08-04-2021, 12:38 AM
Entry of coronaviruses
Coronavirus S proteins are homotrimeric class I fusion glycoproteins that are divided into two functionally distinct parts (S1 and S2) (Fig. 2). The surface-exposed S1 contains the receptor-binding domain (RBD) that specifically engages the host cell receptor, thereby determining virus cell tropism and pathogenicity. The transmembrane S2 domain contains heptad repeat regions and the fusion peptide, which mediate the fusion of viral and cellular membranes upon extensive conformational rearrangements10,11,12. Shortly after the 2002–2003 SARS-CoV outbreak, ACE2 was identified as the functional receptor that enables infection by SARS-CoV13. The high genomic and structural homology between the S proteins of SARS-CoV and SARS-CoV-2 (76% amino acid identity) supported the identification of ACE2 as the cell-surface receptor for SARS-CoV-2 (refs12,14,15,16). Remarkably, essential SARS-CoV contact residues that interact with ACE2 were highly conserved in SARS-CoV-2 as well as in members of the species Severe acute respiratory syndrome-related coronavirus that use ACE2 or have similar amino acid side chain properties14,15,17,18,19. These data were corroborated by the atomic resolution of the interface between the SARS-CoV-2 S protein and ACE2 (refs16,19,20,21). By contrast, the bat Severe acute respiratory syndrome-related coronavirus RaTG13 S sequence (93.1% nucleotide identity to SARS-CoV-2) shows conservation of only one out of six amino acids directly involved in ACE2 binding, even though, based on the entire genomic sequence, RaTG13 is the closest relative of SARS-CoV-2 known to date (96.2%)14 (Box 2).
Viral gene expression and RNA synthesis
Genome translation
The release of the coronavirus genome into the host cell cytoplasm upon entry marks the onset of a complex programme of viral gene expression, which is highly regulated in space and time. The translation of ORF1a and ORF1b from the genomic RNA produces two polyproteins, pp1a and pp1ab, respectively. The latter results from a programmed –1 ribosomal frameshift at the short overlap of ORF1a and ORF1b4. Ribosome profiling revealed that the efficiency of the frameshift between ORF1a and ORF1b lies between 45% and 70% in the case of SARS-CoV-2 (ref.60), similar to that measured for mouse hepatitis virus (MHV)61. This determines the stoichiometry between pp1a and pp1ab, with pp1a being approximately 1.4–2.2 times more expressed than pp1ab60. Sixteen non-structural proteins are co-translationally and post-translationally released from pp1a (nsp1–11) and pp1ab (nsp1–10, nsp12–16) upon proteolytic cleavage by two cysteine proteases that are located within nsp3 (papain-like protease; PLpro) and nsp5 (chymotrypsin-like protease) (Fig. 3). The protease residing in nsp5 is frequently referred to as 3C-like protease (3CLpro), because of its similarities to the picornaviral 3C protease, or as main protease (Mpro), because it is responsible for proteolytic processing of the majority of polyprotein cleavage sites. Proteolytic release of nsp1 is known to occur rapidly62, which enables nsp1 to target the host cell translation machinery63,64,65. Nsp2–16 compose the viral RTC and are targeted to defined subcellular locations where interactions with host cell factors determine the course of the replication cycle66,67,68. Nsp2–11 are believed to provide the necessary supporting functions to accommodate the viral RTC, such as modulating intracellular membranes, host immune evasion and providing cofactors for replication, whereas nsp12–16 contain the core enzymatic functions involved in RNA synthesis, RNA proofreading and RNA modification4,67. RNA synthesis is performed by the nsp12 RNA-dependent RNA polymerase (RdRP) and its two cofactors nsp7 and nsp8, the latter with proposed primase or 3′-terminal adenylyltransferase activity4,67,69,70. Notably, nsp14 provides a 3′–5′ exonuclease activity that assists RNA synthesis with a unique RNA proofreading function71. The coronavirus capping machinery, which is not yet fully elucidated, is composed of nsp10, which functions as a cofactor, nsp13, which provides the RNA 5′-triphosphatase activity, and nsp14 and nsp16, which perform the functions of N7-methyltransferase and 2′-O-methyltransferase, respectively67,72,73,74. Notably, one key enzyme typically involved in the formation of the 5′ cap structure, the guanylyltransferase, has not yet been identified in coronaviruses.
The establishment of the viral RTC is crucial for virus replication and thus a promising target for antivirals against SARS-CoV-2. One such target is Mpro, which resides in nsp5. Mpro releases the majority of nsps from the polyproteins and is essential for the viral life cycle. Furthermore, as Mpro is very sequence specific, compounds that structurally mimic those cleavage sites can specifically target the viral protease with little or no impact on host cellular proteases75,76,77. Based on structural analysis of the protein, multiple research groups have successfully developed lead compounds that block Mpro function in cell culture assays, thus providing frameworks that could aid in rapid drug discovery75,77.
RNA synthesis
Viral genomic replication is initiated by the synthesis of full-length negative-sense genomic copies, which function as templates for the generation of new positive-sense genomic RNA. These newly synthesized genomes are used for translation to generate more nsps and RTCs or are packaged into new virions. A hallmark of coronaviruses and most members of the order Nidovirales is the discontinuous viral transcription process, first proposed by Sawicki and Sawicki78, that produces a set of nested 3′ and 5′ co-terminal subgenomic RNAs (sgRNAs)78,79 (Fig. 4). During negative-strand RNA synthesis, the RTC interrupts transcription following the encounter of transcription regulatory sequences (TRSs) that are located upstream to most ORFs in the 3′ one-third of the viral genome. At these TRS elements, also called TRS ‘body’, the synthesis of the negative-strand RNA stops and is re-initiated at the TRS adjacent to a leader sequence (TRS-L) located at about 70 nucleotides from the 5′ end of the genome78,79,80,81,82. This discontinuous step of coronavirus RNA synthesis involves the interaction between complementary TRSs of the nascent negative strand RNA (negative-sense TRS body) and the positive strand genomic RNA (positive-sense TRS-L). Upon re-initiation of RNA synthesis at the TRS-L region, a negative strand copy of the leader sequence is added to the nascent RNA to complete the synthesis of negative-strand sgRNAs. The discontinuous step of negative strand RNA synthesis results in the production of a set of negative-strand sgRNAs that are then used as templates to synthesize a characteristic nested set of positive-sense sg mRNAs that are translated into structural and accessory proteins. Although the coronavirus sg mRNAs are structurally polycistronic, it is assumed that they are functionally monocistronic and that only the first ORF at the 5′ end, which is absent in the next smaller sgRNA, is translated from each sgRNA78,81.
The TRS elements for SARS-CoV-2 have already been determined by RNA sequencing analyses of viral RNAs80,83. Like for SARS-CoV, the consensus TRS core of SARS-CoV-2 is 5′-ACGAAC-3′ and eight sg mRNAs have been shown to be produced in SARS-CoV-2-infected cells (sg mRNAs 2–9). In addition to canonical sgRNAs, recent reports also determined the existence of numerous non-canonical RNA products of discontinuous transcription, including fusions of the 5′ leader sequence to unexpected 3′ sites, TRS-L independent long-distance fusions, and local fusions resulting in small deletions mainly in the structural and accessory genes60,80. However, it remains to be determined whether all of these non-canonical sgRNAs truly arise by discontinuous transcription or whether they represent RNAs that result from recombination. Nevertheless, similar findings were previously reported for other coronaviruses, including MHV61 and HCoV-229E81, which indicates an enhanced coding potential for coronaviruses80. Overall, these unexpected fusion events may drive coronavirus evolution through variant generation, and novel ORFs could encode additional accessory proteins that are involved in either viral replication or modulation of the host immune response60,80.
The RdRP residing in nsp12 is the centrepiece of the coronavirus RTC and has been suggested as a promising drug target as it is a crucial enzyme in the virus life cycle both for replication of the viral genome but also for transcription of sgRNAs. The structure of the SARS-CoV-2 RdRP nsp12 and its cofactors nsp7 and nsp8 has been elucidated and shows a high degree of conservation to the SARS-CoV structure69,84,85. The amino acid sequence of the SARS-CoV and SARS-CoV-2 RdRPs show a >95% similarity with most changes located in the nidovirus RdRP-associated nucleotidyltransferase domain, which, despite being a genetic marker of Nidovirales, has yet to be functionally elucidated69. The structural similarities of the RdRP active site, including conserved key amino acid residues, with other positive-sense RNA viruses suggest the possibility to repurpose known drugs that are effective against other RNA viruses69. One of the most promising candidates is the phosphoramidate remdesivir (RDV), which, in its triphosphate form, acts as a substrate for viral RdRPs and competes with ATP86. RDV has shown potential as an antiviral agent against a broad range of RNA viruses, including Filoviridae (for example, Ebola virus), Paramyxoviridae (for example, Nipah virus) and Pneumoviridae (for example, respiratory syncytial virus) as well as other coronaviruses, including SARS-CoV and MERS-CoV86,87. The RdRP of SARS-CoV-2 selectively incorporates RDV over ATP, which subsequently results in a delayed-chain termination86,88. In contrast to classic nucleoside analogues that lead to immediate termination of the synthesis reaction after incorporation, the RdRP continues for three nucleotides after RDV has been incorporated before chain termination. Nucleotide analogues like RDV may have limited efficacy owing to the proofreading function of the exonuclease domain contained in nsp14 (ExoN)89. The corrective function that is exerted by ExoN is not only responsible for maintaining the stability of the coronavirus genome but also enables the excision of erroneous mutagenic nucleotides71,89. The mode of action observed for RDV might be an explanation for its increased efficiency over other nucleoside analogues as the delayed-chain termination could lead to improved evasion from the proofreading function of nsp14. The current model suggests steric hindrance as a likely reason for termination, disturbing the positioning of the RNA and thus hampering the translocation to the next position86,88. RDV was shown to reduce virus replication of SARS-CoV-2 in vitro90 and was demonstrated to restrict clinical symptoms of SARS-CoV-2 in rhesus macaques upon early pre-symptomatic treatment91. However, a recent randomized, double-blind, placebo-controlled clinical trial in humans with severe COVID-19 showed limited clinical efficacy of RDV treatment92 and further studies will be necessary. Another promising candidate is the purine analogue favipiravir (FPV), which has been shown to effectively target multiple RNA viruses93. Although the mechanism of action is not yet completely understood, a recent study of the in vitro mechanism of FPV suggested a combination of chain termination, slowed RNA synthesis and lethal mutagenesis as the mode of action against SARS-CoV-2, which indicates that FPV might be used to effectively restrict viral replication93. Indeed, results of an experimental pilot study showed that using FPV as treatment against COVID-19 led to increased recovery and faster viral clearance times in treated patients compared to control treatments94. Clinical studies with both RDV and FPV are currently ongoing and will establish whether these compounds are effective antivirals to treat coronavirus infections93.
[TBC below]
https://www.nature.com/articles/s41579-020-00468-6
Coronavirus S proteins are homotrimeric class I fusion glycoproteins that are divided into two functionally distinct parts (S1 and S2) (Fig. 2). The surface-exposed S1 contains the receptor-binding domain (RBD) that specifically engages the host cell receptor, thereby determining virus cell tropism and pathogenicity. The transmembrane S2 domain contains heptad repeat regions and the fusion peptide, which mediate the fusion of viral and cellular membranes upon extensive conformational rearrangements10,11,12. Shortly after the 2002–2003 SARS-CoV outbreak, ACE2 was identified as the functional receptor that enables infection by SARS-CoV13. The high genomic and structural homology between the S proteins of SARS-CoV and SARS-CoV-2 (76% amino acid identity) supported the identification of ACE2 as the cell-surface receptor for SARS-CoV-2 (refs12,14,15,16). Remarkably, essential SARS-CoV contact residues that interact with ACE2 were highly conserved in SARS-CoV-2 as well as in members of the species Severe acute respiratory syndrome-related coronavirus that use ACE2 or have similar amino acid side chain properties14,15,17,18,19. These data were corroborated by the atomic resolution of the interface between the SARS-CoV-2 S protein and ACE2 (refs16,19,20,21). By contrast, the bat Severe acute respiratory syndrome-related coronavirus RaTG13 S sequence (93.1% nucleotide identity to SARS-CoV-2) shows conservation of only one out of six amino acids directly involved in ACE2 binding, even though, based on the entire genomic sequence, RaTG13 is the closest relative of SARS-CoV-2 known to date (96.2%)14 (Box 2).
Viral gene expression and RNA synthesis
Genome translation
The release of the coronavirus genome into the host cell cytoplasm upon entry marks the onset of a complex programme of viral gene expression, which is highly regulated in space and time. The translation of ORF1a and ORF1b from the genomic RNA produces two polyproteins, pp1a and pp1ab, respectively. The latter results from a programmed –1 ribosomal frameshift at the short overlap of ORF1a and ORF1b4. Ribosome profiling revealed that the efficiency of the frameshift between ORF1a and ORF1b lies between 45% and 70% in the case of SARS-CoV-2 (ref.60), similar to that measured for mouse hepatitis virus (MHV)61. This determines the stoichiometry between pp1a and pp1ab, with pp1a being approximately 1.4–2.2 times more expressed than pp1ab60. Sixteen non-structural proteins are co-translationally and post-translationally released from pp1a (nsp1–11) and pp1ab (nsp1–10, nsp12–16) upon proteolytic cleavage by two cysteine proteases that are located within nsp3 (papain-like protease; PLpro) and nsp5 (chymotrypsin-like protease) (Fig. 3). The protease residing in nsp5 is frequently referred to as 3C-like protease (3CLpro), because of its similarities to the picornaviral 3C protease, or as main protease (Mpro), because it is responsible for proteolytic processing of the majority of polyprotein cleavage sites. Proteolytic release of nsp1 is known to occur rapidly62, which enables nsp1 to target the host cell translation machinery63,64,65. Nsp2–16 compose the viral RTC and are targeted to defined subcellular locations where interactions with host cell factors determine the course of the replication cycle66,67,68. Nsp2–11 are believed to provide the necessary supporting functions to accommodate the viral RTC, such as modulating intracellular membranes, host immune evasion and providing cofactors for replication, whereas nsp12–16 contain the core enzymatic functions involved in RNA synthesis, RNA proofreading and RNA modification4,67. RNA synthesis is performed by the nsp12 RNA-dependent RNA polymerase (RdRP) and its two cofactors nsp7 and nsp8, the latter with proposed primase or 3′-terminal adenylyltransferase activity4,67,69,70. Notably, nsp14 provides a 3′–5′ exonuclease activity that assists RNA synthesis with a unique RNA proofreading function71. The coronavirus capping machinery, which is not yet fully elucidated, is composed of nsp10, which functions as a cofactor, nsp13, which provides the RNA 5′-triphosphatase activity, and nsp14 and nsp16, which perform the functions of N7-methyltransferase and 2′-O-methyltransferase, respectively67,72,73,74. Notably, one key enzyme typically involved in the formation of the 5′ cap structure, the guanylyltransferase, has not yet been identified in coronaviruses.
The establishment of the viral RTC is crucial for virus replication and thus a promising target for antivirals against SARS-CoV-2. One such target is Mpro, which resides in nsp5. Mpro releases the majority of nsps from the polyproteins and is essential for the viral life cycle. Furthermore, as Mpro is very sequence specific, compounds that structurally mimic those cleavage sites can specifically target the viral protease with little or no impact on host cellular proteases75,76,77. Based on structural analysis of the protein, multiple research groups have successfully developed lead compounds that block Mpro function in cell culture assays, thus providing frameworks that could aid in rapid drug discovery75,77.
RNA synthesis
Viral genomic replication is initiated by the synthesis of full-length negative-sense genomic copies, which function as templates for the generation of new positive-sense genomic RNA. These newly synthesized genomes are used for translation to generate more nsps and RTCs or are packaged into new virions. A hallmark of coronaviruses and most members of the order Nidovirales is the discontinuous viral transcription process, first proposed by Sawicki and Sawicki78, that produces a set of nested 3′ and 5′ co-terminal subgenomic RNAs (sgRNAs)78,79 (Fig. 4). During negative-strand RNA synthesis, the RTC interrupts transcription following the encounter of transcription regulatory sequences (TRSs) that are located upstream to most ORFs in the 3′ one-third of the viral genome. At these TRS elements, also called TRS ‘body’, the synthesis of the negative-strand RNA stops and is re-initiated at the TRS adjacent to a leader sequence (TRS-L) located at about 70 nucleotides from the 5′ end of the genome78,79,80,81,82. This discontinuous step of coronavirus RNA synthesis involves the interaction between complementary TRSs of the nascent negative strand RNA (negative-sense TRS body) and the positive strand genomic RNA (positive-sense TRS-L). Upon re-initiation of RNA synthesis at the TRS-L region, a negative strand copy of the leader sequence is added to the nascent RNA to complete the synthesis of negative-strand sgRNAs. The discontinuous step of negative strand RNA synthesis results in the production of a set of negative-strand sgRNAs that are then used as templates to synthesize a characteristic nested set of positive-sense sg mRNAs that are translated into structural and accessory proteins. Although the coronavirus sg mRNAs are structurally polycistronic, it is assumed that they are functionally monocistronic and that only the first ORF at the 5′ end, which is absent in the next smaller sgRNA, is translated from each sgRNA78,81.
The TRS elements for SARS-CoV-2 have already been determined by RNA sequencing analyses of viral RNAs80,83. Like for SARS-CoV, the consensus TRS core of SARS-CoV-2 is 5′-ACGAAC-3′ and eight sg mRNAs have been shown to be produced in SARS-CoV-2-infected cells (sg mRNAs 2–9). In addition to canonical sgRNAs, recent reports also determined the existence of numerous non-canonical RNA products of discontinuous transcription, including fusions of the 5′ leader sequence to unexpected 3′ sites, TRS-L independent long-distance fusions, and local fusions resulting in small deletions mainly in the structural and accessory genes60,80. However, it remains to be determined whether all of these non-canonical sgRNAs truly arise by discontinuous transcription or whether they represent RNAs that result from recombination. Nevertheless, similar findings were previously reported for other coronaviruses, including MHV61 and HCoV-229E81, which indicates an enhanced coding potential for coronaviruses80. Overall, these unexpected fusion events may drive coronavirus evolution through variant generation, and novel ORFs could encode additional accessory proteins that are involved in either viral replication or modulation of the host immune response60,80.
The RdRP residing in nsp12 is the centrepiece of the coronavirus RTC and has been suggested as a promising drug target as it is a crucial enzyme in the virus life cycle both for replication of the viral genome but also for transcription of sgRNAs. The structure of the SARS-CoV-2 RdRP nsp12 and its cofactors nsp7 and nsp8 has been elucidated and shows a high degree of conservation to the SARS-CoV structure69,84,85. The amino acid sequence of the SARS-CoV and SARS-CoV-2 RdRPs show a >95% similarity with most changes located in the nidovirus RdRP-associated nucleotidyltransferase domain, which, despite being a genetic marker of Nidovirales, has yet to be functionally elucidated69. The structural similarities of the RdRP active site, including conserved key amino acid residues, with other positive-sense RNA viruses suggest the possibility to repurpose known drugs that are effective against other RNA viruses69. One of the most promising candidates is the phosphoramidate remdesivir (RDV), which, in its triphosphate form, acts as a substrate for viral RdRPs and competes with ATP86. RDV has shown potential as an antiviral agent against a broad range of RNA viruses, including Filoviridae (for example, Ebola virus), Paramyxoviridae (for example, Nipah virus) and Pneumoviridae (for example, respiratory syncytial virus) as well as other coronaviruses, including SARS-CoV and MERS-CoV86,87. The RdRP of SARS-CoV-2 selectively incorporates RDV over ATP, which subsequently results in a delayed-chain termination86,88. In contrast to classic nucleoside analogues that lead to immediate termination of the synthesis reaction after incorporation, the RdRP continues for three nucleotides after RDV has been incorporated before chain termination. Nucleotide analogues like RDV may have limited efficacy owing to the proofreading function of the exonuclease domain contained in nsp14 (ExoN)89. The corrective function that is exerted by ExoN is not only responsible for maintaining the stability of the coronavirus genome but also enables the excision of erroneous mutagenic nucleotides71,89. The mode of action observed for RDV might be an explanation for its increased efficiency over other nucleoside analogues as the delayed-chain termination could lead to improved evasion from the proofreading function of nsp14. The current model suggests steric hindrance as a likely reason for termination, disturbing the positioning of the RNA and thus hampering the translocation to the next position86,88. RDV was shown to reduce virus replication of SARS-CoV-2 in vitro90 and was demonstrated to restrict clinical symptoms of SARS-CoV-2 in rhesus macaques upon early pre-symptomatic treatment91. However, a recent randomized, double-blind, placebo-controlled clinical trial in humans with severe COVID-19 showed limited clinical efficacy of RDV treatment92 and further studies will be necessary. Another promising candidate is the purine analogue favipiravir (FPV), which has been shown to effectively target multiple RNA viruses93. Although the mechanism of action is not yet completely understood, a recent study of the in vitro mechanism of FPV suggested a combination of chain termination, slowed RNA synthesis and lethal mutagenesis as the mode of action against SARS-CoV-2, which indicates that FPV might be used to effectively restrict viral replication93. Indeed, results of an experimental pilot study showed that using FPV as treatment against COVID-19 led to increased recovery and faster viral clearance times in treated patients compared to control treatments94. Clinical studies with both RDV and FPV are currently ongoing and will establish whether these compounds are effective antivirals to treat coronavirus infections93.
[TBC below]
https://www.nature.com/articles/s41579-020-00468-6