Structure, Function And Evolution of Corinavirus.

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Introduction:

The coronavirus spike protein is a multifunctional molecular machine that mediates coronavirus entry into host cells. It first binds to a receptor on the host cell surface through its S1 subunit and then fuses viral and host membranes through its S2 subunit. Two domains in S1 from different coronaviruses recognize a variety of host receptors, leading to viral attachment. The spike protein exists in two structurally distinct conformations, prefusion and postfusion. The transition from prefusion to postfusion conformation of the spike protein must be triggered, leading to membrane fusion. This article reviews current knowledge about the structures and functions of coronavirus spike proteins, illustrating how the two S1 domains recognize different receptors and how the spike proteins are regulated to undergo conformational transitions. I further discuss the evolution of these two critical functions of coronavirus spike proteins, receptor recognition and membrane fusion, in the context of the corresponding functions from other viruses and host cells.

Coronaviruses belong to the family Coronaviridae in the order Nidovirales. They can be classified into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Among them, alpha-and betacoronaviruses infect mammals, gammacoronaviruses infect avian species, and deltacoronaviruses infect both mammalian and avian species.

Representative alphacoronaviruses include human coronavirus NL63 (HCoV-NL63), porcine transmissible gastroenteritis coronavirus (TGEV), PEDV, and porcine respiratory coronavirus (PRCV). Representative betacoronaviruses include SARS-CoV, MERS-CoV, bat coronavirus HKU4, mouse hepatitis coronavirus (MHV), bovine coronavirus (BCoV), and human coronavirus OC43. Representative gamma-and deltacoronaviruses include avian infectious bronchitis coronavirus (IBV) and porcine deltacoronavirus (PdCV), respectively. Coronaviruses are large, enveloped, positive-stranded RNA viruses. They have the largest genome among all RNA viruses, typically ranging from 27 to 32 kb. The genome is packed inside a helical capsid formed by the nucleocapsid protein (N) and further surrounded by an envelope. Associated with the viral envelope are at least three structural proteins: The membrane protein (M) and the envelope protein (E) are involved in virus assembly, whereas the spike protein (S) mediates virus entry into host cells. Some coronaviruses also encode an envelope-associated hemagglutinin-esterase protein (HE). Among these structural proteins, the spike forms large protrusions from the virus surface, giving coronaviruses the appearance of having crowns (hence their name; corona in Latin means crown). In addition to mediating virus entry, the spike is a critical determinant of viral host range and tissue tropism and a major inducer of host immune responses.

The coronavirus spike contains three segments: a large ectodomain, a single-pass transmembrane anchor, and a short intracellular tail. The ectodomain consists of a receptor-binding subunit S1 and a membrane-fusion subunit S2. Electron microscopy studies revealed that the spike is a clove-shaped trimer with three S1 heads and a trimeric S2 stalk. During virus entry, S1 binds to a receptor on the host cell surface for viral attachment, and S2 fuses the host and viral membranes, allowing viral genomes to enter host cells. Receptor binding and membrane fusion are the initial and critical steps in the coronavirus infection cycle; they also serve as primary targets for human inventions. In this article, I review the structure and function of coronavirus spikes and discuss their evolution.

Receptors Recognized by Corinavirus Proteins:

Coronaviruses demonstrate a complex pattern for receptor recognition. For example, the alphacoronavirus HCoV-NL63 and the betacoronavirus SARS-CoV both recognize a zinc peptidase angiotensin-converting enzyme 2 (ACE2). Moreover, HCoV-NL63 and other alphacoronaviruses recognize different receptors: other alphacoronaviruses such as TGEV, PEDV, and PRCV recognize another zinc peptidase, aminopeptidase N (APN). Similarly, SARS-CoV and other betacoronaviruses recognize different receptors: MERS-CoV and HKU4 recognize a serine peptidase, dipeptidyl peptidase 4 (DPP4) ; MHV recognizes a cell adhesion molecule, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) ; BCoV and OC43 recognize sugar. The alphacoronavirus and PEDV and the gammacoronavirus IBV also use sugar as receptors or coreceptors. Other than their role in viral attachment, these coronavirus receptors have their own physiological functions. The diversity of receptor usage is an outstanding feature of coronaviruses. To further compound the complexity of the issue, the S1 subunits from different genera share little sequence similarity, whereas those from the same genus have significant sequence similarity. Therefore, the following questions have been raised regarding receptor recognition by coronaviruses:

  (a) How do coronaviruses from different genera recognize the same receptor protein? 

  (b) How do coronaviruses from the same genus recognize different receptor proteins?

  (c) What is the molecular basis for coronavirus spikes to recognize sugar receptors and             function as viral lectins?

Two major domains in coronavirus S1, N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD), have been identified. One or both of these S1 domains potentially bind receptors and function as the receptor-binding domain (RBD). S1-NTDs are responsible for binding sugar , with the only known exception being betacoronavirus MHV S1-NTD that recognizes a protein receptor CEACAM1. S1-CTDs are responsible for recognizing protein receptors ACE2, APN, and DPP4. Crystal structures have been determined for a number of S1 domains complexed with their respective receptor . These structures, along with functional studies, have addressed many of the puzzles surrounding receptor recognition by coronaviruses.

Receptors Recognized by Corinavirus S1 CTDS:

The structure of betacoronavirus SARS-CoV S1-CTD complexed with human ACE2 provided the first atomic view of coronavirus S1. SARS-CoV S1-CTD contains two subdomains: a core structure and a receptor-binding motif (RBM). The core structure is a five-stranded antiparallel β-sheet. The RBM presents a gently concave outer surface to bind ACE2. The base of this concave surface is a short, two-stranded antiparallel β-sheet, and two ridges are formed by loops. The ectodomain of ACE2 contains a membrane-distal peptidase domain and a membrane-proximal collectrin domain  Several virus-binding motifs (VBMs) have been identified on the outer surface of the peptidase domain, away from the buried peptidase catalytic site. SARS-CoV binding does not interfere with the enzymatic activity of ACE2, nor does the enzymatic activity of ACE2 play any role in SARS-CoV entry.

Research on SARS-CoV-ACE2 interactions has provided novel insight into cross-species transmissions of SARS-CoV. During the SARS epidemic, highly similar SARS-CoV strains were isolated from both human patients and palm civets from nearby animal markets. Their S1-CTDs differ by only two residues in the RBM region: Asn479 and Thr487 in human viral strains become Lys479 and Ser487 in civet viral strains, respectively. However, human SARS-CoV S1-CTD binds to human ACE2 much more tightly than civet SARS-CoV S1-CTD does. Two virus-binding hot spots have been identified on human ACE2, centering on ACE2 residues Lys31 and Lys353, respectively. Both hot spots consist of a salt bridge buried in a hydrophobic environment and contribute critically to virus-receptor binding. Residues 479 and 487 in SARS-CoV S1-CTD interact closely with these hot spots and are under selective pressure to mutate. Two naturally selected viral mutations, K479N and S487T, strengthened the hot spot structures and enhanced the binding affinity of S1-CTD for human ACE2. Consequently, these two mutations played important roles in the civet-to-human and human-to-human transmissions of SARS-CoV during the SARS epidemic.  Compared to human ACE2, rat ACE2 contains two different residues that disfavor SARS-CoV binding: His353 disturbs the hot spot structure centering on Lys353, whereas Asn82 introduces an N-linked glycan, presenting steric interference with SARS-CoV binding. Mouse ACE2 also contains His353 but does not have the N-linked glycan at the 82 position. Thus, rat ACE2 is not a receptor for SARS-CoV, whereas mouse ACE2 is a poor receptor. Consequently, SARS-CoV does not infect rat cells, and it infects mouse cells inefficiently. SARS-like coronaviruses (SLCoVs) have been identified in bats, and some can infect human cells. Structural details on how these bat SLCoV S1-CTDs interact with ACE2 from different mammalian species still wait to be determined. Overall, these studies on SARS-CoV-ACE2 interactions reveal that (a) one or two mutations in viral RBDs can cause serious epidemic outcomes and (b) one or a few residue variations in receptor homologs from different animal species can form critical barriers for cross-species transmissions of viruses.

Structure of Betacoronavirus:

The structure of betacoronavirus MERS-CoV S1-CTD complexed with human DPP4, when compared with SARS-CoV, presented an interesting example of how two structurally similar viral RBDs recognize different protein receptors. Like SARS-CoV S1-CTD, MERS-CoV S1-CTD also contains two subdomains, a core structure and an RBM. The core structures of MERS-CoV and SARS-CoV S1-CTDs are similar to each other, whereas their RBMs are markedly different. In contrast to the loop-dominated and gently concave surface of SARS-CoV RBM, MERS-CoV RBM consists of a four-stranded antiparallel β-sheet, presenting a relatively flat surface to bind DPP4. On the other hand, DPP4 forms a homodimer and each monomer contains a hydrolase domain and a β-propeller domain. The VBMs are located on the outer surface of the β-propeller domain, away from the peptidase catalytic site. The variations of VBM residues on DPP4 homologs from different mammalian species pose a barrier for cross-species transmissions of MERS-CoV. For example, mouse and rat DPP4 molecules are both poor receptors for MERS-CoV because they each contain a number of VBM residues that disfavor MERS-CoV binding. Camel DPP4 is an effective receptor for MERS-CoV due to its conserved VBM residues. Indeed, MERS-CoV has been isolated from camels, suggesting a camel-to-human transmission of MERS-CoV. Several MERS-related coronaviruses have been isolated from bats . Among them, HKU4 recognizes DPP4 using a structural mechanism similar to that used by MERS-CoV, indicating a bat origin of MERS-CoV. Overall, these studies reveal that viral RBDs with a conserved core structure can recognize different receptors through structural variations in their RBM, and they also reinforce the concept that receptor recognition is a critical determinant of viral host ranges.

Structure of Alphacoronavirus:

The structure of alphacoronavirus HCoV-NL63 S1-CTD complexed with human ACE2, when compared with SARS-CoV, showed how two structurally divergent viral RBDs recognize the same protein receptor. HCoV-NL63 S1-CTD contains a core structure and three RBM loops. The core structure of HCoV-NL63 S1-CTD is a β-sandwich consisting of two three-stranded antiparallel β-sheets. It differs from the core structure of SARS-CoV S1-CTD, which consists of a single-layer, five-stranded β-sheet. The RBMs of HCoV-NL63 S1-CTD are three short, discontinuous loops. They differ from the RBM of SARS-CoV S1-CTD, which is a long, continuous subdomain. Nevertheless, HCoV-NL63 and SARS-CoV S1-CTDs share the same structural topology (connectivity of secondary structural elements). Despite their different structures, HCoV-NL63 and SARS-CoV S1-CTDs bind to the same VBMs on human ACE2 . Between the two SARS-CoV-binding hot spots on human ACE2, the hot spot centering on Lys353 also plays a critical role in HCoV-NL63 binding.  Consequently, as with SARS-CoV, entry of HCoV-NL63 into mouse cells is inefficient due to the presence of His353 on mouse ACE2. These studies demonstrate that viral RBDs with different structures can bind to a common virus-binding hot spot on the same protein receptor.

Nevertheless, HCoV-NL63 and SARS-CoV S1-CTDs share the same structural topology (connectivity of secondary structural elements). Despite their different structures, HCoV-NL63 and SARS-CoV S1-CTDs bind to the same VBMs on human ACE2 . Between the two SARS-CoV-binding hot spots on human ACE2, the hot spot centering on Lys353 also plays a critical role in HCoV-NL63 binding. Consequently, as with SARS-CoV, entry of HCoV-NL63 into mouse cells is inefficient due to the presence of His353 on mouse ACE2 . These studies demonstrate that viral RBDs with different structures can bind to a common virus-binding hot spot on the same protein receptor.

The structure of alphacoronavirus PRCV S1-CTD complexed with procine APN, when compared with HCoV-NL63, presented another example of how two similar coronavirus RBDs bind to different protein receptor. Like HCoV-NL63 S1-CTD, PRCV S1-CTD contains a β-sandwich core structure and three RBM loops. The core structures of PRCV and HCoV-NL63 S1-CTDs are similar to each other, but their RBMs are divergent, leading to different receptor specificities. The ectodomain of APN has a seahorse-shaped structure and forms a head-to-head dimer. The VBMs are located on the outer surface of APN, away from the buried APN catalytic site. Several other alphacoronaviruses, such as TGEV, PEDV, human coronavirus 229E, feline coronavirus, and canine coronavirus, recognize APN from their natural host as their receptor.  These APN-recognizing alphacoronaviruses, except for PEDV, have been shown to also recognize feline APN, suggesting transmission of feline coronavirus from cats to other mammals. These studies showed again that similar viral RBDs with a conserved core structure can recognize different protein receptors through structurally divergent RBMs.

The above studies provide insight into the evolution of coronavirus S1-CTDs. Although the core structures of alpha-and betacoronavirus S1-CTDs are a β-sandwich and a single-layer β-sheet, respectively, they share the same structural topology, suggesting a common evolutionary origin. The S1-CTDs from different genera likely have undergone extensive divergent evolution to attain different core structures. The three RBM loops of alphacoronavirus S1-CTDs might have further diverged into ACE2-binding RBMs in HCoV-NL63 and APN-binding RBMs in PRCV. The RBM subdomain of betacoronavirus S1-CTDs might also have diverged into ACE2-binding RBM in SARS-CoV and DPP4-binding RBM in MERS-CoV. Despite their different structures, alphacoronavirus HCoV-NL63 and betacoronavirus SARS-CoV S1-CTDs bind to a common region on ACE2, possibly driven by the common virus-binding hot spot on ACE2. The tertiary structures of the S1-CTDs from gamma-and deltacoronaviruses are unavailable but are likely related to the folds of alpha-and betacoronavirus S1-CTDs. The complex evolutionary relationships among the S1-CTDs from different genera reflect the heavy evolutionary pressure on this domain, which is discussed in more detail in next post.

Acknowledgement:

This work is supported by Nation Institutes of Health.