key: cord-0840500-kw86z8bs
authors: Mishra, Tarun; Sreepadmanabh, M; Ramdas, Pavitra; Sahu, Amit K; Kumar, Atul; Chande, Ajit
title: SARS CoV-2 nucleoprotein enhances the infectivity of lentiviral spike particles
date: 2021-02-15
journal: bioRxiv
DOI: 10.1101/2021.02.11.430757
sha: 21de58f2abc05b6d67cec7d8823f3d0b31ef55a0
doc_id: 840500
cord_uid: kw86z8bs

The establishment of SARS CoV-2 spike-pseudotyped lentiviral (LV) systems has enabled the rapid identification of entry inhibitors and neutralizing agents, alongside allowing for the study of this emerging pathogen in BSL-2 level facilities. While such frameworks recapitulate the cellular entry process in ACE2+ cells, they are largely unable to factor in supplemental contributions by other SARS CoV-2 genes. To address this, we performed an unbiased ORF screen and identified the nucleoprotein (N) as a potent enhancer of spike-pseudotyped LV particle infectivity. We further demonstrate that this augmentation by N renders LV spike particles less vulnerable to the neutralizing effects of a human IgG-Fc fused ACE2 microbody. Biochemical analysis revealed that the spike protein is better enriched in virions when the particles are produced in the presence of SARS CoV-2 nucleoprotein. Importantly, this improvement in infectivity is achieved without a concomitant increase in sensitivity towards RBD binding-based neutralization. Our results hold important implications for the design and interpretation of similar LV pseudotyping-based studies.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has provided a strong impetus for 33 studies aimed at discovering and characterizing neutralizing antibodies or small molecule 34 inhibitors targeted against the severe acute respiratory syndrome coronavirus 2 (SARS 2). A major focus of these efforts has been the spike (S) glycoprotein of the virus, which 36 mediates the viral entry into target cells by recognition and binding to the cell-surface receptor 37 angiotensin-converting enzyme 2 (ACE2) 1, 2 . 38 39 Numerous studies have demonstrated that the spike protein may be employed to generate 40 stably pseudotyped lentiviral, retroviral, and vesicular stomatitis virus particles 3-7 . Given that 41 live SARS CoV-2 is a biosafety level-3 agent, these pseudotyping approaches have greatly 42 facilitated investigations undertaken within lower containment facilities. Importantly, this has also 43 allowed for the screening and characterization of neutralizing antibodies against SARS CoV-2, 44 as these primarily show reactivity against the spike protein 8 . Furthermore, such pseudoviruses 45 are deployable as platforms for the large-scale screening of small molecule inhibitors and 46 pharmacological agents which possess therapeutic potential against COVID-19 9 . 47 as well as the ability of ACE2-IgFc to dimerize under the experimental conditions that retained 98 disulphide linkage, were detected by Western blotting (Fig. 3B) . These experiments also 99 confirmed the presence of an intact Histidine tag. Furthermore, we tested the specific interaction 100 of the ACE2-IgFc with spike-pseudotyped LV particles by a pull-down experiment wherein the 101 on-bead capture of viral particles using protein G-bound ACE2-IgFc resulted in a ~10,000-fold 102 enrichment of spike pseudotyped viruses over VSV-G pseudotyped or non-enveloped (bald) 103 lentiviral particles (Fig. 3C) . This also suggests that Fc configuration remained intact after fusion 104 with ACE2 thereby facilitating the immobilization of the microbody on the protein G magnetic 105 beads for virion capture. In sum, these results established that the ACE2-IgFc molecule is stably 106 expressed and demonstrates a high affinity specifically towards the S glycoprotein-laden 107 particles. 108 109 Following this, we wished to ascertain the relative titer of ACE2-IgFc required to effectively 110 restrict the infection of ACE2+ target cells, under the influence of the N protein. Briefly, lentiviral 111 particles either pseudotyped with S protein (with and without co-transfection with N) or bald 112 particles lacking envelope were generated, normalized to the RT units, incubated with ACE2-113 IgFc according to the indicated concentrations, and used to transduce ACE2+ HEK293T target 114 cells. Luciferase activity assay, as a quantitative and sensitive indicator of transduction events, 115 revealed the ability of ACE2-IgFc to impair the infectivity of spike pseudotyped viruses in a 116 dose-dependent manner. Furthermore, results obtained herein demonstrate the requirement for 117 at least one log higher neutralizing titer of ACE2-IgFc microbodies in the case of N-enhanced 118 particles as opposed to those generated solely by the spike glycoprotein in order to elicit similar 119 levels of inhibition (Fig. 4A) . The higher requirement of neutralizing agent was not a result of 120 higher particle counts as the inoculum was normalized to the reverse transcriptase units prior to 121 the target cell challenge. 122 123 Based on this, we envisaged a modification of virus particles by N that plausibly impacted the 124 virion quality, rather than the quantity. To better understand this, we asked if N protein directly 125 impacted the spike protein itself in order to effect infectivity enhancement. Accordingly, the viral 126 particles produced under indicated conditions were pelleted on a sucrose cushion as described 127 earlier 14 . Biochemical analysis of the virus pellet and the corresponding cell lysates by Western 128 blotting revealed that while N modestly improved the steady-state levels of spike in the producer 129 cell lysates, the spike signal was noticeably more prominent from the virions (Fig. 4B) . The components do not show any apparent changes in expression. Altogether, these experiments 132 indicated that N protein likely enhances the incorporation of spike protein into the virions, 133 thereby improving the particle quality and its infectivity. The list of plasmids that were used in this study is provided below (refer to "Table S1: List of 190

Plasmids") and are available upon a reasonable request. pScalps-Luciferase-Zsgreen was 191 generated by cloning a firefly luciferase gene that was PCR amplified with primers incorporating 192 the XhoI/EcoRI restriction sites. The resultant PCR product was digested and ligated in the 193 pScalps ZSgreen plasmid using identical sites and the inserts were confirmed by Sanger 

For Western blot-based analysis, samples were prepared in a 4X Laemmli buffer, boiled for 5 261 min at 95°C, and run on either 8% or 12.5% tricine gels for electrophoresis depending upon the 262 molecular weight range being detected by this method. Following this, gels were electro blotted 263 on the PVDF membrane (Immobilon-FL, Merck-Millipore). Blocking of membranes was carried 264 out by incubation with either a 5% BSA solution or the proprietary Odyssey Blocking Buffer (LI-for one hour each at room temperature, each of which were followed by three washes for five 267 minutes. 268

Detection of p24, beta actin, SARS CoV-2 spike glycoprotein, SARS CoV-2 genes, and ACE2-269

IgFc was carried out using mouse anti-p24 (NIH ARP), rabbit anti-beta actin (LI-COR generate the final Fc fragment, which was cloned into pTZ57R. 296 combined construct was ligated into the ACE2 containing plasmid using a EcoRI/XbaI digestion 302 to yield the final ACE2-IgFc construct. The construct was verified with Sanger sequencing (refer 303 to " Table S2 : Sequences of ACE2-IgFc"). 304 305 Biochemical characterization of ACE2-IgFc microbody 306 HEK293T cells were transfected with the plasmid encoding ACE2-IgFc (5 µg plasmid in a 35 307 mm dish). As a control, 5 µg of pcDNA3.1BS(-) was also transfected. Media was changed 308 twelve hours post-transfection and fresh DMEM (without FBS) was added to the dishes. Culture 309 media was collected both 48-and 72-hours post-transfection, and samples were prepared in 310 either 8% or 2% SDS-containing 4X Laemmli buffer (with and without TCEP added, 311 respectively) for SDS-PAGE. 8% tricine gels were run, and analyzed using Western Blotting. 312

The amount of ACE2 present in the supernatant was determined by comparison with standard 313 concentrations of pure BSA as ascertained by band density analysis following silver staining of 314 SDS-PAGE gels. For Western Blotting, the proteins were transferred to a PVDF membrane, 315 blocked in a 5% BSA solution in TBS, and primary mouse-derived anti-Histidine antibodies were 316 incubated with the blot in a 1:4000 dilution, followed by goat-derived anti-mouse antibodies in a 317 1:5000 dilution. 318 319 Pull-down assay using ACE2-IgFc 320 A 30 µl of protein-G Dynabeads (Thermofisher Scientific Cat #10003D) were first equilibrated by 321 washing with 10% FBS-containing DMEM. Following this, these were incubated with gentle 322 mixing by inversion for 15 minutes with 100 µl of ACE2-IgFc containing supernatant (equivalent 323 of 10 µg/ml). This was split into three equal fractions of 10 µl each. The beads-containing 324 mixtures then were immobilized on a magnetic rack, supernatant was aspirated off, and 325 replaced with 100 µl (10mU equivalent/mL RT) of culture supernatant carrying either bald 326 viruses or viruses pseudotyped with VSV-G or Spike glycoproteins. Mixing was carried out for a 327 period of 15 minutes, following which these were placed back on the magnetic rack, supernatant 328 was aspirated, and the beads washed twice with SGPERT Core Buffer -first with 500 µl and 329 then with 100 µl. Finally, 20 µl of RT Lysis Buffer was added and the reaction was incubated at 330 room temperature before mixing with TritonX diluted 10-fold using core buffer. The resulting 331 supernatant was collected after immobilizing the beads and processed for an SGPERT Assay. Viruses Cells -+ + -+ Spike N p24 N Actin S2 S1 +S2 S2 S1 +S2 -

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