Characterization of Critical Determinants of ACE2-RBD Interaction

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. Utilizing our recently developed NanoBiT technology-based biosensor, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, before and after the addition of competitive inhibitors, as well as neutralizing antibody activity. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.


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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent 48 responsible for the COVID-19 pandemic and is an ongoing worldwide public health threat. As of 49 October 16 th 2020, there have been over 39 million confirmed cases, and over 1 million confirmed 50 deaths resulting from SARS-CoV-2 infection (WHO reports,51 https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports).

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Tremendous efforts are currently underway to develop rapid drug screening methodologies and 53 novel vaccines. The large variability of disease severity among individuals infected with SARS-

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CoV-2 continues to be investigated [1] and new findings in this field may shed light on strategies 55 to tailor these new therapeutics to patients.

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Entry of SARS-CoV-2 is mediated by interaction of the viral Spike glycoprotein (S) with 57 its main target receptor angiotensin converting enzyme 2 (ACE2), found on the surface of 58 mammalian cells, primarily in the lower respiratory tract [2]. The S protein is composed of two 59 subunits, S1 and S2, which play cooperatively a role in viral entry and fusion of the viral membrane 60 with host cell membrane. Binding to ACE2 is mediated by the receptor binding domain (RBD), 61 located in the C-terminus of the S1 subunit [3]. The identification of amino acid residues that are 62 crucial for the interaction of RBD with ACE2 is of great interest to gain a better understanding of 63 the interplay between viral entry and host genetic factors which may contribute to the observed 64 variability in disease pathogenesis. As such, characterizing functional mutants of RBD and ACE2 65 may provide critical insights for the development of drugs and vaccines.

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The development of biosensor technology is a highly valuable and sensitive analytical tool 67 with a broad spectrum of applications, such as diagnosis and drug development [4,5]. Biosensors 68 designed to emit bioluminescence often rely on luciferase, a class of enzymes that catalyze 69 substrate to produce a bioluminescent signal. One such tool is the NanoLuc Binary Technology 70 (NanoBiT), which enables rapid analysis of protein-protein interactions through use of 71 Nanoluciferase, a small luciferase reporter [5][6][7][8][9][10][11][12]. By exploiting this technology, we have recently 72 developed an assay to rapidly investigate RBD and ACE2 interactions. In addition, this assay can 73 be used to elucidate the impact of both RBD and ACE2 amino acid mutations on their binding 74 abilities, as well as their potential implications for drug development and evaluating immune 75 responses.
Previous studies have examined crucial residues in ACE2 and the SARS-CoV-1 Spike 77 domain [13,14]. Similarly, recent studies have begun to unravel important interactions in SARS-

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CoV-2 RBD and ACE2 [15]. While some progress has been made towards examining the impact 79 of specific mutations within the SARS-CoV-2 RBD and ACE2, there remain much to be studied 80 with regards to their impact on binding, infectivity and host susceptibility to viral infection. In 81 addition, the impact of specific RBD mutations on the efficacy of potential therapeutics has not 82 been explored.

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The first crucial step of SARS-CoV-2 viral entry is mediated by binding of RBD to ACE2, 96 its main cognate receptor expressed on the surface of the human airway epithelium ( Figure 1A).

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In this study, we aim to investigate whether selected mutations in both SARS-CoV-2 S protein 98 RBD and its host receptor ACE2 could impact their interactions with one another. We also examine 99 how specific mutations in ACE and RBD may alter the efficacy of drugs and neutralizing 100 antibodies being developed for treatment and disease prevention purposes, respectively. To 101 accomplish these aims, we use a NanoBiT SARS-CoV-2-RBD and ACE2 biosensor, previously 102 developed by our lab, to initially characterize the RBD: ACE2 interaction ( Figure 1B) [17]. The another; however, both subunits interact to produce a bioluminescent signal in the presence of 107 furimazine, the substrate for Nanoluciferase, if the fusion proteins interact with each other.

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Mutations within the ACE2 ectodomain alter binding affinity of ACE2 with SARS-CoV-2 109 RBD, SARS-CoV-2 S1 subunit, and SARS-CoV-1 RBD 110 ACE2 is a key interacting partner involved in SARS-CoV-2 viral entry, thus we first 111 performed in silico mutagenesis analysis to assess putative ACE2 mutants that could be potentially 112 defective at interacting with SARS-CoV-2 RBD. Based on the overall 3D crystal structure analysis 113 of ACE2 bound to RBD (Figure 2A immunoblotting ( Figure 3A). 119 We then investigated the binding affinity of ACE2 wild-type and mutants with both SARS-120 CoV-2-RBD and SARS-CoV-2 S1 as complementary binding partner. The rationale for also 121 including an S1-based NanoBiT binding partner in our assays was that RBD is encompassed within 122 the S1 subunit of the S glycoprotein. Hence, including S1 would more closely mimic SARS-CoV-2 123 S behavior in the context of viral infection. Thus, we proceeded to compare whether S1 fused to 124 LgBiT and RBD-LgBiT constructs have similar binding affinity for ACE and its mutants. When SARS-CoV-2 S1 ( Figure 3C). In summary, we found that the same 12 ACE2 mutants that showed 132 decreased binding to SARS CoV-2 RBD also has impaired interactions with SARS CoV-2 S1, 133 with the addition of mutant Y83A that reported a reduced affinity for SARS-CoV-1 S1 only.
Since structural analysis has shown that certain residues in SARS-CoV-2 RBD are well 135 conserved within SARS-CoV-1 RBD [17], we decided to also combined the various ACE2-SmBiT 136 mutants with a LgBiT-SARS-CoV-1 RBD construct. This allowed us to also evaluate how ACE2 137 mutations could impact ACE2 : SARS-CoV-1 RBD interaction. In figure 3D, we further show that To reinforce the ACE2 mutational findings observed with our biosensor technology 148 described above, we then decided to utilize a lentiviral-based pseudovirus infectivity assay. We

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Utilizing ACE2 sequences as a predictor for the SARS-CoV-2 susceptibility of various 160 species and mutation prevalence in humans 161 Our findings suggest that identification of ACE2 amino acid sites which impact RBD 162 binding may provide insights as to SARS-CoV-2 susceptibilities of different individuals or species 163 based on their genetics. Using multiple sequence alignments of the 12 target mutation sites 164 identified to be essential for RBD binding (Figure 3), we propose a method to predict SARS-CoV-165 2 virus susceptibility ( Figure 5A). It is plausible to hypothetize that any species with an identical 166 ACE2 amino acid sequence to the human ACE2 receptor would theoretically be highly susceptible 167 to the virus, and possibly act as a source of transmission, such as the common chimpanzee (Pan 168 troglodytes; Figure 5A). With the presence of only one or two differences at key ACE2 binding  A key concern surrounding RNA viruses, and viruses in general, is their capacity to evolve  Figure 6J). Importantly, this data may indicate that these receptor binding mutations do not 219 provide an immune evasive advantage to the virus, and as a result, should not pose a serious threat 220 to the efficacy of developed vaccines, and immunity, to viruses harboring these mutations. 222 We used multiple sequence alignment tools to further investigate amino acid conservation    For all analyses, *P<0.05, **P<0.01, ***P<0.005, n.s. = not significant.   Table 1 Insert DNA sequence Amino Acid  mutation (no effect on the protein sequence) is green and missense mutations are red. (each SARS-CoV-2 AA is green, the AA from the same chemistry is orange, and AA with different 473 chemistry is red. WebLogo (http://weblogo.threeplusone.com) representation shows a relative