NMR-Based Analysis of Nanobodies to SARS-CoV-2 Nsp9 Reveals a Possible Antiviral Strategy Against COVID-19

Abstract

Following the entry into the host cell, SARS-CoV-2 replication is mediated by the replication transcription complex (RTC) assembled through a number of nonstructural proteins (Nsps). A monomeric form of Nsp9 is particularly important for RTC assembly and function. In the present study, 136 unique nanobodies targeting Nsp9 are generated. Several nanobodies belonging to different B-cell lineages are expressed, purified, and characterized. Results from immunoassays applied to purified Nsp9 and neat saliva from coronavirus disease (COVID-19) patients show that these nanobodies effectively and specifically recognize both recombinant and endogenous Nsp9. Nuclear magnetic resonance analyses supported by molecular dynamics reveal a composite Nsp9 oligomerization pattern and demonstrate that both nanobodies stabilize the tetrameric form of wild-type Nsp9 also identifying the epitopes on the tetrameric assembly. These results can have important implications in the potential use of these nanobodies to combat viral replication.

Keywords: COVID-19; NMR spectroscopy; Nsp9; SARS-CoV-2; nanobodies.

Figure 1

Llama derived nanobodies specifically cross‐react with Nsp9 in COVID‐19 saliva samples. A) Decreasing amount of purified recombinantly expressed Nsp9 (purified rec wtNsp9) preincubated with BSA were separated by SDS PAGE, immunostained with nanobodies 2NSP23 and 2NSP90. Detection was with HRP‐conjugated secondary antibodies to 6xHis tag (aHis‐HRP) or to the VHH domain (aVHH‐HRP). B) RTqPCR analysis of Sars‐Cov‐2 N2 mRNA levels as proxy for viral load. mRNA levels were normalized against human RNase P mRNA. Each red dot represents a single measurement value. Each column represents a mean value from at least 4 independent measurements (n ≥ 4). Error bar represents the standard deviation from the mean value C) 15 µg of saliva protein samples from COVID‐19 negative and positive individuals were loaded together with 50 and 10 ng of purified NSP9 which served as a positive control. Top panel, SDS PAGE, bottom panel, corresponding immunoblots with nanobodies 2NSP23 and 2NSP90.

Figure 2

A–C) ¹⁵N‐¹H HSQC NMR spectrum of SARS‐CoV‐2 Nsp9 (138 × 10⁻⁶ m in phosphate buffer, pH 7.03, 298 K). A moderately strong resolution enhancement weighing function (45°‐shifted squared sinebell) was applied prior to 2D Fourier transform. For the red‐highlighted region, the right panels show the difference between the signals without (B) and with (C) the same resolution enhancement as applied in (A). D) ¹⁵N‐¹H HSQC NMR spectrum of SARS‐CoV‐2 triSer‐Nsp9 (131 × 10⁻⁶ m in aqueous acetate, pH 4.7, 298 K). Similar spectra are obtained also at pH 3.7 and 6.1. E) Overlay of the HSQC maps of triSer‐Nsp9 (red contours) and wild‐type Nsp9 (black contours) from SARS‐CoV‐2. Green highlighting marks the missing backbone amide cross‐peaks in the mutant spectrum, whereas blue highlighting indicates the missing connectivities in the wild‐type spectrum. The assignments of the missing signals in the spectrum of triSer‐Nsp9 are reported in black. The assignment of some of the additional signals present only in the spectrum of triSer‐Nsp9 is shown in red and is tentative for G100 and G104. All assignments were from Biological Magnetic Resonance Bank (BMRB 6501, 50 513, 50 622).

Figure 3

A) Crystal structure of SARS‐CoV‐2 Nsp9 tetramer (PDB ID: 7BWQ). The red regions locate fragments 67–69, 17–22, and residue 37, namely the interdimer contact surface that results highlighted by the different NMR pattern observed with triSer‐Nsp9 and Nsp9. The yellow regions show the positions of G100, G104, and A108 at the intradimer contact surface. Also, these residues exhibit a responsive pattern when comparing the spectra of the mutant and wild type. The pink regions (fragments 30–32 and 44–46) respond with a similar pattern as the red regions in the mutant spectrum, most likely revealing effects that occur more distantly with respect to the contact areas. B) Overlay of the ¹⁵N‐¹H HSQC maps of SARS‐CoV‐2 Nsp9 recorded at 278 K, in the absence (black contours) and presence of 2NSP23, at protein:nanobody ratio 1:0.43 (green contours) and 1:0.63 (cyan contours). C) ¹⁵N‐¹H HSQC spectrum of Nsp9 and 2NSP23 at protein:nanobody ratio 1:2. Similar patterns were obtained also with 2NSP90. D) Overlay of ¹⁵N‐¹H HSQC regions of Nsp9 recorded at 276 K, in the absence (black contours) and presence of 2NSP90, at protein:nanobody ratio 1:0.43 (blue contours) and 1:0.74 (magenta contours). Analogous chemical shift changes were observed also with 2NSP23.

Figure 4

A) The SARS‐CoV‐2 Nsp9 tetramer with the interdimer and intermonomer contact surfaces highlighted in red and yellow, respectively. The blue surfaces indicate the location of the tetramer epitopes interacting with nanobodies 2NSP23 and 2NSP90. An analogous epitope pair is present on the opposite face of the tetramer. The first epitope is comprised of the surfaces e1, e2a, and e2b formed by segment [Q11‐M12‐S13‐C14] with residue L29, residue N27 and residue K86, respectively, B) and the additional contributions from L45 and S46 that are already part of the tetramer interface. The second epitope is comprised of the surfaces e3 and e4 formed by the segments C) [D50‐L51‐K52‐W53] and D) [C73‐R74‐F75‐V76 + Y87‐L88‐Y89], respectively.