of these proteins is necessary to be able to assess any impact on the virus life cycle.(21)

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U274 This is the largest additional ORFs (ORF30) and the second largest sub-genomic mRNA encoded protein, termed U274 because of its length of 274 amino acids. (65) The sub-genomic mRNA encoding U274 has been found in high quantities in infected cells and also has a strong match to the transcription regulating consensus sequence close to and upstream of its first ORF.(49-50)(64) The exact function of this protein is unknown but analysis of sera taken from patients infected unit the corona viruses has shown that the sera contains antibodies against the U274 protein which indicate that the protein may play a role in the biogenesis of corona viruses.(66) Interactions also observed between U274 protein and other structural proteins (M, E and S-Protein) suggest that the U274 protein may play a role in virus assembly.(65) U122 This protein is another group specific gene product encoded by ORF7a (also known ORFX4 or ORF8) partially. U122 name also derived from its amino acids length (122 amino acids). U122 protein found to the peri-nuclear region and is associated with the ER of infected cells. (49-50)(63) It play role through over expression to induce apoptosis.(67)

V.

VIRAL GENOME ORGANIZATION

Corona virus genomes are capped, poly-adenylated, positive sense, single stranded RNAs of 27-32 kb in length.(68) Because of positive sense genome, they are able to serve as mRNA and it has been shown that the purified genomic RNA is infectious.(2)(21) The leader RNA is a sequence of 65-98 nucleotides that is present at the 5’ end of the genome as well as at the end of 5’ ends of all sub-genomic mRNAs.(69) The leader sequence is followed by an untranslated region of approximately 200-400 nucleotides. There is another untranslated region of approximately 200 to 500 nucleotides at the 3' end of the genome which is followed by a poly (A) tail that varies in length. The sequences that make up both the 5' and 3' untranslated region are essential for RNA transcription and replication. The remaining genome is made up of 7 to 10 ORFs that encode the genes necessary for virus replication. Specifically for corona viruses, the genome contains 10 ORFs encoding the replicase gene, 4 structural proteins, and 5 potential nonstructural genes that are more than 50 amino acids in length. (27) The first ORF makes up two thirds of the viral genome (about 20 - 22 kb in length) and encodes a polyprotein that is the precursor of the viral polymerase. This gene actually consists of two over lapping ORF that is effectively combined into a single ORF through ribosomal frame shifting. The order in which the polymerase polyprotein and the four structural proteins found in all corona viruses is 5'-Pol-S-E-M-N-3'. Several other ORFs that encode a variety of nonstructural proteins can be found interspersed between the known structural proteins. (21) Some corona viruses also contain a gene that encodes for a hemagglutinin-esterase protein. The number of nonstructural genes present as well as their order in the viral genome, sequence, and method of expression vary widely among corona viruses.(70) The functions of most of these nonstructural proteins are widely unknown; some are even absent in genomes of some corona viruses.(71)

VI.

CORONA VIRUS LIFECYCLE

The initial step in any virus life cycle is the binding of virions to the plasma membrane of the host cell. The viral protein that is primarily responsible for the binding of the virion to the plasma membrane of the host cell is the S-Protein.(2)(10) It is the S-Protein that drives infection of the host cell by facilitating viral attachment to the target cell and promoting fusion between the host cell plasma membrane and the viral membrane. This fusion of the two membranes allows the insertion of the viral genome into the cellular cytoplasm. Viral attachment is accomplished through the interaction of the S-Protein and specificreceptor glycoproteins on the cell surface. Given its role in viral attachment, the S-Protein is the main determinant of viral tropism.(21) After binding to the specific cellular receptor, the virus enters the cell through the fusion event between the viral envelope and the plasma membrane or the endosomal membrane of the host cell. The pH of the environment has been shown to affect the efficiency of membrane fusion for some corona viruses such as Beta CoV whose optimum pH for cell-cell fusion is either neutral or slightly alkaline.(72) Given that these viruses may cause fusion of the viral envelop with www.irjmets.com

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the plasma membrane at the typical physiological pH of the extracellular environment, it is likely that they probably enter cells by virus-cell fusion at the plasma membrane. However, the efficiency of virus infectivity has been shown to be reduced through the use of lysosomotropic drugs which suggest that the virus utilizes the endosomal pathway for entry. (73) Further experimental evidence indicates that different corona viruses can enter cells either by the acidic pH-dependent endocytosis or by pH independent fusion at the plasma membrane.(74) Once the virus has entered the host cell, the next step in life cycle is the uncoating of the virus and the release of the genomic RNA into the cytoplasm. The mechanism for this uncoating and release is not currently clear and may require specific cellular proteins in addition to the incorporated viral elements.(75) Interactions between the S-Protein on the virion particle and specific receptor glycoproteins or glycans found on the surface of the target host cell facilitate virion binding to the plasma membrane of the host cell.(21) S-Protein mediated fusion between plasma membrane or endosomal membrane with the viral envelop allows penetration of the cell by the virus. Once inside the cell, gene one encoded by the +ssRNA genome is translated into a large polyprotein.(2)(21) Co-translational or posttranslational processing of this large polyprotein produces an RNA-dependent RNA polymerase as well as several other proteins that are involved in viral RNA synthesis. The genomic RNA is then used as a template by the RNA-dependent RNA polymerase and the other polyprotein products to synthesize negative stranded RNAs. (10) These negative stranded RNAs are subsequently used to produce genomic and sub-genomic mRNAs. These mRNAs have an overlapping nested set of 3' coterminal RNAs that possess a common leader sequence at the 5' end.

Each of the sub-genomic mRNAs, with few exceptions, only has their 5' most ORF translated into viral proteins effectively making them monocistronic.(21) Proteins produced include the S-Protein, M-Protein, E-Protein and N-Protein as well as several nonstructural proteins. Also the HE-Protein is produced in a small subset of corona viruses. Once translated, the N-Protein interacts in the cytoplasm with newly synthesized genomic RNA in order to form helical nucleocapsid. The M and E-Proteins are inserted in the ER and anchored in the GB.(2) The helical nucleocapsid, produced through N-Protein and RNA interactions, most likely first joins with M-Protein at the budding compartment which is located between the rough ER and the GB. Interactions between the M and E-Proteins elicit the budding of virions which encloses the helical nucleocapsid. Also associated in the GB, the S and HE-Proteins are translated on membrane-bound polysomes, inserted into the rough ER and then transported to the GB.(21) During protein transport, some of the S and HE-Proteins interact with the M-Protein and are incorporated into maturing virion particles. S and HE-Proteins not incorporated into virions are transported to the cellular surface where they may play a role in mediating cell-cell fusion or hem-adsorption, respectively.(10) Virions seem to be released by exocytosis-like fusion of smooth-walled vesicles that contain the virion particles with the plasma membrane. Virions also may remain attached to the plasma membranes of infected cells. The entire corona virus replication cycle takes place solely in the cytoplasm of the host cell.(21)

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Fig.2: Corona virus life cycle mechanism

VII.

VIRAL RECEPTORS

Receptors have been elucidated for several corona viruses. The cellular receptor for SARS-CoV and SARS CoV-2 was originally found to be the metallopeptidase angiotensin-converting enzyme 2 (ACE2) whereas for MERS CoV cellular receptor Dipeptidyl Peptidase-4 (DPP4) was recognized.(2)(21) ACE2 ACE2 is a type I transmembrane protein that is made up of 805 amino acids. It contains a single metalloprotease active site with an HEXXH zinc binding domain. (76) The ACE2 protein is synthesized in the human heart muscle, kidneys, testis, gastrointestinal tract, and the lungs. (77) In particular, high levels of ACE2 expression have been found by immunehistochemical examinations in the endothelium of intramyocardial and intrarenal vessels and in the renal tubular epithelium.(2)(32) The enzyme appears be the physiological counterweight of the related angiotensin-converting enzyme (ACE) which is known to cleave the inactive peptide angiotensin I in order to produce the highly potent vasoconstrictor angiotensin II.(10)(76) Specifically, ACE2 has been shown to cleave angiotensin I to the metabolite angiotensin, which in turn is cleaved to angiotensin.(78) There is significant overlap between the tissues that express the ACE2 protein and those tissues that are most correlated with CoV replication and symptomatic manifestation.(77) An obvious example of this overlap would be the lungs. The lungs are the primary site of CoV infection, and the lungs express the ACE2 protein. (79) A less obvious example is the gastrointestinal tract and kidneys. These areas have high levels of ACE2 expression and have also been shown to be an active site of CoV infection.(77-78) CoV has not, however, been found to replicate in the human heart which is a site a high expression of ACE2.(80) DPP4 DPP4 is the MERS CoV receptor, thus possessing advantages in mediating the infection of a corona virus.(10)(21)

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CD209L (L-SIGN) It is a homologue of CD209 and a type II transmembrane glycoprotein in the C-type lectin family that serves as adhesion receptors for ICAM-2 and ICAM-3.(81) The isoform that has been identified as the receptor for the CoV is composed of about 376 amino acids.(21) Structurally, the protein has a short cytoplasmic tail, a transmembrane domain, an extracellular stalk that contains seven repeats of a 23 amino acid sequence (KAAVGELXEKSKXQEIYQELTXL), and a large carboxyl terminal carbohydrate recognition domain.(82) This carbohydrate recognition domain has been shown to bind specifically to high mannose glycans on glycoproteins.(83) It has not yet been determined if it is this carbohydrate recognition domain that participates in viral interactions with the CoV S-Protein.(21)

VIII.

GENOME EXPRESSION

Once released into the cytoplasm, the viral RNA serves as a template for the synthesis of the viral RNA dependent RNA polymerase. This RNA dependent RNA polymerase first synthesizes negative stranded RNAs which are used as templates for the synthesis of multiple subgenomic RNAs as well as full length copies of the genome.(21) It has been shown that there are comparable levels of negative polarity genomic and subgenomic RNA to levels of corresponding positive sense genomic and subgenomic mRNA in infected cells.(84-85) Although levels were comparable, there was no singlestranded negative sense RNA found in the infected cell, only double-stranded.(85) These subgenomic RNAs account for all of the viral proteins except for the ORF 1ab polyprotein. Depending on the strain of the virus, coronaviruses produce five to seven subgenomic mRNAs. All of the mRNAs form a nested set that have common 3′ ends followed by a poly(A) tail. Each of these mRNAs, except for the smallest, contain two or more ORFs in which only the 5′ most ORF, with few exceptions, is translated. This effectively makes each subgenomic mRNA functionally monocistronic. The coronavirus subgenomic RNAs and the genomic RNA share an identical 5′ end leader sequence that is 65 to 98 bases long.(69) The leader sequence is a unique part of the viral genome and only shares partial homology with sequences found between each gene that have been termed both the transcription-associated sequence and the intergenic sequence.(86) The core intergenic sequence shares a common homology with the seven to eighteen nucleotides found at the 3′ end of the leader.(69) Depending on which cell type is used, coronavirus RNA synthesis occurs at membranous structures associated with the ER, late endosomes, or Golgi complex.(87) Studies have shown that in addition to the RNA dependent RNA polymerase, the N protein as well as other host components may be involved in viral RNA synthesis.(88-89)

IX.

REPLICATION OF GENOMIC RNA

The majority (95%) of the genome sized RNA in the infected cell is packaged into nucleocapsids and virions while the other 5% is used to synthesize mRNA encoding the polymerase polyprotein.(90) The replication of the full length RNA would seemingly require a different mechanism than transcription of the subgenomic mRNAs since replication of the full length RNA relies on continuous transcription rather than the discontinuous transcription of subgenomic mRNA synthesis.(21) This mechanism is also utilized in subgenomic mRNA synthesis. The quantity of UCUAA repeats at the 3’ end of the leader have been shown to undergo rapid evolution and it is at these regions that high levels of RNA recombination occur.(91-92) These studies suggest that genomic RNA replication uses the leader dissociation discontinous synthesis involved in mRNA transcription. (21) Defective Interfering (DI) RNAs have been used to delineate which cis-acting sequences are required for the replication of the genomic RNA.(93)

X.

TRANSLATION OF VIRAL PROTEINS

Transcription of the viral genome results in multiple subgenomic mRNAs which, with the exception of the smallest mRNA, contain two or more ORFs in which only the 5’ most ORF, with few exceptions, is translated. Structural proteins are translated by a cap-dependent ribosomal scanning mechanism from separate mRNAs.(94) Translation in virus infected lysates is enhanced by a 5’ leader sequence found in all of the subgenomic mRNAs. This enhancement of translation may give viral mRNAs an advantage as host www.irjmets.com

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cell translation is being shut off by the viral infection. Besides the structural proteins, the virus encoded polymerase is encoded within two large overlapping ORFs (ORF1AB) that utilize a ribosomal frameshifting mechanism in order to synthesize the entire polyprotein. (95) This translation is initiated by the conventional cap-dependent translation mechanism. For several coronaviruses, the second or third ORF has the highest efficiency of translation. This is due to the presence of an internal ribosomal entry site just before the ORF that allows the ribosome to bypass the preceding ORFs. This allows the translation of the targeted ORF through a cap-independent translation mechanism.(96) Other than the virus encoded polymerase, there are other proteins in the genome that contain more than one ORF. These proteins are translated by unknown mechanisms.(97-98)

XI.

ASSEMBLY AND RELEASE OF VIRIONS

The initial step in virion assembly is the formation of helical nucleocapsids by the nucleocapsid proteins binding to the viral RNA. This binding of viral RNA to nucleocapsid protein is facilitated by a sequence of nucleotides found in ORF 1B which is only present in the genomic-length RNA. This sequence has been shown to bind the nucleocapsid protein, and is thought to facilitate packaging of viral genomes into virions.(99) This putative packaging signal maps to a region within ORF 1b and has been shown to function by maintaining secondary structure,(100) which allows interaction between the packaging signal and the RNA being packaged.(101) Packaging of reporter RNAs has also been accomplished by fusing a homologous region of the bovine corona virus genome to the non-viral RNAs.(102) Once the nucleocapsid forms, it interacts with the matrix protein at cellular membranes of the ER or the Golgi complex. The nucleocapsid protein can only be incorporated into virions when complexed with the viral RNA; no unbound protein is able to be packaged into virions.(103) This suggests that the M protein must interact with the viral genome directly. Alternatively, a conformational change may occur when the N protein interacts with the viral genome promoting the interaction between the N and M proteins. This interaction may enable the nucleocapsid to be packaged into budding virus particles formed on the membranes of the ER and the Golgi while simultaneously allowing the formation of the spherical internal core shell surrounding the nucleocapsid.(104) Viruses like particles are able to be formed by the expression of the M and E proteins without the presence of any other additional viral proteins. (103) This suggests, for most corona viruses, that the interactions between the M and E proteins facilitate the formation of virion particles. Mutations introduced into the E and M proteins have shown altered virus morphology and abrogated virus like particle formation, respectively.(105) Regardless of the corona virus type, these experiments show that the spike protein is dispensable in virus particle formation. The budding compartment, located between the ER and Golgi, is the site at which virus budding is first detected. (106) Interaction between the M protein, which accumulates at the budding compartment and the E protein is thought to trigger virus budding. (106) Because virus budding has only been shown to occur at the budding compartment and the E protein has been detected at sites other than that of virus budding, it is thought that the M protein dictates the location of virus budding.(107) Although the E protein is required for particle formation, it may only serve as a scaffolding protein which is not essential part of virus maturation. This is because the E protein is present in such low quantities compared to that of the M protein.(103) It may function in the pinching off of the budding virions at the budding compartment because it has been shown to induce curvature of intracellular membranes containing M. (103) Incorporation of the spike protein and the HE protein into virions is directed by interactions with the M protein which occurs in the pre-Golgi complex.(108) The glycoproteins S and HE are processed as the virions pass through the Golgi where they may undergo additional morphological changes resulting in the compact, electron dense internal core typical of the mature virus particle.(109) After the Golgi, mature virions accrue in large, smooth-walled vesicles that fuse with plasma membrane in order to release the virions into the extracellular space. Although virus release is restricted to certain areas of the cell, the exact mechanism dictating this site restriction is poorly understood.(110)

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CONCLUSION

Overall this is the review article which shows the some aspects of the Corona viruses with valuable disclaimers. The present scenario of this article based on Corona virus structure and functional status that helpful to known morphology and mechanism of action for academic and research area.

XIII. [1] [2]

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