Coronavirus ORF1ab polyprotein associated nsp16 protein is a RlmE Methyltransferase and may methylate 21S mitochondrial rRNA of host cells inhibiting protein synthesis

Covid-19 infections are rapidly spreading worldwide with more than 100000 death and thus understanding the molecular mechanism of tropism of human cells is an urgent need for drug design. We have described here a bioinformatics approach to predict the functional aspects of non-structural nsp16 protein of Corona virus. The covid-19 7098 AA large polyprotein was degraded into sixteen proteins and last nsp16 protein was found an RlmE type rRNA methyltransferase. Nsp16 has no similarity to bacterial RlmABCD but has 25 percent similarity to the bacterial RlmE protein which methylates the U2551 2-hydroxy group of Ribose. The nsp16 proteins of different corona viruses like covid-19, bat-coronavirus, SARS and MERS have strong homology. Mrm2 and Dim1 like yeast and mammalian rRNA methyltransferases have 26-33 percent homologies but not with 2-O-capping MTase as reported previously. Rrp8 MTases also has no similarity to nsp16. We postulated that mitochondrial rRNA methylation of bronchial cells were mediated by the nsp16 protein causing inhibition of protein synthesis due to poor assembly of aminoacyl-tRNA or mRNA and peptidyl transferase at the PTC. This is one of the new molecular mechanism of corona virus cellular tropism and different than ACE-2 mediated blockage of cellular signalling to inhibit aldesterone biosynthesis with abnormal Na ions in cells. We also designed primers based on nsp16 cDNA sequence (nt 20659-21552, accession no MT121215) specific for Covid-19 diagnosis by RT-PCR.


INTRODUCTION
Coronaviruses (family Coronaviridae) are enveloped viruses with a largest positive sense, single-stranded RNA genome of 30kb ). On genetic and antigenic criteria, CoVs have been organised into three groups: α-CoVs, β-CoVs, and γ-CoVs (Dominguez et al. 2014;Lau et al. 2015;Lu et al. 2015). Coronaviruses primarily infect birds, mammals and human, causing a variety of lethal respiratory diseases resembling the common cold, to lower respiratory tract infections such as bronchitis, pneumonia, and even severe acute respiratory syndrome (SARS). In recent years, coronaviral research must be augmented due to pandemic severe respiratory illnesses outbreaks claiming >100000 deaths (Liu et al. 2014).  virus enter cells through ACE2 receptor-mediated endocytosis. The receptor ACE2, was abundant in lungs AT2 alveolar epithelial cell as well as cells in the kidney, heart and blood vessels (Zhao et al. 2020). One of the known regulators of endocytosis is the AP2-associated protein kinase-1 implicated novel target for therapeutic intervention. Figure  nsp16 methylates 21S rRNA in mitoribosome inhibiting protein synthesis. Hypothesis is due to loss of mitochondrial proteins oxidative phosphorylation may be prevented causing sudden death of Corona-infected patients.
Discovery of new molecular target is urgent need for covid-19 and we target here rRNA methylase. There are more than 20 different classes of rRNA methyltransferases that modify 16S rRNA, 23S rRNA and tRNAs in bacteria but mammalian methyltransferases are more diverged (Bauerle et al. 2015). Cfr and Erm methyltransferases are known for erythromycin and linezolid drug resistance in bacteria and others like Rmt, ArmA and Rlm methyltransferases are involved in ribosome biogenesis as well as modulator of drug resistance (figure-2). Different isomers of Rlm (A-N) rRNA methyltransferases methylate at various positions of bacterial 23S rRNA conferring multi-resistant to macrolides and ketolides like erythromycin, telithromycin, and solithromycin. RlmA II MTase has preference to N1 of G748 of 23S rRNA (Jiang et al. 2018). RlmB MTase (protein id. BAI33654) modifies G2251 of 23S rRNA (Lovgren & Wikstrom PM, 2001;Michel et al. 2002) while RlmC modifies m5U747 in 23S rRNA, and RlmD is specific for m5U1939 (Madsen et al. 2003 (Toh et al. 2008). RlmK/L (protein ids. MHY78137 and BAI29841) and both recombined enzyme (YcbY) adds the m7G2069 and m2G2445 methylations in However, some 2'-O-MTases were also implicated in capping of mRNA at the 5'-end by methyl N 7 -methylguanosine linked via an inverted 5′-5′ triphosphate bridge to the 5′-terminal nucleoside of the transcript (Smietanski et al. 2014). Uncapped RNAs, such as nascent viral transcripts, may be detected as 'non-self' by the host cell, triggering an antiviral innate immune response through the production of interferons. Therefore, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2′-O-methyltransferases (2′-O-MTases) to autonomously modify their mRNAs (Egloff et al. 2002;Werner et al. 2011). Virus-encoded 2'-O MTase enzymes involved in the synthesis of the RNA cap structure are different from those of host cells. As a consequence, these pathogenic cap-forming enzymes are potential targets for antimicrobial drugs (Belanger et al. 2010). Such human and rat capping MTases were distinct and no similarity to nsp16 or covid-19 RlmE MTase (Mungall et al. 2003). Thus the report on capping 2'-O methylase activity of nsp16 (nsp13 in case of SARS) is interesting and likely a contamination of other methylase activities (von Grotthuss et al. 2003) but nsp16 ha s no similarity to mammalian 2'capping methyltransferase (Smietanski et al. 2014

MATERIALS & METHODS
The BLAST search was done using web portal www.ncbi.nlm.nih.gov/blast and retrive of covid-19 and other corona viruses 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.

RESULT
We have analyzed the covid-19 genome and the proteins expressed by (+) sense 30kb rNA genome (Figure-4A) (Table-2          Further, we designed the covid specific primers for the RT-PCR to detect the virus load during pathogenesis. The cDNA sequence nt. 20659-21552 (accession no. MT121215) was used to design four pairs of primers using NCBI Primer Design Software (figure-8).
Individual forward and reverse primer were BLAST searched and primer pair-4 appeared more specific for the diagnosis of covid-19 ( figure-9). The primers were further analyzed by     Decroly et al. 2008;(Ferron et al. 2012). From such analysis we concluded that nsp16 RlmE was not involved in mRNA capping (Bollati et al. 2009;Zust et al. 2011). But if human mitoribosome (21S and 12S rRNAs) be methylated by nsp16 protein is an interesting question but 23S rRNA proto-type rRNA in human nucleus is 28S rRNA and some MTases (cfr and emrB) have 23S rRNA specificity rather than 16S rRNA (Figure-3).
We claim that our study is important for unknown microbe like covid-19 that is claiming >90000 lives due to lack of drug. Non-structural viral proteins are ideal for drug discovery understanding molecular mechanism of pathogenicity. It was demonstrated that P7 protein of hepatitis C virus was the target of hexamethylene amiloride (Premkumar et al. 2014) where as the rotavirus NSP4 viroporin domain was shown as calcium-conducting ion channel (Pham et al. 2017 mRNA genome release into cell cytoplasm (Prentice et al. 2004). Natural products have shown to regulate microbes and became good drugs controlling superbug bacteria, malaria and cancer (Chakraborty, 2019;Newman & Cragg , 2012;. The 7a protein of Coronavirus has shown to induce apoptosis by caspase-dependent pathways and thus antibody against 7a protein may be a drug against viral pathogenesis (Tan et al. 2004). Interferon-alpha2b and ribavirin improve outcome in MERS-CoV-infected rhesus macaques giving a hope for the treatment of Covid-19 (Falzarano et al.2013).
We have clearly demonstrated that nsp16 is a RlmE type methyl transferase that may methylates 21S rRNA ofmitochondria but may be 28S rRNA of human bronchial cells also be methylated by nsp16 protein. Bronchial cells with ACE-2 receptors are target for Covid-19 and methylation of rRNAs in such target cells as well as kidney and heart cells may be possible causing abnormal protein synthesis. Such methylation may favours viral protein synthesis inhibiting cellular protein synthesis that have shown in many viral infections (figure-4). In truth stopping Rennin-Angiotensin pathway (figure-1) for aldosterone synthesis causes abnormal Na + ions in cells lowering blood pressure and kidney function. Covid-19 RlmE type MTase has never be implicating in pathogenicity and thus we have discussed a new direction of cellular tropism of Corona viruses. Nsp16 MTase has some similarity to the mmr2 and Dim1 rRNA methyl transferase of yeast and mammals (figure-7). Yeast Rrp8 23S rRNA MTase has poor similarity to nsp16 but no similarity with human Rrp8 protein (data not shown). The primers designed (figure-8) using Coronavirus rlmE gene sequence may be used for diagnosis and therapeutic discovery in the future. Heterogeneous phyto-antibiotics, gene therapy, anti-sense and ribozyme technology and nano-drug carriers may be future medicine against Covid-19 (Cao et al. 2015;Chakraborty, 2019).
ACKNOWLEDGEMENT I thank Dr. Parthapratin Chakravorty of Raja N L Khan Women's College for encouraging covid-19 research.