08-04-2021, 12:50 AM
Expression of structural and accessory proteins
The ORFs encoding the structural proteins (that is, S protein, envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein) are located in the 3′ one-third of coronavirus genomes. Interspersed between these ORFs are the ORFs encoding for so-called accessory proteins. The structural proteins of SARS-CoV-2 have not yet been assessed in terms of their role in virus assembly and budding. In general, coronavirus structural proteins assemble and assist in the budding of new virions at the endoplasmic reticulum (ER)-to-Golgi compartment that are suggested to exit the infected cell by exocytosis95,96,97. However, recent evidence shows that betacoronaviruses, including MHV and SARS-CoV-2, rather egress infected cells via the lysosomal trafficking pathway98. During this process, viral interference with lysosomal acidification, lysosomal enzyme activity and antigen presentation was demonstrated.
At least five ORFs encoding accessory genes have been reported for SARS-CoV-2: ORF3a, ORF6, ORF7a, ORF7b and ORF8 (GenBank entry NC_045512.2) as well as potentially ORF3b99 and ORF9b100, the latter of which is probably expressed as a result of leaky scanning of the sgRNA of the nucleocapsid protein80,99,101. In addition, ORF10 has been postulated to be located downstream of the N gene. However, not all of these ORFs have been experimentally verified yet and the exact number of accessory genes of SARS-CoV-2 is still debated80,102. For example, in the case of ORF10, recent sequencing data questioned whether ORF10 is actually expressed, as the corresponding sgRNA could only be detected once in the entire dataset80. Furthermore, using proteomics approaches, the ORF10 protein has not been found in infected cells100,102, whereas ribosome profiling data suggested that ORF10 may be translated60.
The accessory genes display a high variability among coronavirus groups and usually show no sequence similarity with other viral and cellular proteins. Although they are not required for virus replication in cell culture4,5, they are, to some extent, conserved within the respective virus species and are suspected to have important roles in the natural host. Indeed, in the case of SARS-CoV, it was shown that at least ORF3b, ORF6 and ORF9b function as interferon antagonists6,102,103,104. There are some notable differences between the accessory genes of SARS-CoV-2 and SARS-CoV, with the latter having a total of eight described accessory genes (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8a, ORF8b and ORF9b). In SARS-CoV-2, ORF3b contains a premature stop codon and is thus substantially shorter than the SARS-CoV variant. Although there are indications that ORF3b could exhibit its interferon antagonistic function also in a truncated form99, it has not yet been found to be expressed at the protein level in virus-infected cells100,102. SARS-CoV-2 ORF8 shows an especially low homology to SARS-CoV ORF8. The coding sequence of SARS-CoV ORF8 went through a gradual deletion over the course of the SARS-CoV epidemic. Whereas the early isolates from human patients contained a full-length ORF8, a deletion of 29 nucleotides was observed in all SARS-CoV strains during the middle-to-late stages. This deletion caused the split of one full-length ORF8 into two truncated gene products, ORF8a and ORF8b. Furthermore, less frequent deletion events were also observed, including an 82-nucleotide deletion and a 415-nucleotide deletion, which led to a complete loss of ORF8 (refs105,106), suggesting a possible benefit of SARS-CoV ORF8 deletions in vivo. Notably, however, reconstitution of SARS-CoV ORF8 by reverse genetics was associated with slightly increased fitness in cell culture106. Recently, SARS-CoV-2 ORF8 was reported to bind to major histocompatibility complex and mediate its degradation in cell culture107. This indicates that SARS-CoV-2 ORF8 might mediate a form of immune evasion, which is not the case for the split SARS-CoV ORF8a or ORF8b107. Interestingly, a mutant SARS-CoV-2 strain found in Singapore displayed a deletion of 382 nucleotides in the region of ORF8, indeed spanning most of the ORF and the adjacent the TRS. This may indicate a tendency towards host adaption and decreased pathogenicity108 or, alternatively, that the ORF8 protein is dispensable in humans, whereas it is required in the natural host.
Replication compartments
Primary interactions between nsps and host cell factors during the early coronavirus replication cycle initiate the biogenesis of replication organelles66,109,110. Although mechanisms underlying replication organelle formation are not fully understood, the concerted role of the membrane-spanning nsp3, nsp4 and nsp6 has been implicated in diverting host endomembranes into replication organelles111,112,113. Detailed electron microscopy investigations have described the phenotypic appearance and extent of membrane modifications induced by coronaviruses to accommodate viral replication. Coronavirus infection, like many other positive-sense RNA viruses, manifests in the generation of ER-derived and interconnected perinuclear double-membrane structures such as double-membrane vesicles (DMVs), convoluted membranes and the recently discovered double-membrane spherules112,114,115,116. Interestingly, these structures are highly dynamic and develop during the viral life cycle114,117. Even though replicase subunits — notably SARS-CoV nsp3, nsp5 and nsp8 — have been shown to be anchored on convoluted membranes, to date, the specific location of viral RNA synthesis remains the most intriguing unanswered question114,117. Double-stranded RNA (dsRNA), commonly considered as viral replication intermediates, segregates into the DMV interior97,114,118. Consistently, viral RNA synthesis was shown to occur within DMVs by using metabolic labelling of newly synthesized viral RNA in the context of SARS-CoV, MERS-CoV and infectious bronchitis virus infections116. Although, until recently, no openings towards the cytosol have been observed97,114, molecular pores involving nsp3 were demonstrated to span DMVs in MHV-infected cells118. These newly identified structures, which were also observed in SARS-CoV-2-infected cells, provide a connection between the dsRNA-containing DMV interior and the cytosol, thereby hypothetically rendering newly synthesized viral RNAs available for translation and encapsidation into nascent virions118. They also provide new opportunities to experimentally address the origin, fate and trafficking routes of viral RNAs contained in DMVs.
Replication organelles are a conserved and characteristic feature of coronavirus replication and, consistent with suggested roles of rewired intracellular membranes in the context of other positive-sense RNA virus infections, they provide a propitious niche with adequate concentrations of macromolecules necessary for RNA synthesis while preventing the exposure of viral replication intermediates to cytosolic innate immune sensors95,119. The functional dissection of coronavirus replication organelles has proven challenging as their contributions to viral fitness and pathogenesis are indistinguishable from functions provided by enzymes of the RTC, which are anchored on the membranes of the replication organelle120,121,122. Nevertheless, recent studies revealed the overall composition of the coronavirus RTC, with nsp2–nsp16 and the nucleocapsid protein comprising the viral components68,123. Moreover, several genetic and proteomic screening approaches aimed at deciphering essential coronavirus–host interactions and the RTC microenvironment identified supportive roles of the ER and the early secretory system as well as related vesicular trafficking pathways for efficient replication68,124,125,126 and provided a comprehensive list of cellular proteins that are in close proximity to the coronaviral RTC68,127,128,129. Collectively, these studies, in combination with advanced electron microscopy, provide ground for future studies to dissect the microarchitecture of the coronaviral RTC in relation to remodelled ER-derived membranes and to functionally link those structures to processes taking place in close proximity to the RTC such as translation, replication and transcription of viral RNA.
[TBC below]
https://www.nature.com/articles/s41579-020-00468-6
The ORFs encoding the structural proteins (that is, S protein, envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein) are located in the 3′ one-third of coronavirus genomes. Interspersed between these ORFs are the ORFs encoding for so-called accessory proteins. The structural proteins of SARS-CoV-2 have not yet been assessed in terms of their role in virus assembly and budding. In general, coronavirus structural proteins assemble and assist in the budding of new virions at the endoplasmic reticulum (ER)-to-Golgi compartment that are suggested to exit the infected cell by exocytosis95,96,97. However, recent evidence shows that betacoronaviruses, including MHV and SARS-CoV-2, rather egress infected cells via the lysosomal trafficking pathway98. During this process, viral interference with lysosomal acidification, lysosomal enzyme activity and antigen presentation was demonstrated.
At least five ORFs encoding accessory genes have been reported for SARS-CoV-2: ORF3a, ORF6, ORF7a, ORF7b and ORF8 (GenBank entry NC_045512.2) as well as potentially ORF3b99 and ORF9b100, the latter of which is probably expressed as a result of leaky scanning of the sgRNA of the nucleocapsid protein80,99,101. In addition, ORF10 has been postulated to be located downstream of the N gene. However, not all of these ORFs have been experimentally verified yet and the exact number of accessory genes of SARS-CoV-2 is still debated80,102. For example, in the case of ORF10, recent sequencing data questioned whether ORF10 is actually expressed, as the corresponding sgRNA could only be detected once in the entire dataset80. Furthermore, using proteomics approaches, the ORF10 protein has not been found in infected cells100,102, whereas ribosome profiling data suggested that ORF10 may be translated60.
The accessory genes display a high variability among coronavirus groups and usually show no sequence similarity with other viral and cellular proteins. Although they are not required for virus replication in cell culture4,5, they are, to some extent, conserved within the respective virus species and are suspected to have important roles in the natural host. Indeed, in the case of SARS-CoV, it was shown that at least ORF3b, ORF6 and ORF9b function as interferon antagonists6,102,103,104. There are some notable differences between the accessory genes of SARS-CoV-2 and SARS-CoV, with the latter having a total of eight described accessory genes (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8a, ORF8b and ORF9b). In SARS-CoV-2, ORF3b contains a premature stop codon and is thus substantially shorter than the SARS-CoV variant. Although there are indications that ORF3b could exhibit its interferon antagonistic function also in a truncated form99, it has not yet been found to be expressed at the protein level in virus-infected cells100,102. SARS-CoV-2 ORF8 shows an especially low homology to SARS-CoV ORF8. The coding sequence of SARS-CoV ORF8 went through a gradual deletion over the course of the SARS-CoV epidemic. Whereas the early isolates from human patients contained a full-length ORF8, a deletion of 29 nucleotides was observed in all SARS-CoV strains during the middle-to-late stages. This deletion caused the split of one full-length ORF8 into two truncated gene products, ORF8a and ORF8b. Furthermore, less frequent deletion events were also observed, including an 82-nucleotide deletion and a 415-nucleotide deletion, which led to a complete loss of ORF8 (refs105,106), suggesting a possible benefit of SARS-CoV ORF8 deletions in vivo. Notably, however, reconstitution of SARS-CoV ORF8 by reverse genetics was associated with slightly increased fitness in cell culture106. Recently, SARS-CoV-2 ORF8 was reported to bind to major histocompatibility complex and mediate its degradation in cell culture107. This indicates that SARS-CoV-2 ORF8 might mediate a form of immune evasion, which is not the case for the split SARS-CoV ORF8a or ORF8b107. Interestingly, a mutant SARS-CoV-2 strain found in Singapore displayed a deletion of 382 nucleotides in the region of ORF8, indeed spanning most of the ORF and the adjacent the TRS. This may indicate a tendency towards host adaption and decreased pathogenicity108 or, alternatively, that the ORF8 protein is dispensable in humans, whereas it is required in the natural host.
Replication compartments
Primary interactions between nsps and host cell factors during the early coronavirus replication cycle initiate the biogenesis of replication organelles66,109,110. Although mechanisms underlying replication organelle formation are not fully understood, the concerted role of the membrane-spanning nsp3, nsp4 and nsp6 has been implicated in diverting host endomembranes into replication organelles111,112,113. Detailed electron microscopy investigations have described the phenotypic appearance and extent of membrane modifications induced by coronaviruses to accommodate viral replication. Coronavirus infection, like many other positive-sense RNA viruses, manifests in the generation of ER-derived and interconnected perinuclear double-membrane structures such as double-membrane vesicles (DMVs), convoluted membranes and the recently discovered double-membrane spherules112,114,115,116. Interestingly, these structures are highly dynamic and develop during the viral life cycle114,117. Even though replicase subunits — notably SARS-CoV nsp3, nsp5 and nsp8 — have been shown to be anchored on convoluted membranes, to date, the specific location of viral RNA synthesis remains the most intriguing unanswered question114,117. Double-stranded RNA (dsRNA), commonly considered as viral replication intermediates, segregates into the DMV interior97,114,118. Consistently, viral RNA synthesis was shown to occur within DMVs by using metabolic labelling of newly synthesized viral RNA in the context of SARS-CoV, MERS-CoV and infectious bronchitis virus infections116. Although, until recently, no openings towards the cytosol have been observed97,114, molecular pores involving nsp3 were demonstrated to span DMVs in MHV-infected cells118. These newly identified structures, which were also observed in SARS-CoV-2-infected cells, provide a connection between the dsRNA-containing DMV interior and the cytosol, thereby hypothetically rendering newly synthesized viral RNAs available for translation and encapsidation into nascent virions118. They also provide new opportunities to experimentally address the origin, fate and trafficking routes of viral RNAs contained in DMVs.
Replication organelles are a conserved and characteristic feature of coronavirus replication and, consistent with suggested roles of rewired intracellular membranes in the context of other positive-sense RNA virus infections, they provide a propitious niche with adequate concentrations of macromolecules necessary for RNA synthesis while preventing the exposure of viral replication intermediates to cytosolic innate immune sensors95,119. The functional dissection of coronavirus replication organelles has proven challenging as their contributions to viral fitness and pathogenesis are indistinguishable from functions provided by enzymes of the RTC, which are anchored on the membranes of the replication organelle120,121,122. Nevertheless, recent studies revealed the overall composition of the coronavirus RTC, with nsp2–nsp16 and the nucleocapsid protein comprising the viral components68,123. Moreover, several genetic and proteomic screening approaches aimed at deciphering essential coronavirus–host interactions and the RTC microenvironment identified supportive roles of the ER and the early secretory system as well as related vesicular trafficking pathways for efficient replication68,124,125,126 and provided a comprehensive list of cellular proteins that are in close proximity to the coronaviral RTC68,127,128,129. Collectively, these studies, in combination with advanced electron microscopy, provide ground for future studies to dissect the microarchitecture of the coronaviral RTC in relation to remodelled ER-derived membranes and to functionally link those structures to processes taking place in close proximity to the RTC such as translation, replication and transcription of viral RNA.
[TBC below]
https://www.nature.com/articles/s41579-020-00468-6