Further possible functions of this protein were suggested when we observed that RNA fragments copurify with nsp3a from Escherichia coli cell lysates. We characterized the RNA fragments by using mass spectrometry, NMR spectroscopy, and electrophoretic gel mobility shift assays and identified the short sequence adenosine-uridine-adenosine (AUA) as a key structural element. NMR chemical shift perturbation studies to identify the amino acid residues of nsp3a most strongly affected by RNA binding (Fig. 2) indicated that these residues correspond to 2 helical secondary structure elements that represent a feature unique to nsp3a, one that has not been observed in other ubiquitin-like proteins. It thus appears that nsp3a may have evolved a new function of the ubiquitin-like fold that involves RNA binding or RNA processing. The sequence AUA occurs several times in the 5′-untranslated region of the SARS-CoV genome, further indicating that this binding or processing activity may be related to subgenomic RNA synthesis, which is an essential part of CoV replication. Overall, these data provide a novel lead that perturbation of the nsp3a-RNA interaction may be a promising strategy for the design of therapeutic agents to treat SARS.
Nonstructural Protein 3C
The third domain from residues 366–722 of nsp3, termed nsp3c, is unique to SARS-CoV and does not occur in other known coronaviruses. One-dimensional 1H NMR screening of constructs provided by the FSPS consortium showed that a construct spanning residues 451–651 contained a globular domain. We established purification protocols for this construct but found that it was chemically unstable, subject to proteolytic degradation after purification by nickel-affinity and size-exclusion chromatography. Edman degradation of the resulting 15.5-kD fragment indicated a high-frequency proteolytic cleavage site between residues 512 and 513. We cloned the fragment composed of residues 513–651 and found that it was amenable to NMR structure determination, with long-term stability of several weeks in aqueous solution. The solution structure revealed a mixed α/β fold with similarity to nucleic acid helicases and other nucleotide-binding proteins, suggesting a possible physiologic function in RNA unwinding during genome replication, a function that may be linked to those of other parts of nsp3, in particular, nsp3a.
We are currently refining the analysis of the structure of nsp3c, including the spatial arrangement of potential active-site residues that may be responsible for nucleotide or RNA binding. NMR structure determinations of additional components of the SARS proteome are at various stages of completion. Combined with the data on other parts of the SARS-CoV proteome accrued within and outside of the FSPS, our research can be expected to contribute to a broad scientific foundation for the development of strategies for handling potential future infections caused by SARS-CoV.
Almeida, M.S., Johnson, M.A., Herrmann, T., Geralt, M., Wüthrich, K. Novel β-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus. J. Virol. 81:3151, 2007.
Chatterjee, A., Johnson, M.A., Serrano, P., Pedrini, B., Wüthrich, K. NMR assignment of the domain 513-651 from the SARS-CoV nonstructural protein nsp3. Biomol. NMR Assign. 1:191, 2007.
Serrano, P., Johnson, M.A., Almeida, M.S., Horst, R., Herrmann, T., Joseph, J.S., Neuman, B.W., Subramanian, V., Saikatendu, K.S., Buchmeier, M.J., Stevens, R.C., Kuhn, P., Wüthrich, K. Nuclear magnetic resonance structure of the N-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus. J. Virol. 81:12049, 2007.