Ribosomal proteins homologies and methylation of mitoribosome by Corona virus Nsp9, Nsp10 and previously described Nsp13-16 proteins suggested their roles in the inhibition of host protein synthesis: Discovery of new therapeutic targets against Corona virus infections

Multi-Alignment method coupled with phylogenetic analysis we disclosed the Nsp9 and Nsp10 non-structural proteins of Corona Virus as rRNA RlmH/K methyltransferases with similarities with bin recombinase and int-core integrase fold. Further, Nsp9 has similarities to S8 ribosomal protein and Nap10 has similarity to S10 ribosomal protein. Previously, we showed Nsp13, Nsp14, Nsp15 and Nsp16 are also different types of rRNA RlmE/N and Cfr-like methyltransferases-ribonuclease with RNA helicase domains. Two domains of Nsp13 astonishingly have similarities to ribosomal proteins L6 and L9. Taken together, Nsp9/10 and Nsp13-16 proteins could mimic host ribosome assembly and also could methylate rRNA of mitobibosome preventing mitochondrial protein synthesis and oxidative phosphorylation. Low ATP synthesis causes lowering blood pressure following coma but very ATP concentration (1-10nM) surely induces platelets aggregation through vWA, collagen and GpIIb/IIIa proteins followed by fibrin formation and blood clotting as recently have seen in the lung of many Corona virus infected patients. We have also postulated that two polyproteins itself resemble like 28S and 38S mitoribosome subunits and compete with rRNAs inhibiting the ribosome turnover and new protein synthesis due to their similarities with many ribosomal proteins. Such finding may be valuable in computer-based novel drug design against Corona virus. protein synthesis inhibition, ATP clotting,


Introduction
Corona viruses (family Coronaviridae) are enveloped viruses with a largest positive sense, single-stranded RNA genome of ~30kb (Woo et al. 2009;Marra et al. 2014). On genetic and antigenic criteria, CoVs have been organised into three different groups: α-CoVs, β-CoVs, and γ-CoVs (Lau et al. 2015;Dominguez et al. 2014;Lu et al. 2015). Coronaviruses primarily infect birds, mammals and human, causing many lethal respiratory syndromes resembling the common cold, such as bronchitis, pneumonia, and even severe acute respiratory syndrome (SARS). Recently coronaviral research augmented due to pandemic severe respiratory illnesses outbreaks claiming >100000 deaths due to human Corona virus (Liu et al. 2014). COVID-19 virus enters cells through ACE2 receptor-mediated endocytosis in lungs alveolar epithelial cell as well as cells in the heart and kidney (Zhao et al. 2020).
The function of Nsp2 was proposed by me as RNA topoisomerase and Nsp13 was determined as 2'-O-ribose capping Guanosine methyltransferase. By bioinformatics approach comparing 200 DNA and MTases, Ligases, RNases, DNases, Ribosomal proteins as well as some RNA virus associated non-structural proteins we wanted to determine the functions of nsp9 and Nsp10 of Corona virus (Decroly et al. 2008;Bollati et al. 2009). We proposed Nsp16 as RlmE-type 2'-O-ribose Uridine methyltransferase and Nsp14 as N7 Guanidine methyltransferase where as now we proposed Nsp9 as RlmG-like and Nsp10 as ErmD-type methyltransferase accounting total five rRNA methyltransferases in Corona virus genome.
We investigated the homology profiles of many recombinases and transposases using CLUSTAL-Omega software. Several thousand IS elements were sequenced but could be classified into ∼20 families (IS2, IS30, Tn10, Tn21 etc) on the basis of the sequences of their transposases and terminal inverted repeats as well as associated antibiotic resistant, drug efflux and metal resistant genes as found in integrons and transposons (Blakely et al. 1993;Calos & Miller, 1980;Jacques & Michael, 1998). Int1I, int and Rci are different integrases (Polard & Chandler, 1995). Escherichia coli RecA, RecB and recC have roles in homologous recombination, DNA repair, and the induction of the SOS response (Kowalczykowski & Eggleston, 1994;Taylor & Smith, 1985). Bacterial and mammalian RNases are also RNA modifying enzymes and exonucleases are also compared (Beintema et al. 1988;Gotte & Menegazzi, 2019;Bechhofer & Deutscher, 2019). We also compared sixty 30S plus 50S ribosomal proteins as they are powerful RNA binding proteins (Hammerling et al. 2020). We have compared amino acid sequences of those 100-150 proteins with Nsp9 and Nsp10 non-structural proteins related to polyprotein ORF1ab of coronavirus. BLAST search and Multi-alignment analysis are important tools to find the function of Unknown viral protein like Nsp9/10 of Coeonavirus. Previously, Nsp2 and Nsp13 functions were predicted as RNA helicase-topoisomerase but and capping Guanine 2'-O-Ribose methyltransferase respectively (Chakraborty, 2020;Belanger et al. 2010).

Materials and Methods
The BLAST search was done using web portal www.ncbi.nlm.nih.gov/blast and retrive of covid-19 and other Coronaviruses cDNA sequences were done using web portal www.ncbi.nlm.nih.gov/nucleotide or protein. NCBI Primer Design Software was used for primer selection and Oligoanalyzer 3.2 software was used to analyze primer dimmer and hairpin structure. Multalin Software and CLUSTAL Omega Software were used to multiple align of protein sequences and NCBI BLAST seq-2 analysis portal used to analyze homology between two sequences. NCBI pubmed portal (www.ncbi.nlm.nih.gov/pubmed) used to retrieve references and papers. CLUSTAL Omega Phylogenetic tool used to determine the closer structural similarities among the proteins and Seq-2 BLAST was confirmed percentage of sequence homology between two related proteins (Chakraborty et al. 2017;Chakraborty, 2020). The structure and localization of Corona proteins were demonstrated in Figure- It was clearly indicated that Nsp9 share a relation with ermD and Nsp10 with exonuclease or RlmK. Figure-3 showed the multi-alignment and phylogenetic relation of Nsp9 and Nsp10 with seventy five Escherichia coli ribosomal proteins. It is clearly indicated that Nsp9 has close similarity to S8 and Nsp10 to S10 and L22 ribosomal proteins.
We detected about 20%-25% scattered homologies between Nsp9 and S8 ribosomal protein Nsp10 vs. int-core integrase. The ErmD has also similarities to RmtH and int integrase but such similarities are not great and expected for distance search between viruses vs, Bacteria.
The result might be confusing but problem solved when we did homology search between RlmG vs. bin-recombinase and RlmG vs. int-core integrase (figure-6AB) showing homology profile of those enzymes. The problem is BLAST multi-alignment hardly able to catch such similarities and only gave maximum homology at certain point of the sequence. Similarly, CLUSTAL multi-alignment search drastically made gaps and hard to interpret homology data unless we made phylogenetic conversion.
Previously, Nsp13 RNA helicase was shown having multiple domains and functions and ideal for targets for anti-viral drugs (Ivanov et al. 2004;Jang et al. 2008;Jia et al. 2019). We confirmed the sequence similarities of L6 and L9 ribosomal proteins with Nsp13 proteins as well as Nsp2 has a relation to L1 etc supporting such view. We are much confused with the data but it appears that many non-structural proteins of Corona virus have significant homologies with the Ribosomal proteins and thus they could interfare with the ribosomal assembly correctly. Other words, Corona virus ribosomal proteins could interact with the Human ribosome preferably in the mitochondria to methylate as well as topological changes of mRNA or rRNA to alter the translational profile of host mRNA for protein synthesis or enhanced translation of viral structural genes (Zust et al. 2011). Whatever the situation, we predicted that COX1 and COXII types proteins deficiency in mitochondria lead to low oxidative phosphorylation at the ETC and low ATP synthesis. This could cause many physiological abnormalities in host like sudden low blood pressure, coma and heart attack as found in many Corona patients. If ATP concentration in blood reached below 10nM that could lead to blood clotting which was again shown recently in lung and brain of the many Corona patients in the Netherlands.

Discussion
Mechanism of Corona virus pathogenesis is not known and no drug or vaccine yet approved today claiming every day >1000 peoples worldwide and within four months about 30 millions infections and >210000 deaths occurred mostly in the developed countries like USA, England, France, Italy and Spain. Asian and African countries also greatly have affected but due to early lock down technology death rate has restricted to hundreds only. Recently, Russia, Canada and Netherland also have affected to some extent. Sadly Corona pandemic created 100 millions jobless and worldwide panic and WHO has suggested that the pandemic may cause 20-50 millions death due to lack of food and sanitation. We must act to prevent the spread of Corona virus. Thus, the present bioinformatics analysis of Nsp9 and Nsp10 unknown proteins of Corona virus is important and may through some highlights to its control measures targeting those suspected ribosome antagonist and rRNA methyltransferases (von Grotthuss et al. 2003;Albert et al. 2019;Hammerling et al. 2020). We previously showed that Nsp2 is a RNA topoisomerase, a great target for DNA topoisomerase inhibitors (Chakraborty, 2020b). As well as Nsp13-16 may be other RlmE and Cfr-like methyltransferases although previous report has suggested Nsp13 as ATP-dependent RNA helicase (Adedeji et al. 2012;supra, 2016;Hao et al. 2017;Subissi et al. 2014;Chakraborty, 2020c).
Many enzyme has multiple domains and thus they participate in multiple enzymatic activities as Nsp13 and Nsp9/10 which also have recombinase and transposase types domains (Jiang et al. 2018 (Falzarano et al. 2013).

Conclusion
We proposed a model where Nsp9, Nsp10 as well as previously reported Nsp13-16 have similarities to the ribosomal proteins and assembled in mitoribosome to methylate 21S and 12S rRNAs inhibiting protein synthesis. Low synthesis of COXI and COXII to types ETC enzymes cause low ATP synthesis which accelerates low blood pressure and heart attach.
Further, very low ATP concentration also favours activation of platelets aggregation following factor XIII/IX-mediated thrombin-stimulated fibrin formation leading to blood clot in the lung and brain of COVID-19 patients. So targeting those RlmE, ErmD and RlmG types methyltransferases with phytochemicals, antisense, ribozyme and Crispr-Case6 technologies may be gene medicine for Corona virus pathogenesis (Cao et al. 2015;Chakraborty, 2019). However, major focus for Corona-specific drug development was pinpointed to the ACE-2 receptor antagonist, RNA-dependent RNA polymerase (Nsp12), c3 protease (Nsp3) inhibitors (Hu et al. 2020). Similarly, for vaccine development major focus molecules was studied likely spike protein of Corona virus (Zhao et al. 2020). I have alternate thought like Nsp2 RNA topoisomerase and rRNA methyltransferases (Chakraborty, 2020abc;Ferron et al. 2012;Gorbalenya et al. 1988;Smietansk et al. 2014). We have also designed peptide vaccine candidates and RT-PCR primers . We and other have developed many methods of toxic drug nanocarriers and plant extracts may be valuable source for superbugs and Corona virus control , Chakraborty, 2018Kilianski et al. 2014;Ulasli et al. 2014). Never the less, we clearly predicted the functions of Nsp9/10 proteins of Corona virus. Acknowledgement I thank Sir Narendra Modi, prime Minister of India for stimulation through his Mon Ki Bath programme to fight against COVID-19.

Conflict of Interest
I have no conflict of interest to any company and any organization.

Ethical Issues
No patient data was used in this study. PDB Database and GenBank Database were analyzed.

Funding
This is lock down research from my home computer and no funding from any agency