Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation

Nonstructural protein 1 (Nsp1) is a major pathogenicity factor of SARS-CoV-2. It inhibits host-cell translation, primarily through a direct interaction between its C-terminal domain and the mRNA entry channel of the 40S small ribosomal subunit, with an N-terminal β-barrel domain fine-tuning the inhibition and promoting selective translation of viral mRNA. SARS-CoV-2 nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. To provide the biochemical basis for this, it is essential to characterize the efficiency of translational inhibition by the said protein variants. Here, we use an in vitro translation system to investigate the translation inhibition capacity of a series of clinically observed Nsp1 deletion variants. We find that a frequently observed deletion of residues 79-89 destabilized the N-terminal domain (NTD) and severely reduced the capacity of Nsp1 to inhibit translation. Interestingly, shorter deletions in the same region have been reported to effect the type I interferon response but did not affect translation inhibition, indicating a possible translation-independent role of the Nsp1 NTD in interferon response modulation. Taken together, our data provide a mechanistic basis for understanding how deletions in Nsp1 influence SARS-CoV-2 induction of interferon response and COVID-19 progression.


Summary 24
Nonstructural protein 1 (Nsp1) is a major pathogenicity factor of SARS-CoV-2. It inhibits host-cell 25 translation, primarily through a direct interaction between its C-terminal domain and the mRNA entry 26 channel of the 40S small ribosomal subunit, with an N-terminal β-barrel domain fine-tuning the 27 inhibition and promoting selective translation of viral mRNA. SARS-CoV-2 nsp1 is a target of recurring 28 deletions, some of which are associated with altered COVID-19 disease progression. To provide the 29 biochemical basis for this, it is essential to characterize the efficiency of translational inhibition by the 30 said protein variants. Here, we use an in vitro translation system to investigate the translation inhibition 31 capacity of a series of clinically observed Nsp1 deletion variants. We find that a frequently observed 32 deletion of residues 79-89 destabilized the N-terminal domain (NTD) and severely reduced the capacity 33 of Nsp1 to inhibit translation. Interestingly, shorter deletions in the same region have been reported to 34 effect the type I interferon response but did not affect translation inhibition, indicating a possible 35 translation-independent role of the Nsp1 NTD in interferon response modulation. Taken together, our 36 data provide a mechanistic basis for understanding how deletions in Nsp1 influence SARS-CoV-2 37 induction of interferon response and COVID-19 progression. 38 Results 111 Circulating deletions in SARS-CoV-2 Nsp1 differ in their inhibitory effect on translation 112 Several amino acid deletions are reported in SARS-CoV-2 Nsp1: Δ85, Δ82-83, 85 and Δ79-89 in the N-113 terminal domain (NTD) (25) and Δ141-143 in the C-terminal domain (CTD) (26) (Fig. 1A). These 114 deletions have been correlated to clinical characteristics, but the mechanistic biochemical basis of why 115 they alter the course of COVID-19 has not been determined. We wished to assess the effect of these 116 deletions on Nsp1's ability to shut down host translation. As a first step towards this analysis, we purified 117 wild-type Nsp1 and a series of deletion mutants to homogeneity and monodispersity (Fig. S1). We then 118 established a translation assay based on lysates of the human cell line HEK293F. Since Nsp1 has been 119 reported to reduce translation of viral mRNAs to a lesser degree than cellular mRNAs (14,17,21), we 120 reasoned that a reporter mRNA resembling a viral mRNA would provide a more sensitive assay. Thus, 121 we measured translation efficiency of a reporter mRNA encoding firefly luciferase, equipped with the 122 SARS-CoV-2 5' UTR, a 5' cap, and 3' poly(A) sequence, in the presence of increasing concentrations of 123 both wild-type recombinant Nsp1 and its deletion variants (Fig.1B). 124 In agreement with earlier reports (17,21), wild-type Nsp1 efficiently abrogates production of firefly 125 luciferase in concentration-dependent manner. Already at 0.1 μM, wild-type (wt) protein reduces the 126 translational efficiency by more than 95% (Fig. 1C). An effect similar to the wt is observed for the NTD 127 deletion variant ∆85 and the CTD deletion variant ∆141-143. The NTD deletion variant ∆82-83,85 128 shows slightly reduced suppression of translation compared to the wild type. In contrast, the longest 129 deletion in the NTD, ∆79-89, is substantially weakened in its ability to inhibit translation as compared 130 to wt Nsp1 (Fig. 1C). We observed that even at 3 μM protein concentration, the translation efficiency 131 reduces only to 40-50 %, establishing that the ∆79-89 protein is less effective in translation shutdown 132 than the wild-type and other deletion variants. Taken together, these data establish a biochemical basis 133 for correlating COVID-19 disease progression to the translational shutdown efficiency of circulating 134 deletion mutants in Nsp1. 135

136
Deletions in Nsp1 lead to altered protein stability that correlates with translation inhibition 137 None of the investigated nsp1 mutations was in the region previously reported to interact with the 138 ribosome, and yet the longer deletions in the NTD were clearly altered in their translation shutdown 139 capacity. We wanted to investigate if this effect could be due to the destabilizing effects of the deletions 140 on the NTD. We first noted that all proteins were soluble and monodisperse when purified from E. coli, 141 but the Δ79-89 construct eluted at a lower volume in size exclusion chromatography, indicting a larger 142 hydrodynamic radius (Fig. S1). To probe the effects of the deletions on protein stability, we assessed 143 the thermal stability of wt Nsp1 and deletion variants by thermal shift assay (TSA) (29) (Fig. 2). In this 144 assay, SYPRO orange dye is used as a probe to estimate the extent of unfolding of the protein with increasing temperature, and the melting temperature (Tm) from each curve is determined as described in 146 the experimental procedure section. Raw TSA data ( Fig. 2A, B) demonstrates that all mutants of SARS-147 CoV-2 Nsp1 showed similar, wild-type-like unfolding profile, apart from the longest deletion variant 148 (∆79-89). Wt Nsp1 and all altered proteins, with a notable exception of ∆79-89, allowed for fitting of a 149 simple melting curve (Fig. 2C) indicating a single transition temperature that is comparable to that of 150 the wt protein (Fig. 2E). The thermal denaturation curve of ∆79-89 ( Fig. 2A,B) was shallow and could 151 not be perfectly recapitulated by fit to a single melting temperature (Fig. 2D). However, using the first 152 part of the curve gave a fit that is consistent with the qualitative appearance of the curve (Fig. 2D) and 153 a lowered thermal stability. This suggests that already at room temperature the structural integrity of 154 ∆79-89 variant is compromised. In summary, our results establish a correlation between structural 155 stability of Nsp1's NTD and its ability to inhibit translation. 156 157 Structure predictions suggest that NTD -barrel destabilization causes the decreased stability of 158

79-89 nsp1 159
We next wanted to explore the possible structural basis for the decreased translation inhibition and 160 thermal stability of the altered Nsp1 proteins. To investigate how the Nsp1 deletions impact its structure 161 we predicted the structures of wt, 141-143, 85, 82-83, 85 and 79-89 Nsp1 using AlphaFold 2 (30). 162 Since there are available experimental structure of the free SARS-CoV-2 Nsp1 NTD (residues 10-126) 163 (27), as well as the ribosome-inserted CTD (15-17), we could compare them to the prediction of the wt 164 Nsp1 structure as a baseline. The predicted Nsp1 structure aligns very well with the experimentally 165 determined N-terminal -barrel domain, with average positional shifts (root-mean-square deviation, 166 RMSD) of the respective C atoms of only 0.64 Å for residues 10 to 126 (Fig. 3A, Fig. S2A). In the 167 CTD, two α-helices are correctly predicted where they are observed in the experimental structure, albeit 168 at a different angle to each other than in the 40S-bound structures (Fig. S2A). Overall, the per-residue 169 prediction quality score (pLDDT) correlated with the rigidity of the fold across Nsp1, with the NTD β-170 barrel having higher scores than the CTD which was reported to be flexible in solution ( Fig. S2B)

(19). 171
Supported by the accurate prediction of the wt Nsp1 structure, we thus reasoned that structure 172 predictions for the altered Nsp1 proteins may shed light on the differential effects of the deletions on 173 protein stability. Unsurprisingly, the deletion 141-143, predicted to be located in a disordered region 174 of the CTD, had no effect on the predicted fold of other parts of Nsp1 (Fig. S2). The other three deletions 175 (85, 82-83, 85 and 79-89) are all located at the beginning of the fourth -strand of the -barrel (4) 176 ( Fig. 3B, D). Interestingly, despite being located in a secondary structure element, the two shorter 177 deletions are not predicted to affect the-barrel integrity (Fig. S2C), likely given that the hydrogen 178 bonding between -strands is mediated by the protein backbone and can be rescued by the residues 179 "next-in-line" to the deleted residues. The predicted integrity of the fold of these deletions correlates well with their unaltered thermal stability (Fig. 2E). The only striking effect on the integrity of the -181 barrel domain is predicted to stem from the longest deletion. The 79-89 structure prediction suggests 182 a near-complete dissolution of the 3-5 strands leading to a break in the -barrel domain, severely 183 affecting its integrity (Fig. 3C, E). This prediction is in line with the drastic reduction in thermal stability 184 for the 79-89 protein from 46°C to ~27°C (Fig. 2E). Taken  For expression of wild-type Nsp1, the nsp1 gene was amplified from the construct pLVX-EF1alpha-225

SARS-CoV-2-nsp1-2xStrep-IRES-Puro (Addgene) (31) and inserted into a 1B vector (Macrolab, UC 226
Berkeley) using In-Fusion cloning kit (Takara Bio). Deletion mutations were generated from this 227 plasmid by standard site-directed mutagenesis methods. The reporter plasmid T7-5'UTR-Firefly 228 luciferase for RNA synthesis was generated by inserting the T7 promoter upstream of SARS-CoV2 5' 229 UTR fused to the firefly luciferase coding sequence into the destination vector pUC19. All plasmids 230 were sequenced to confirm cloning of the correct sequence. 231 232

Protein expression and purification 233
Wt Nsp1 and all deletion variants were expressed and purified as follows. The plasmid was transformed 234 into E. coli BL21(DE3) cells for overexpression. An overnight culture was grown at 37℃ to inoculate 235 the secondary culture. Cells were grown at 37℃ until the OD600 reached 0.4, after which the incubator 236 temperature was changed to 25℃ to let the cells cool down to induction temperature 25℃. At OD600 of 237 around 0.8-0.9 the protein expression was induced by addition of 0.5 mM Isopropyl β-d-1-238 thiogalactopyranoside (IPTG) and the protein was expressed at 25℃ overnight. Cells were harvested by 239 centrifugation at 6,000 rpm (rotor JLA-8.1000 Beckman Coulter, Brea, USA) for 60 minutes. After 240 discarding the supernatant the cell pellet was washed with lysis buffer (50 mM HEPES-NaOH, pH 7.4, 241 300 mM NaCl, 0.1 mM THP, 10 mM Imidazole and 5 % glycerol) and stored at -80℃. In vitro translation lysates were prepared from HEK293F cells using a previously described protocol 283 (32-34). Cells were scraped and collected by centrifugation for 5 minutes at 600 rpm at 4°C. Cells were 284 washed once with cold PBS (137 mM NaCl, 2.7mM KCl, 100mM Na2HPO4, 2mM KH2PO4) and re-285 suspended in Lysolecithin lysis buffer (20 mM HEPES-KOH, pH 7.4, 100 mM KOAc, 2.2 mM 286 Mg(OAc)2, 2 mM DTT, and 0.1 mg/ml lysolecithin), using 1 ml for 8x10 6 cells. Cells were incubated for 1 min on ice, then immediately centrifuged for 10 sec at 10,000 g at 4℃. The pellet was re-suspended 288 in cold hypotonic extraction buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KOAc, 1 mM Mg(OAc)2, 4 289 mM DTT, and Complete EDTA-free protease inhibitor cocktail (Roche) at an equal volume to the size 290 of the pelleted cells. After 5 min of incubation on ice, cells were transferred into a pre-cooled Dounce 291 homogenizer and lysed by 20-25 strokes. The lysate was centrifuged at 10,000*g for 10 min at 4 °C, 292 and the supernatant transferred to a fresh tube. Aliquots were flash frozen in liquid N2 and stored at -293

80°C. 294 295
In vitro translation assays 296 In vitro translation reactions were performed as previously described ( plate (4titudE 4ti-07 10/C) sealed afterward with PCR optical Seal (4titudE). Thermal scanning (10 to 315 95℃ at 1.5 ℃/min) was performed using a real-time PCR instrument C1000 Touch Thermal Cycler 316 (CFX96 from Bio-Rad) and fluorescence intensity was measured after every 10 seconds. According to 317 the described protocol, raw data were truncated in Microsoft Excel to remove post-peak quenching (35). 318 A non-linear fitting of the truncated dataset to a Boltzmann Sigmoidal equation was performed to obtain 319 the melting temperature (Tm) using Prism 9 (GraphPad Software). 320 321 322 323