Dodecamer Structure of Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein nsp10

Abstract

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. We report the crystal structure of nsp10 from SARS-CoV at 2.1-Å resolution. The nsp10 structure has a novel fold, and 12 identical subunits assemble to form a unique spherical dodecameric architecture. Two zinc fingers have been 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 threedimensional structures to be revealed experimentally. The zinc finger sequence motifs are conserved among all three coronavirus antigenic groups, implicating an essential function for nsp10 in all coronaviruses. Based on the structure, we propose that nsp10 is a transcription factor for coronavirus replication/transcription.Coronaviruses (CoVs) are positive-stranded RNA viruses known to cause highly prevalent respiratory and enteric diseases in humans and animals. About two-thirds of their genomes encode the viral replicase that mediates viral RNA synthesis (19). The replicase gene is comprised of two open reading frames at the 5Ј end of the genome, termed ORF1a and ORF1b (26). The upstream ORF1a encodes polyprotein pp1a (450 to 500 kDa), while ORF1a and ORF1b together encode pp1ab (750 to 800 kDa). Expression of the ORF1b-encoded half of pp1ab requires a Ϫ1 ribosomal frameshift during translation. The polyproteins pp1a and pp1ab undergo extensive proteolytic processing by viral proteases to produce 16 functional subunits known as nonstructural proteins, which then assemble to form the replicase complex required for viral replication and transcription.Prior to the global severe acute respiratory syndrome (SARS) outbreak in 2003, scant attention was paid to coronaviruses by researchers, as this genus of viruses causes severe diseases predominantly in animals and only comparatively mild diseases in humans. In the wake of the SARS outbreak, greater attention has been focused on the replicase proteins with a view to understanding the replication/transcription machinery and to identify new therapeutic targets. To date, the threedimensional structures of a series of nsp proteins have been reported (1). nsp5, also called the main protease or 3C-like protease, was the first SARS protein structure determined in 2003 (24) and has since been the focus of concerted efforts for the design of antiviral inhibitors. Last year, a broad-spectrum inhibitor was reported with efficient in vitro inactivation of multiple coronavirus main proteases, potent antiviral activity, and extremely low cellular toxicity (23). The structure of nsp9 was determined in 2004, and it was found to be a singlestranded RNA binding protein (4, 17). More recently, the complex structure between nsp7 and nsp8 revealed a hexadecameric assembly that should constitute a processivity factor for nsp12, an RNA-dependent RNA pol...

Biogenesis and Dynamics of the Coronavirus Replicative Structures

“…In addition to the nsps mentioned above, other CoV nsps are involved in RNA binding (nsp9 and nsp10; [48,49]) or in evasion of the antiviral response of the host (nsp1 and nsp3; [50,51,52,53,54,55,56,57]). The function of nsp2 is not yet known, although this protein was shown not to be essential for virus replication [58,59].…”

Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

“…The crystal structure of nsp10 shows that it belongs to the zinc finger protein family (22,28,29). nsp10 has no known enzymatic activity but may have a role in the regulation of enzymatic activities at different steps of the viral transcription/replication or by playing an architectural role.…”

Oscillation, cooperativity, and intermediates in the self-repressing gene

“…SARS-CoV nsp10, for instance, forms a dodecameric structure (n ¼ 12) [19]; Rhizobium leguminosarum NodD has been shown to bind to DNA preferentially in octamer form [20]; and k phage CI, while it has a small autoregulatory effect with tetramer and even dimer forms, only fully represses itself when an octamer form of the protein links distant sites on DNA together [21]. Therefore, we have attempted a less complete search of parameters considering additional steps (instead of monomer to n-mer, monomer to dimer to tetramer, etc.).…”