Dynamics of SARS-Cov-2 ORF7a Gene Deletions and Fate of Downstream ORF7b and ORF8 Genes Expression

SARS-CoV-2 small transactivator proteins like ORF7a, ORF7b and ORF8 were implicated as modulator of cellular genes like STAT1/2, Tom70, IRF3 to down regulate IFN?, IL6 and IgG production. Genetic locus of those genes was frequently deleted and mutated with no expression of those small proteins and virus was cleared by host immune response more easily. We searched the ORF7a and ORF7b mutants in the NCBI database to characterize the genes and their presence in different variant and sub-variants were addressed to check if extinction of such mutated corona viruses with time. The ORF7a deletions were reported first in 2020 in early corona virus lineages but no further study was made. Thus, we reviewed the occurrence of such ORF7a gene deletions in alpha, delta and omicron corona viruses. Specific oligonucleotide at the deletion boundary was used to BLAST-N search for identical genes in the Database. Multi-alignment found ORF7a 60-63nt deletions in the carboxy-terminus which sometime also deleted the TAG termination codon of ORF7a as well as ATG initiation codon of ORF7b gene with no production of both functional proteins. In some situation a chimera ORF7a protein may be formed due to inactivation of TGA codon. More surprisingly, ORF7a/b deletion mutants also associated with creation of downstream TAA termination codon in the ORF8 gene which was shown as potential hotspot of at least one dozen mutations. We also detected two omicron BA.4 variant with three deletions in the ORF7a gene that also created two early termination codons. Such inactivation mechanisms were found dominant in omicron, alpha and few delta corona virus variants. The hairpin structures of ORF7a gene locus were changed (hairpin 6) in more than one deletion mutant and stiff hairpin 2 structure at the NH2 terminus might driver such deletion process. The associated important changes in the ORF1ab and spike proteins were also analysed to pinpoint variant status. Absence of expression of ORF7a, ORF7b and ORF8 proteins likely enhance SARS-CoV-2 clearance in host and may be one of the mechanisms of lower pathogenicity with time.

SARS-CoV-2 is a large positive-stranded RNA virus with~30000 nucleotides genome. It has structural proteins Membrane (M), Envelope (E), Nucleocapsid (N), Spike (S) coded from 3’-1/3 part of the virus independently but RNA-dependent RNA polymerase was coded from nsp12 domain of ORF1ab polyprotein coded from 2/3 of the 5’-part of the genome [1]. ORF1ab polyprotein was degraded into sixteen polypeptides (nsp1-nsp16) (Figure 1). The nsp2 protein is RNA topoisomerase whereas Nsp3 and nsp5 are proteases [2]. The nsp6, nsp7, nsp8, nsp9 and nsp10 were small accessory proteins involved in RNA polymerase replication complex. The nsp14 and nsp15 are nucleases and nsp16 is methyltransferase and as well as nsp13 is RNA helicase [3-5]. Nsp11 is a small peptide and function was not known. The ORF3a, ORF6, ORF7a/b, ORF8, ORF9 and ORF10 small proteins also coded from 3’ end of the SARS-CoV-2 genome and have roles in regulating cellular genes [6]. Many drugs were discovered against proteases and RNA polymerases but vaccines were only important remedy that halted the corona virus spread [7]. The most frequent mutation that occurred in most corona virus isolates was 3037C>T which is a synonymous change that usually accompanied 3 other mutations that include 241C>T, 14408C>T (P323L in RdRp) and 23403A>G (D614G in S-protein). ORF7a gene is a hotspot for deletion mutations but mechanism of such changes was poorly understood [8]. More 29 mutations in spike of omicron corona viruses with concurrent D614G and N501Y mutations caused antibody escape with mild diseases. Those mutations were: A67V (V67), T95I (I93), N211I (I206), L212V (V207), V215P (P210), R216E (E211), G341D (D336), S373L (L368), S375P (P370), S377F (F372), K419N (N414), N442K (K437), G448S (S443), S479N (N474), E486A (A481), Q495R (R490), G498S (S493), Q500R (R495), Y507H (H502), T549K (K544), H657Y (Y652), P683H (H678), N766K (K761), D798Y (Y793), N858K (K853), Q956H (H951), N971K (K966), and L983F (F978) [in sate values for omicron virus positions] [9].  The ORF7a is a type-I transmembrane protein of 121 amino acid residues with an N-terminal signal peptide (residues 1–15), an immunoglobulin (Ig)-like ectodomain (residues 16–96), a transmembrane domain (residues 97–116), and an ER retention motif (residues 117–121)  (see, figure-14). About 872nt deletion in the ORF7a/b locus was reported expressing stable corona virus isolates [10,11]. Sequencing of SARS-CoV-2 led to discovery of 382nt deletion in the ORF7a/b-ORF8 genes (see, accession no. MT374101) [12-14]. Further study indicated two medium (81nt and 96nt) deletion mutants at the NH2 terminus of ORF7a and two other medium deletion (40nt and 47nt) in the carboxy terminus of ORF7a gene [15-17]. ORF7a subverts phosphorylation of STAT2, suppressing transcriptional activation of antiviral IFN-stimulated genes that can otherwise restrict viral replication [18]. Although the proposed function for ORF7a is intracellular, antibodies against ORF7a are elevated in the serum of COVID-19 patients. Preliminary studies indicated that recombinant ORF7a protein interacts with CD14+ monocytes and triggers expression of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-a). However, it was unclear if those mutants interacted badly in COVID-19 patients. We extended such studies and reviewed in details how similar ORF7a gene deletions (~63nt) were incorporated in recent corona virus variants. In Vero cell culture ORF7a defective viruses grew slow indicating ORF7a protein was necessary for viral replication [19]. We deeply characterized the different ORF7a deletion mutants to demonstrate their spread into alpha, delta and omicron corona virus variants.

We searched PubMed to get idea on published papers on ORF8 and also searched SARS-CoV-2 NCBI database using BLAST-N and BLAST-X search methods. Multi-alignment of protein was done by MultAlin software and multi-alignment of DNA by CLUSTAL-Omega software, EMBL-EBI [20]. The ORF8 mutants was obtained by BlastN search of deletion boundary of ~120nt sequence and then analyzing the sequences with 90-100% similarities. Blast-X search of ORF7a deleted gene used to get more deletion mutants. Then, the other ORF7a mutants were detected by Blast-N search with selected deletion boundaries. Hairpin structure of ORF7a gene~ 200nt sequence was done by OligoAnalyzer 3.1 software (Integrated DNA Technologies). The protein 3-D structure was determined by SWISS-Model software [21].

We got the idea of ORF7a deletion (accession no. OP711842) from our earlier work on ORF8 inactivation with creation of TAA termination mutation (Figure 2). BlastN search with deletion boundary gave ten sequences with 100% homology and 100% cover (Figure 3). The oligonucleotides of few sequences at the deletion boundaries were given in Table 1. The sequences with accession nos. MW309830, MW309829 and MW309831 were obtained from earlier studies in 2020 whereas we found here only one sequences belonged to omicron variant (Table 1). Table 1 showed that only ORF7a deletion mutants with accession numbers OK249834, OL447329, MT628273, ON926042 and MW309829 were spread into omicron variants. The figure 4 showed the Blast-2 search between oligonucleotides with mutant genomes to confirm the authenticity of oligonucleotides (Figure 4). Few ORF7a deletion abolished ATG codon of ORF7b gene (accession numbers OL522556, OK249824, OK192887, MZ727798, OP334687, OW352845) while in few ORF7a mutants ATG codon of ORF7b was retained (accession numbers OP711837, OL447329, OU556481, OP398207, OK439931, ON926042) (Figure 5). In (Figure 6), we demonstrated that few ORF7a deletion mutants had consecutive three deletions (accession nos. OP174404 and OP490708; Omicron BA.4 variant). The first deletion was 85nt at 27471 (5’-aca aca gta ctt tta aaa gaa cct tgc tct tct gga aca tac gag ggc aat tca cca ttt cat cct cta gct gat aac aaa ttt g-3’), followed by second 45nt deletion at 27608 (5’-aca cgt cta tca gtt acg tgc cag atc agt ttc acc taa act gtt-3’) and a third deletion of 63nt at 27687 (5’-cca att ttt ctt att gtt gcg gca ata gtg ttt ata aca ctt tgc ttc aca ctc aaa aga aag-3’) but no change in ORF7b and ORF8 genes. Such truncation also created TGA and TAA two termination codons but nullifying its own TGA termination codon facilitating no ORF7a protein synthesis (Figure 6). We demonstrated the BlastX data of ORF7a/b-ORF8 locus of two mutants. In mutant -1 (accession no. ON940286) ORF7a gene fusion protein may be obtained but ORF7b and ORF8 protein could made normal (Table 2). Whereas in mutant-2 (accession no. OK249834), no ORF7a and also ORF7b due to deletion of ATG codon as well as ORF8 due to creation of TAA termination codon. Thus, we clearly demonstrated the hotspot deletion site of the ORF7a/b-ORF8 locus. In figure-7, we presented few ORF7a 96nt deletion mutants belong to omicron BA.2 and BA.4 corona viruses as well as B.1.1.7 lineage (Table 3) (Figure 7). We made primers to amplify such deletion sequences and made restriction analysis to characterize the mutant sequences (Figure 9). We showed the multi-alignment of spike protein of ORF7a deletion mutants to demonstrate their variant status. The OM966459 sequence was omicron BA.1 as it had 143VYY deletion and 215EPE insertion in the spike. The MT628273, ON577132, ON577132 sequences were early lineages as no ~30 mutations in the RBD domain of spike.

The ON940286, ON608606, and ON926042 sequences were omicron BA.2 with spike 24LPP deletion but no 69HV deletion. The OP174404 and OP490708 sequence were omicron BA.4 variant having 141KSF ORF1ab protein deletion (data not shown). The OP791425, OP721043, ON852734, OP753941, OP543868 and OP405203 sequences were likely omicron BA.5 variants. These findings clearly demonstrated the spread of ORF7a deletion mutants into omicron corona virus. Note that all omicron variants had 31ERS N-protein deletion (Figure 10). The accession numbers beginning with OX, OV, OU and OD (UK origin) had no protein expression data. Thus, our protein multi-alignment data was limited. We showed ORF1ab polyprotein deletions that frequently found in different corona virus isolates. The GHVMV, 141KSG and 3675SGF were important deletions and we found ORF7a deletion mutants also had such genetic imprint. Never-the-less, we have no idea regarding the functional consequence in nsp1 transactivator protein for GHVMV and KSF deletion and in nsp6 accessory protein for SGF deletion. Finally, we BlastN searched the full length ORF7a sequence which had three deletions to trap more diverse ORF7a deleted sequences (Figure 11). The accession number OP174404 had first 85nt deletion at 27468, followed by second 45nt deletion at 27608 and a third deletion of 63nt at 27713 position as described before. The ORF7a mutant (accession no. ON482431) had 7nt first deletion at 27468 followed by second 83nt deletion at 27509. Whereas, the ORF7a mutant (accession no. ON611111) had 85nt one deletion at 27468 position and in another mutant (accession no. OP077998) had also one 100nt deletion at the same position. Thus, we showed the dynamics of ORF7a gene deletions with time since 2020 to 2022. It should be noted that few sequences (OV426369, OM356217, and ON482431) were Delta variant with 157FR deletion in the spike whereas one sequence was B.1.1.7 origin with 69HV and 145Y amino acids deletions in the spike. On the other hand, 24LPP deletion only suggested that ON611111 and OX031117 sequences were omicron BA.2 variant which had also 3675SGF ORF1ab deletion. Whereas with 141KSF deletion in nsp1 domain of ORF1ab polyprotein and 24LPP plus 69HV spike deletions suggested BA.4 omicron corona virus (see, accession numbers OP077998 and OP174404). The accession number OK235146 was classified as omicron BA.5 variant with no 141KSF mutation but spike 24LPP plus 69HV spike deletions. But the expression of ORF8 gene was hampered in this virus due to creation of CAA=TAA termination codon and only 26AA length ORF8 might be produced. Anyway, a R52I mutation in the ORF8 gene was also identified in this virus as we had reported earlier (in press).  The ORF7a deletion mutants belong to accession numbers OV426369, OM356217, ON482431 and OL835581 were Delta variants with 157FR deletion in the spike but we also found 119DF deletion in the ORF8 gene (data not shown here). Thus, we showed the transmission and propagation of ORF7a deletion mutants across early B.0 and B.1 lineages to B.1.1.7 and B.1.617.2 most deadly variants and then also to mild pathogenic but highly transmissible omicron corona virus variants. We classified omicron variant into sub-variants BA.1, BA.2, BA.4 and BA.5 and found ORF7a deletions in all omicron sub-variants (Figure 12). We showed the hairpin structure of ORF7a gene locus which was changed in three deletion mutants (hairpin loop 6 was abolished) but not in one deletion mutant. We postulated that stiff hairpin loop-2 may have role in driving deletion in the ORF7a gene. We performed a protein multi-alignment of 25 sequences from different corona virus variants and only one I88S point mutation detected. Overall, ORF7a gene deletions found consistently in B.1.1.7 and B.1.617.2 and then B.1.1.529 variants. But point mutation in the ORF7a protein was very limited (Figure 13).

The ORF7a deletion B.1 lineage corona viruses were appeared in 2020 and necessary for viral replication and immunity. We showed that the 40-100nt deletions in the ORF7a gene were rampant and transmitted from B.1.1.7 to B.1.617.2 and B.1.1.529 corona viruses. Three deletions in the same gene was limited and we found only two such sequence by searching NCBI database. The ORF7a truncations were defective in suppressing the host immune response, which might explain why these mutations quickly disappeared in the immunocompetent population [22]. The ORF8 is downregulating antigen presentation. Other corona virus accessor protein Nsp6 binds TANK binding kinase 1(TBK1) to supress interferon regulatory factor 3 (IRF3) phosphorylation similar to Nsp13. Such cellular interacting protein binds importin Karyopherin ?2 to limit IRF3 nuclear translocation. A Delta variant SARS-CoV-2 originated in Poland with deletion of 872nt ORF7a/b and ORF8 genes (GISAID database accession no. hCoV-19/Poland/PL_ P3686/2021; hCoV-19/Poland/PL_P3688/2021; hCoV19/Poland/PL_P3693/2021). Similar to other RNA viruses, the SARS-CoV-2 genome evolves quite rapidly, indicated by a 1.1 × 103   substitution/site/year mutation rate. Our data clearly presented the propagation of ORF7a deletion mutants in most VOV of corona viruses with or without damaging expression of 43 AA ORF7b protein. The ORF8 protein is also 121 AA and its deregulation is due to creation of termination codon (CAA=TAA; C>T at 27972) with or without S24L mutation but not with L84S mutation (Figure 14). Extensive spike protein mutations occurred in omicron variants and such deletion and point mutation were used to identify different variant type. We clearly demonstrated the ORF7a deletion happened in all corona virus variants including BA.1, BA.2, BA.4 and BA.5 which were spreading now with mild symptoms [23-36].

Dr Asit thanks NCBI database and BLAST program and CLUSTAL-Omega software for free distribution worldwide.

Competent Interest

The author declared no conflict of interest. The data was generated by computer analysis of NCBI database.

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