SARS basic research

Crystal structure of the SARS-CoV main protease (Mpro)

 

The 33.8-kDa SARS-CoV main protease (also called the 3C-like protease) plays a pivotal role in mediating viral replication and transcription functions through extensive proteolytic processing of two replicase polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). The crystal structures of the SARS-CoV main protease were determined at different pH values and in complex with a specific inhibitor (1). This work provided the first crystal structure of any protein from the SARS coronavirus. The protease structure has a fold that can be described as an augmented serine-protease, but with a Cys-His at the active site. This series of crystal structures, which was the first to our knowledge of any protein from the SARS virus, reveal substantial pH-dependent conformational changes, and an unexpected mode of inhibitor binding, providing a structural basis for rational drug design. The crystal structure of the SARS-CoV main protease was published in PNAS (1).

 

Design of wide-spectrum inhibitors targeting the SARS Mpro

 

In a follow-up study to the SARS Mpro structure, Zihe Rao’s group proposed a strategy for preventing infection by existing and possible future emerging coronaviruses. After analyzing the critical subsites of Mpro substrate binding pocket from representatives of the three groups of CoVs Mpros, all of those subsites were found to be highly conserved. A compound targeting this conservative region was designed and found to rapidly inactivate multiple Mpros covering all three groups of CoVs in vitro. The compound, termed N3, also shows potent antiviral activity with extremely low cytotoxicity. A uniform inhibition mechanism was elucidated from the structures of Mpro-inhibitor complexes from SARS-CoV and TGEV. This compound could rapidly inactivate the Mpro of the SARS-CoV, as well as the Mpros of HCoV-NL63 and HCoV-HKU1, two recently identified human coronaviruses associated with bronchiolitis and pneumonia, and is currently in the pre-clinical testing stage. Further modification of this potent inhibitor could rapidly lead to the discovery of a single agent with clinical potential against existing and possible future emerging CoV-related diseases. This work was published in PLoS Biology (2) and was described as a “Crowning achievement” in a Research Highlight in Nature (2005, 437, 454-455).

 

Crystal structure of the SARS-CoV spike protein fusion core

 

The coronavirus spike protein, an enveloped glycoprotein essential for viral entry, belongs to the class I fusion proteins and is characterized by the presence of two heptad repeat (HR) regions, HR1 and HR2. These two regions form a fusion-active conformation similar to those of other typical viral fusion proteins. This hairpin structure likely juxtaposes the viral and cellular membranes, thus facilitating membrane fusion and subsequent viral entry. Zihe Rao’s group determined the crystal structures of the spike fusion core proteins of severe acute respiratory syndrome coronavirus (3) and mouse hepatitis virus (4). The fusion core is a six-helix bundle with three HR2 helices packed against the hydrophobic grooves on the surface of the central coiled coil formed by three parallel HR1 helices in an oblique antiparallel manner. The structures share significant similarity with other viral fusion proteins, suggesting a conserved mechanism of membrane fusion. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation, which have been successful in HIV-1 inhibitor development, should be applicable to the inhibition of severe acute respiratory syndrome coronavirus on the basis of the SARS spike protein fusion core structure.

 

Crystal structure of the SARS-CoV nsp7-nsp8 supercomplex

 

The SARS-CoV replicase gene encodes 16 non-structural proteins (nsp) with multiple enzymatic functions, yet little is known about the mechanism of the coronavirus replication/transcription machinery, particularly the interactions between the various non-structural protein components, and further work is needed to identify viable targets. Zihe Rao’s group determined the structure of the complex between two non-structural proteins, nsp7 and nsp8. Eight copies of nsp7 and eight copies of nsp8 together form an intricate scaffold that resembles a hollow cylinder. The inner dimensions and electrostatic properties of the cylinder suggest that it should encircle nucleic acid, and an interaction was demonstrated with dsRNA by EMSA and mutagenesis. The architecture and electrostatic properties are strongly reminiscent of PCNA or the β-subunit ring, the processivity factors of DNA polymerase, suggesting that the nsp7-nsp8 complex should be a processivity factor for the RNA-dependent RNA polymerase (nsp12). The structure of the nsp7-nsp8 complex was published in Nature Structural and Molecular Biology (5).

 

More recently, Zihe Rao’s group identified a new isoform of nsp8, suggesting that SARS-CoV nsp8 undergoes resectioning in the cell. This smaller nsp8 isoform, termed nsp8C, consists of the C-terminal globular domain of nsp8 only. Following proteolysis, nsp8C may regulate RNA synthesis through remodeling the components of the primase (6).

 

Crystal structure of SARS-CoV nsp10

 

The severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural proteins nsp1 to nsp16 have been implicated by genetic analysis in the assembly of a functional replication/transcription complex. Zihe Rao’s group determined the crystal structure of nsp10 from SARS-CoV at 2.1-A resolution (7). The nsp10 structure has a novel fold, and 12 identical subunits assemble to form a unique spherical dodecameric architecture. Two zinc fingers were identified from the nsp10 monomer structure with the sequence motifs C-(X)2-C-(X)5-H-(X)6-C and C-(X)2-C-(X)7-C-(X)-C. The nsp10 crystal structure is the first of a new class of zinc finger protein three-dimensional structures to be revealed experimentally. The zinc finger sequence motifs are conserved among all three coronavirus antigenic groups, implying an essential function for nsp10 in all coronaviruses, possibly as a putative transcription factor.

 

Crystal structure of MHV nsp15

 

Zihe Rao’s group determined the crystal structure of mouse hepatitis virus strain A59 (MHV-A59) nsp15 is reported at 2.15-Å resolution (8). Nsp15 is an XendoU endoribonuclease and was the first from this family to have its three-dimensional structure unveiled. The MHV-A59 nsp15 monomer structure has a novel protein fold. Two nsp15 trimers form a back-to-back hexamer that is believed to be the functional unit. The structure reveals the catalytic site, which is supported by mutagenesis analysis. Gel filtration and enzyme activity assays confirmed that the hexamer is the active form for nsp15 and demonstrate the specificity of nsp15 for uridylate. The high sequence conservation of nsp15 in coronaviruses, including severe acute respiratory syndrome, suggests that this protein may provide a new target for the design of antiviral therapeutics.

 

Structural analysis of the coronavirus ADRP domain

 

In coronaviruses, nsp3 includes a domain with a macroH2A-like fold and ADP-ribose-1"-monophosphatase (ADRP) activity, which is proposed to play a regulatory role in the replication process. However, the significance of this domain for the coronaviruses is still poorly understood due to the lack of structural information from different lineages. Rao’s group determined the crystal structures of viral ADRP domains, from the group I human coronavirus 229E and the group III avian infectious bronchitis virus, as well as their respective complexes with ADP-ribose (9). Together with data from a previous analysis of the ADRP domain from the group II SARS-CoV and from other related functional studies of ADRP domains, a systematic structural analysis of the coronavirus ADRP domains was realized for the first time to provide a structural basis for the function of this domain in the coronavirus replication process.

 

Crystal structure of the C-terminal cytoplasmic domain of nsp4

 

RNA replication of positive-strand RNA viruses is typically associated with the cytoplasmic membranes of infected cells. The replication of coronaviruses takes place on cytoplasmic double membrane vesicles (DMVs) originating in the endoplasmic reticulum (ER). Three trans-membrane non-structural proteins: nsp3, nsp4 and nsp6 are proposed to be membrane anchors of the coronavirus replication complex. Nsp4 is located to the ER membrane when expressed alone, but is recruited into the replication complex in infected cells. Rao’s group determined the crystal structures of the C-terminal hydrophilic domain of nsp4 (nsp4C) from MHV strain A59 and its C425S site-directed mutant (10). The highly conserved 89 amino acid region from T408 to Q496 is shown to possess a new fold. The wild-type (WT) structure, featuring two monomers linked by a Cys425-Cys425 disulfide bond in one asymmetric unit, exists as dimer in solution and can dissociate easily into monomers in a reducing environment. As nsp4C is exposed in the highly reducing cytosol, the monomer of nsp4C should be physiological. This structure may serve as a basis for further functional studies of nsp4.

 

Other basic research

 

During the SARS outbreak, Zihe Rao’s group used the BJ01 strain of SARS to clone the genes of all 4 structural and 16 non-structural proteins, and to perform expression and purification of 48 proteins or important functional fragments thereof. A serological study confirmed that SARS-positive patient sera contained strong antibodies against the S, N and M structural proteins, but not the E protein. The neutralizing ability of antisera directed against the expressed M protein and S protein fragments were tested and found to be greater than that of convalescent patient antisera, confirming that the former as immunogens induce a strong neutralizing antibody response (11). Furthermore, the immune response of the expressed M protein was examined against SARS patient sera, and it was shown that the M protein has strong antigenicity (12). These results together have vital significance for the development of an effective protein-based vaccine against SARS coronavirus infection.

 

With more than 20 protein and complex structures from SARS-CoV and other coronaviruses, together with wide-spectrum inhibitor design targeting the coronavirus main protease, Zihe Rao’s group has proven to be the most productive in coronavirus structural biology. He currently holds eight innovation patents for his SARS research.

 

 

 

1. Yang, H., M. Yang, Y. Ding, Y. Liu, Z. Lou, Z. Zhou, L. Sun, L. Mo, S. Ye, H. Pang, G. F. Gao, K. Anand, M. Bartlam, R. Hilgenfeld, and Z. Rao*. 2003. The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor. Proc Natl Acad Sci U S A 100:13190.

2. Yang H., W. Xie, X. Xue, K. Yang, J. Ma, W. Liang, Q. Zhao, Z. Zhou, D. Pei, J. Ziebuhr, R. Hilgenfeld, K. Y. Yuen, L. Wong, G. Gao, S. Chen, Z. Chen, D. Ma, M. Bartlam, and Z. Rao*. 2005. Design of wide-spectrum inhibitors targeting coronavirus main proteases. PLoS Biology 3(10):e324.

3. Xu, Y., Z. Lou, Y. Liu, H. Pang, P. Tien, G. F. Gao, and Z. Rao*. 2004. Crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core. J Biol Chem 279:49414.

4. Xu, Y., Y. Liu, Z. Lou, L. Qin, X. Li, Z. Bai, H. Pang, P. Tien, G. F. Gao, and Z. Rao*. 2004. Structural basis for coronavirus-mediated membrane fusion. Crystal structure of mouse hepatitis virus spike protein fusion core. J Biol Chem 279:30514.

5. Zhai, Y., F. Sun, X. Li, H. Pang, X. Xu, M. Bartlam, and Z. Rao*. 2005. Insights into SARS-CoV transcription and replication from the structure of the nsp7-nsp8 hexadecamer. Nat. Struct. & Mol. Biol. 12: 980-6.

6. Li, S., Q. Zhao, Y. Zhang, Y. Zhang, M. Bartlam, X. Li, and Z. Rao. 2010. New nsp8 isoform suggests mechanism for tuning viral RNA synthesis. Protein & Cell 2:198-204.

7. Su, D., Lou, Z., Sun, F., Zhai, Y., Yang, H., Zhang, R., Joachimiak, A., Zhang, X. C., Bartlam, M. & Rao, Z. (2006). Dodecamer structure of severe acute respiratory syndrome coronavirus nonstructural protein nsp10. J Virol 80, 7902-8.

8. Xu, X., Zhai, Y., Sun, F., Lou, Z., Su, D., Xu, Y., Zhang, R., Joachimiak, A., Zhang, X. C., Bartlam, M. & Rao, Z. (2006). New antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp15. J Virol 80, 7909-17.

9.  Xu, Y., L. Cong, C. Chen, L. Wei, Q. Zhao, X. Xu, Y. Ma, M. Bartlam, and Z. Rao, 2009. Crystal structures of two coronavirus ADP-ribose-1''-monophosphatases and their complexes with ADP-Ribose: a systematic structural analysis of the viral ADRP domain. J Virol 83: 1083-92.

10. Xu, X., Z. Lou, Y. Ma, X. Chen, Z. Yang, X. Tong, Q. Zhao, Y. Xu, H. Deng, M. Bartlam, and Z. Rao, 2009. Crystal structure of the C-terminal cytoplasmic domain of non-structural protein 4 from mouse hepatitis virus A59. PLoS One 4: e6217.

11. Pang, H., Y. Liu, X. Han, Y. Xu, F. Jiang, D. Wu, X. Kong, M. Bartlam, and Z. Rao*. 2004. Protective humoral responses to severe acute respiratory syndrome-associated coronavirus: implications for the design of an effective protein-based vaccine. J Gen Virol 85:3109.

12. Han, X., M. Bartlam, Y. H. Jin, X. Liu, X. He, X. Cai, Q. Xie, and Z. Rao*. 2004. The expression of SARS-CoV M gene in P. Pastoris and the diagnostic utility of the expression product. J Virol Methods 122:105.