SARS-CoV-2, the virus causing the Covid-19 pandemic, belongs to the beta coronaviruses as do SARS- and MERS-CoV. These coronaviruses are made of an exceptionally large single-stranded RNA genome ( 30kb), which encodes 16 non-structural proteins (nsp1 to nsp16), 4 viral structural proteins (S, M, E, and N) and several accessary proteins (6). The nonstructural genes are more highly conserved than the structural genes among coronaviruses. Although beta coronaviruses caused outbreaks of the severe acute respiratory syndrome (SARS) in 2002/2003 and the Middle East respiratory syndrome (MERS) in 2012, to date there is still no effective vaccine or drug to prevent or treat the infections. The nsp1-16 proteins are encoded in two open reading frames (ORF1a and ORF1b), between which there is a -1 frame shift (Fig. 1B). The polyproteins are processed by a virally encoded main protease (Mpro, nsp5) and a papain-like protease (PLpro, nsp3) (6). In addition to proteases, at least five more proteins (nsp12-16) have enzymatic activities. Nsp12 is the main and catalytic subunit of RdRp, and nsp13 is an RNA helicase. Nsp7 and nsp8 form accessary subunits of RdRp and stabilize nsp12. Given the large RNA genome to be copied, RdRp likely depends on the helicase to untangle the RNA. It has been reported that RdRp increases the helicase activity by 2-fold (7). The pandemic this year has inspired greater efforts to understand the molecular mechanisms of these deadly coronaviruses. Atomic structures of apo Covid-19 RdRp (nsp7, nsp8 and nsp12 complex) have been determined by cryoEM (3) (Fig. 2A). The C-terminal half of Covid-19 RdRp is homologous to a wide variety of small RNA viral RdRp, including those of poliovirus, foot-and-mouse disease virus, human rhinovirus, Hepatitis C (HCV), Dengue, and yellow fever viruses (Fig. 2A). But the first 350 residues of RdRp are unique among coronaviruses, as are nsp7 and nsp8, which are suspected to compose an RNA primase to initiate the RNA synthesis. Remdesivir was modeled into the apo Covid-19 RdRp structure (3). However, this doesnt explain why Remdesivir should have specificity toward coronaviruses but not against many related RNA viruses. Moreover, the modeling has not led to improvement or new designs of more effective Covid-19 inhibitors. No detailed structure of Remdesivir bound to Covid-19 RdRp has been determined. MERS and SARS helicase structures were recently solved using X-ray crystallography (4, 5) (Fig. 2B). They share 72% sequence identity, and SARS and SARS-CoV-2 helicases are 100% identical. These helicases belong to the well-known Superfamily 1 (SF1) and translocate along single-stranded RNA in the 5 to 3 direction (8). My group elucidated how the SF1 helicase UvrD unwinds dsDNA by a coupled wrench and inchworm mechanism (9). We anticipate that Covid-19 needs to have special mechanism to unwind RNA, which has more complex structure than a DNA double helix. Our initial plan of in vitro assembly of the viral RTC has been scratched because the goal was accomplished already by other groups. So currently we are making nsp poly proteins in HEK293 cells and solving the problems of poor cleavage of polyproteins to individual nsp proteins. Meanwhile we are assaying the RTC activity with mixed individually expressed and purified proteins as well as polyproteins. In the infected cells, it has been reported that viral RNA replication and transcription occurs in the double-membrane vesicles (DMV), which are formed by viral nsp3 and nsp4. So we are making DMVs and testing polyprotein localization, processing on DMV now. The next step is to assemble RTC on DMVs in vivo. In preparation of nsp proteins for characterizing viral RTC, we found the viral polyprotein (nsp5) proessing cannot be tagen for granted and hence delved into understanding how the main viral protease sequentially release each nsp from the polyprotein. We are determining the structure of nsp5 in the polyprotein form. References 1. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe Acute Respiratory Syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 5, 536-544 (2020). 2. S. S. Jean, P. I. Lee, P. R. Hsueh, Treatment options for COVID-19: The reality and challenges. J Microbiol Immunol Infect 10.1016/j.jmii.2020.03.034 (2020). 3. Y. Gao et al., Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science 10.1126/science.abb7498 (2020). 4. Z. Jia et al., Delicate structural coordination of the Severe Acute Respiratory Syndrome coronavirus Nsp13 upon ATP hydrolysis. Nucleic Acids Res 47, 6538-6550 (2019). 5. W. Hao et al., Crystal structure of Middle East respiratory syndrome coronavirus helicase. PLoS Pathog 13, e1006474 (2017). 6. Y. Chen, Q. Liu, D. Guo, Emerging coronaviruses: Genome structure, replication, and pathogenesis. J Med Virol 92, 418-423 (2020). 7. A. O. Adedeji et al., Mechanism of nucleic acid unwinding by SARS-CoV helicase. PLoS One 7, e36521 (2012). 8. A. O. Adedeji, K. Singh, S. G. Sarafianos, Structural and biochemical basis for the difference in the helicase activity of two different constructs of SARS-CoV helicase. Cell Mol Biol (Noisy-le-grand) 58, 114-121 (2012). 9. J. Y. Lee, W. Yang, UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127, 1349-1360 (2006).