Prior the development of any anti-viral agents or vaccine, the initial step requires comprehensive understanding of viral replication process that augments the number of viral copies in the host cell. The replication of typical virus has following step in their lifecycle are (1):
1. Attachment and entry
2. Uncoating
3. Gene expression
4. Genome replication
5. Assembly and maturation
6. Egress and release
2. Ion channel blockers e.g. Amantidine, Rimantidine
3. Polyprotein processing inhibitors e.g. Boceprevir, Telaprevir
4. Polymerase inhibitors e.g. Acyclovir, Zidovudine, Nevirapine
5. Integrase inhibitors e.g. Raltegravir
6. Protease inhibitors e.g. Saquinavir, Ritonavir, Lopinavir
7. Neuraminidase inhibitors e.g. Zanamivir, Oseltamivir
8. Nucleotide analog e.g. Remdesivir
1. Attachment and entry
2. Uncoating
3. Gene expression
4. Genome replication
5. Assembly and maturation
6. Egress and release
Anti-viral agents target the sites and inhibits the above steps (1). Based on the steps of drug inhibition, the anti-viral agents can be of the following types:
1. Attachment and entry inhibitors e.g. Enfuvirtide, Maraviroc2. Ion channel blockers e.g. Amantidine, Rimantidine
3. Polyprotein processing inhibitors e.g. Boceprevir, Telaprevir
4. Polymerase inhibitors e.g. Acyclovir, Zidovudine, Nevirapine
5. Integrase inhibitors e.g. Raltegravir
6. Protease inhibitors e.g. Saquinavir, Ritonavir, Lopinavir
7. Neuraminidase inhibitors e.g. Zanamivir, Oseltamivir
8. Nucleotide analog e.g. Remdesivir
This section attempts to discuss on the anti-viral agents strategies with reference to the replication cycle of the SARS-CoV-2, which is an enveloped virus belonging to the family Coronaviridae. The lifecycle of SARS-CoV-2 is explained for the purpose of illustration. The steps leading to the multiplication of viral copies in the human host has following steps.
1. Attachment and Entry: SARS-CoV-2 is an enveloped positive sense virus capable of exhibiting illness ranging from mild to moderate and in some cases severity leads to death. The host cell receptor is present in the alveoli of the lungs called as Angiotensin Converting Enzyme 2 (ACE2) which is attached to the spike protein present in the virus. The spike protein is club-like structure and contains two subunits (S1 and S2) which is capable to bind in the specific host cells having ACE2 receptor in it. Two modes of viral entry have been recognized, one being the entry via endosomes and the other by plasma membrane fusion. However, spike proteins (S1 and S2) of SARS-CoV-2 mediate attachment to the membrane of the host cell engaging ACE2. The steps in the invasion of virus to the host cell in given in the figure 1.
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| Figure 1. Step in viral attachment and entry of SARS-CoV-2 *ACE2- Angiotensin Concerting Enzyme 2 *TMPRSS2- Transmembrane protease serine 2 |
1.1 Entry via endosomes: Following attachment of the virus with ACE2, the virus is internalized inside the endosome in the low pH.
1.2 Entry via fusion of plasma membrane: Plasma membrane fusion is facilitated by the transmembrane protease serine 2 (TMPRSS2) that lies in close proximity to ACE2. This type of fusion is more efficient for viral replication than the endosomal membrane fusion pathway. It is because this type of fusion does not activate the host cell antiviral immune response (2) . Besides SARS-CoV-2, viruses found to use the TMPRSS2 include Influenza virus and other human coronaviruses HCoV-229E, MERS-CoV and SARS-CoV (4).
2. Uncoating: The viruses release it genomic material either by endosomal fusion or by plasma membrane fusion at low pH. Proteolytic cleavage of S protein and fusion of viral and endosomal membranes trigger release of viral RNA into host cell cytoplasm (22).
3. Translation and processing of polyproteins: After the viral RNA is released into the host cell, polyproteins are translated. The genome encodes non-structural proteins (NSPs) and structural proteins. Two-third portion of the viral genome are reponsible for the genes encoding non-structural proteins (7). The non-structural proteins (NSPs) are responsible for viral RNA synthesis while the structural proteins are important for viral assembly. Like other beta-coronaviruses, the genome of SARS-CoV-2 is arranged in the order of 5'-replicase(ORF1a/b)-spike(S)-envelope(E)-membrane(M)--nucleocapsid(N)-poly(A)-3' (3). The 5' to 3' end of the genome encodes polyproteins, Replicase polyprotein 1a (pp1a) and Replicase polyprotein 1ab (pp1ab); Spike glycoprotein (S); ORF3a protein (NS3a); Envelope small membrane protein (E); Membrane protein (M); ORF6 protein; ORF7a protein; ORF7b protein; ORF8 protein; Nucleoprotein (N); ORF9b protein; ORF14 and Hypothetical ORF10 protein (5). Thus, NSps, structural proteins as well as several accessory protein together function in the viral multiplication and release from the host cell.
Initially, polyproteins pp1a and pp1ab are translated which are cleaved by the Papain-like protease (PLpro) and 3-Chymotrypsin-like protease (3CLpro) to form functional non-structural proteins (NSPs) such as RNA dependent RNA polymerase (RdRp) and form the replication complex.
4. Transcription and Replication: RdRp is resposible for replication of structural proteins RNA. Structural proteins S1,S2, Envelope (E), Membrane (M) are translated by ribosomes that are bound to the endoplasmic reticulum (ER). These are presented on the surface of ER as preparation of viral assembly. The nucleocapsid (N) remains in cytoplasm.
5. Assembly and Maturation: The nucleocapsid (N) fuse together with the genomic RNA fuse with viral structural proteins from ER through the Golgi apparatus to the cell surface via small vesicles.
6. Release: The mature virus is released from the cell and the new virus again invades other healthy cells of the body.
The overall process of replication of SARS-CoV-2 is illustrated in figure 2.
2. Uncoating: The viruses release it genomic material either by endosomal fusion or by plasma membrane fusion at low pH. Proteolytic cleavage of S protein and fusion of viral and endosomal membranes trigger release of viral RNA into host cell cytoplasm (22).
3. Translation and processing of polyproteins: After the viral RNA is released into the host cell, polyproteins are translated. The genome encodes non-structural proteins (NSPs) and structural proteins. Two-third portion of the viral genome are reponsible for the genes encoding non-structural proteins (7). The non-structural proteins (NSPs) are responsible for viral RNA synthesis while the structural proteins are important for viral assembly. Like other beta-coronaviruses, the genome of SARS-CoV-2 is arranged in the order of 5'-replicase(ORF1a/b)-spike(S)-envelope(E)-membrane(M)--nucleocapsid(N)-poly(A)-3' (3). The 5' to 3' end of the genome encodes polyproteins, Replicase polyprotein 1a (pp1a) and Replicase polyprotein 1ab (pp1ab); Spike glycoprotein (S); ORF3a protein (NS3a); Envelope small membrane protein (E); Membrane protein (M); ORF6 protein; ORF7a protein; ORF7b protein; ORF8 protein; Nucleoprotein (N); ORF9b protein; ORF14 and Hypothetical ORF10 protein (5). Thus, NSps, structural proteins as well as several accessory protein together function in the viral multiplication and release from the host cell.
Initially, polyproteins pp1a and pp1ab are translated which are cleaved by the Papain-like protease (PLpro) and 3-Chymotrypsin-like protease (3CLpro) to form functional non-structural proteins (NSPs) such as RNA dependent RNA polymerase (RdRp) and form the replication complex.
4. Transcription and Replication: RdRp is resposible for replication of structural proteins RNA. Structural proteins S1,S2, Envelope (E), Membrane (M) are translated by ribosomes that are bound to the endoplasmic reticulum (ER). These are presented on the surface of ER as preparation of viral assembly. The nucleocapsid (N) remains in cytoplasm.
5. Assembly and Maturation: The nucleocapsid (N) fuse together with the genomic RNA fuse with viral structural proteins from ER through the Golgi apparatus to the cell surface via small vesicles.
6. Release: The mature virus is released from the cell and the new virus again invades other healthy cells of the body.
The overall process of replication of SARS-CoV-2 is illustrated in figure 2.
 |
| Figure 2. Replication of SARS-CoV-2 |
Based on the life-cycle of the SARS-CoV-2, there are various target sites which help in inhibition of maturation of virus inside host cell. Various antiviral-drugs are repurposed which were efficient in the past and targets to the sites of the virus that shares close nucleotide seqeunce proximity to SARS-CoV-2 (such as MERS-CoV, SARS-CoV). The anti-viral drugs and their mode of action are discussed in this section.
1. Inhibition of viral entry: Griffithsin is an inhibitor that bind to the spike glycoprotein preventing the viral entry (6) .
2. Protease inhibitors:
i) Inhibition of TRMPSS2: TRMPSS2 is an extracellular protease that helps in the proteolytic cleavage of ACE2 receptor during viral uptake and also the cleavage of the spike glycoprotein (7). Inhibitor to TRMPSS2 can be another target for inhibition of virus entry during the event of plasma membrane fusion.
ii) Inhibition of PLpro and 3-CLpro: PLpro and 3-CL-pro are essential protease that regulates further process of viral replication. Inhibitors of these protein can be a probable target for inhibition of viral replication.
PLpro cleaves N-terminus of the replicase polyprotein to release Nsp1, Nsp2 and Nsp3 (12). Likewise, 3CLpro (also called Nsp5) mediates maturation of non-structural proteins. It is at first cleaved during poly-protein processing and ultimately cleaved to release Nsp4-Nsp16 (3).
3. Polyprotein processing inhibitors: Without proper processing of the viral genome, functional structural proteins cannot be generated by the virus. Inhibitors to polyprotein processing such as pp1a and pp1ab can help prevent the replication of virus in host cell.
4. Ion channel blockers: Virus requires an acidic pH in the fusion process so that it can expose its genomic materials (e.g. single stranded positive sense RNA in case of SAR-CoV-2). Without release of genetic materials further protein synthetic machinery cannot be carried out. Blocking of the porin channels that increase the pH can also be done preventing virus to release its genome in the cytoplasm.
5. RdRp inhibitor: The viral replication is mediated in general by kinase signaling pathway. Protein kinases are the enzymes that covalently modify proteins. Modification is done by attaching the phosphate group from ATP molecule to serine, threonine and/or tyrosine residues. Inhibiting this pathway can inhibit the function of RdRp in virus (9). In-vitro activity of Src-family of tyrosine kinases (SFK) have been identified in MERS-CoV (a coronavirus that shares 50% nucleotide sequence homology) (10). The core structure of RdRp are conserved and structure resembles that of cupped right hand and consists of fingers, palm and thumb subdomains (11). Moreover, the RdRp encoded by NSP12 has been reported to have unique β-hairpin domain at its N-terminus. This structure can be utilized to design the new antiviral option for viral RdRp (23).
6. NSP9 disruption: NSP9 is a RNA binding protein when present in dimeric form induces the viral infection. Disruption of this protein can be a probable target to overcome the infection.
Functions of other non-structural proteins are summarized in Table 1.
1. Inhibition of viral entry: Griffithsin is an inhibitor that bind to the spike glycoprotein preventing the viral entry (6) .
2. Protease inhibitors:
i) Inhibition of TRMPSS2: TRMPSS2 is an extracellular protease that helps in the proteolytic cleavage of ACE2 receptor during viral uptake and also the cleavage of the spike glycoprotein (7). Inhibitor to TRMPSS2 can be another target for inhibition of virus entry during the event of plasma membrane fusion.
ii) Inhibition of PLpro and 3-CLpro: PLpro and 3-CL-pro are essential protease that regulates further process of viral replication. Inhibitors of these protein can be a probable target for inhibition of viral replication.
PLpro cleaves N-terminus of the replicase polyprotein to release Nsp1, Nsp2 and Nsp3 (12). Likewise, 3CLpro (also called Nsp5) mediates maturation of non-structural proteins. It is at first cleaved during poly-protein processing and ultimately cleaved to release Nsp4-Nsp16 (3).
3. Polyprotein processing inhibitors: Without proper processing of the viral genome, functional structural proteins cannot be generated by the virus. Inhibitors to polyprotein processing such as pp1a and pp1ab can help prevent the replication of virus in host cell.
4. Ion channel blockers: Virus requires an acidic pH in the fusion process so that it can expose its genomic materials (e.g. single stranded positive sense RNA in case of SAR-CoV-2). Without release of genetic materials further protein synthetic machinery cannot be carried out. Blocking of the porin channels that increase the pH can also be done preventing virus to release its genome in the cytoplasm.
5. RdRp inhibitor: The viral replication is mediated in general by kinase signaling pathway. Protein kinases are the enzymes that covalently modify proteins. Modification is done by attaching the phosphate group from ATP molecule to serine, threonine and/or tyrosine residues. Inhibiting this pathway can inhibit the function of RdRp in virus (9). In-vitro activity of Src-family of tyrosine kinases (SFK) have been identified in MERS-CoV (a coronavirus that shares 50% nucleotide sequence homology) (10). The core structure of RdRp are conserved and structure resembles that of cupped right hand and consists of fingers, palm and thumb subdomains (11). Moreover, the RdRp encoded by NSP12 has been reported to have unique β-hairpin domain at its N-terminus. This structure can be utilized to design the new antiviral option for viral RdRp (23).
6. NSP9 disruption: NSP9 is a RNA binding protein when present in dimeric form induces the viral infection. Disruption of this protein can be a probable target to overcome the infection.
Functions of other non-structural proteins are summarized in Table 1.
Table 1. Function of non-structural proteins
| Non-structural Proteins | Function | Reference |
| NSP1 and NSP2 | Suppression of host gene expression | 13 |
| NSP3 and NSP5 | Formation of M protease | 13 |
| NSP4 and NSP6 | Transmembrane protein | 15 |
| NSP7 and NSP8 | Acts as Primase | 16 |
| NSP9 | RNA-binding protein; dimeric form helps in viral infection | 16 |
| NSP10 | Co-factor for replicative enzymes | 17 |
| NSP12 | RdRp activity. | 18 |
| NSP13 | Shows helicase activity | 19 |
| NSP14 and NSP15 | Exoribonuclease activity | 20 |
| NSP16 | Methyl-tranferase activity in conjunction with NSP10 | 21, 24 |
7. Nucleotide analog: The nucleotide analogue terminates the protein synthesis. Currently, Remdesivir has been extensively used to manage the patient with SARS-CoV-2 infection.
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