Nucleocapsid phosphoprotein, SARS-CoV-2

Five de-identified samples that had been assessed as positive by other methods were obtained from the New Zealand Auckland District Health Board. An aliquot of 80 µl of Viral Transport Media that had previously stored a nasopharyngeal swab from a patient infected with SARS-CoV-2 was used for RNA isolation using the QIAamp Viral RNA Mini spin kit (Qiagen, Cat No./ID: 52904) according to manufacturer specifications, with the following modifications: sample volume was brought up from 80 to 140 µl using 1× PBS, and RNA was eluted using two elutions with 40 µl Buffer AVE for a final volume of ∼80 µl.
To determine C_q values, we performed quantitative RT-PCR. We used the 2× SensiFAST Probe No-ROX One-Step Mix (Bioline, Cat No./ID: BIO-76005), according to manufacturer specifications using 2.5 µl RNA template. Primers and probes were from the CDC 2019-nCoV CDC Assay (IDT, Cat No./ID: 10006606) targeting the N1 and N2 regions of the nucleocapsid phosphoprotein of SARS-CoV-2. NP targets the RNase P gene for detection of human nucleic acids and serves as an internal control for sample integrity. Reactions were run at a total volume of 20 µl on a real-time PCR device. The results are shown in Table 2.

The structure of the N-NTD in complex with the 7mer RNA duplex was calculated using HADDOCK [17 (link)]. The RNA homology model was prepared by mutating the native 7mer RNA duplex (PDB 4U37) [19 (link)] in Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.) that was subsequently subjected to an energy minimization in YASARA [18 (link)]. For the actual docking, we used a representative structure from the set of obtained structures and followed a standard protocol. As active were selected those N-NTD residues with CSP > 0.05 ppm and at least 20% solvent accessibility (A50, T57, H59, R92, I94, S105, R107, R149, A152 and Y172), while as passive were additionally selected adjacent solvent exposed residues (T49, T54, L55, R88, A90, K102, L104, Y109, Y111, P151, A155, A156, E174 and G175). On the RNA side, all 14 nucleobases were defined as active for the experimentally driven docking protocol. In addition, in all three regions within the N-NTD were defined as fully flexible segments for the advanced stages of the docking calculation (the N-terminal G1-T9, the central loop I54-M61 and the C-terminal S136-S140). The final set of 200 water-refined structures was clustered using a Fraction of Common Contacts approach [32 (link)] with a default cut-off 0.75 and a minimal cluster size = 4. The resulting structures were sorted into 7 clusters and the most populated cluster (n = 30) that also exhibited the lowest interaction energy was selected for detailed analysis. The structure of the N-NTD in complex with the ssRNA-10mer was calculated in YASARA [18 (link)]. The significantly perturbed backbone amide groups from residues (CSP > 0.06 ppm) N47, S51, F53, L56, G60, K61, K65, F66, A90, R93, I94, R95, G97, D98, K100, K102, D103, L104, G129, R149, A152, A156, I157, L159, Q160, T165, T166, L167, Y172, G175 and R177 outlined the U-shaped binding epitope for the ssRNA-10mer molecule. In addition, the signal from arginine side-chain NH^ε groups were significantly perturbed for residues 88, 89, 92 and 177 but remained at their original positions for 68, 93, 95. We combined this information in generating the inter-molecular distance restraints used in YASARA docking calculation that consisted of an initial energy minimization followed by a 100 ns of molecular dynamics using the default md_fast.mrc macro. The 3.9 Å upper distance limits were set between K65 NZ–U1 P, K61 NZ–U2 P, R88 NH1 –U5 P, R89 NH1 –U4 P, R92 NH1 –A6 P, K102 NZ–A7 P, R107 NH1 –A8 P, T166 CG2 –G10 C5, and R177 NH1 –C9 P and R177 NH2 –C10 P.

The SARS-CoV-2 Research Grate Test Material (RGTM 10169; National Institute of Standards and Technology, Gaithersburg, MD), consisting of two synthetic RNA fragments from the SARS-CoV-2 genome (including SARS-CoV-2 sequences 25949–29698 and 12409–15962 of isolate USA-WA1/2020) in a background of 5 ng/µL human Jurkat RNA, was diluted over six serial log dilutions with nuclease-free water for use as calibration standards. RNA template of SARS-CoV-2 Nucleocapsid Phosphoprotein (N protein) encapsulated in the MS2 bacteriophage construct (PerkinElmer, Waltham, MA) was prepared following manufacturer’s instructions for use as a positive control (PC). Single-use aliquots of reference RNA were stored in LoBind microcentrifuge tubes (Eppendorf, Hamburg, Germany) at –80°C.