nsp10

Nsp10 is a small novel coronaviral protein located at the junction of PP1a/1b. It found in all coronaviruses and is highly conserved (51 – 56% identity). The structure of nsp10 solved by two groups (including ours) reveals that it adopts a novel fold and contains two novel Zn2+ fingers. The two Zn fingers, along with a well-defined positively charged patch on the protein surface, and the physical location of nsp10 in the SARS proteome within a cluster of proteins which interact with RNA, hinted at a possible RNA binding function. However, RNA-binding experiments indicate that while nsp10 binds to both single-stranded and double-stranded DNA and RNA, it does so with only micromolar affinity. This does not rule out the possibility that it may bind specifically to a hitherto undetermined RNA sequence and/or bind nucleic acid as part of a larger complex that confers processivity during replication of the genomic or sub-genomic RNA of the virus.  In MHV, there is evidence that nsp10 plays a critical role in the replicase complex: a temperature-sensitive Gln65Glu mutation caused a defect in minus-strand RNA synthesis at the non-permissive temperature of 40ºC while plus strand synthesis appeared to be unaffected. When modeled on the SARS nsp10 structure, mutation of Gln65 (absolutely conserved in all coronavirus nsp10 proteins) to a Glu residue generates a large contiguous negatively charged surface patch, which may unfavorably alter the binding interface with an interacting protein and/or RNA. Immunofluorescence studies using confocal microscopy in MHV-infected cells, have demonstrated that p15 (MHV counterpart of SARS nsp10) co-localizes with p28, p65, 3CL protease, p10, p22, p12, the RNA-dependent RNA polymerase, the helicase, and the nucleocapsid protein (counterparts of SARS nsp1, 2, 5, 7, 8, 9, 12, 13 and sars9a, respectively), at the site of replication complexes , . Further, specific interactions between MHV p15, p28 and p10 were seen in yeast two-hybrid and co-immunoprecipitation experiments suggesting a direct role of nsp10 in the replicase complex . Genetic analysis in MHV also indicates that homologues of nsp10, nsp4, and nsp16 are involved in replicase complex assembly .

Nsp10 also appears to contribute significantly to the cytopathogenic effects observed in SARS infection in lung tissue by interacting with the oxido-reductase system in mitochondria. Yeast two-hybrid experiments screening a human embryo lung cDNA library for interactions, using SARS nsp10 as a bait revealed two subunits of cellular RNA polymerase B complex (BTF3 and ATF5), the NADH 4L subunit and cytochrome oxidase II as hits . Specific interaction with cytochrome oxidase II was supported by a GST pull-down assay and Western blotting. Interestingly, transient expression of SARS nsp10 in KMB-17 cells inhibited replication of human coronavirus (HCoV) 229E, perhaps by competitively interfering with the HCoV replicase complex. 

Recently, Su et al.  determined the structure of a construct spanning nsp10 and nsp11. This crystallized as a dodecamer, but is not thought to have any physiological significance. In this structure, the region corresponding to nsp11 was disordered.

 

Joseph JS, Saikatendu KS, Subramanian V, Neuman BW, Brooun A, Griffith M, Moy K, Yadav MK, Velasquez J, Buchmeier MJ, Stevens RC, Kuhn P. Crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs. J Virol. 2006 Aug;80(16):7894-901.

Donaldson EF, Sims AC, Graham RL, Denison MR, Baric RS. Murine Hepatitis Virus replicase protein nsp10 is a critical regulator of viral RNA synthesis.J Virol. 2007 Mar 28; [Epub ahead of print]

Su D, Lou Z, Sun F, Zhai Y, Yang H, Zhang R, Joachimiak A, Zhang XC, Bartlam M, Rao Z. Dodecamer structure of severe acute respiratory syndrome coronavirus nonstructural protein nsp10. J Virol. 2006 Aug;80(16):7902-8.

Siddell S, Sawicki D, Meyer Y, Thiel V, Sawicki S. Identification of the mutations responsible for the phenotype of three MHV RNA-negative ts mutants. Adv Exp Med Biol. 2001;494:453-8.Siddell et al., Adv Exp Med Biol 494:453-458, 2001

Bost AG, Carnahan RH, Lu XT, Denison MR. Four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly. J Virol. 2000 Apr;74(7):3379-87.

Brockway SM, Lu XT, Peters TR, Dermody TS, Denison MR. Intracellular localization and protein interactions of the gene 1 protein p28 during mouse hepatitis virus replication. J Virol. 2004 Nov;78(21):11551-62.

Li Q, Wang L, Dong C, Che Y, Jiang L, Liu L, Zhao H, Liao Y, Sheng Y, Dong S, Ma S. The interaction of the SARS coronavirus non-structural protein 10 with the cellular oxido-reductase system causes an extensive cytopathic effect. J Clin Virol. 2005 Oct;34(2):133-9.

M.M.C. Lai and K.V. Holmes, Coronaviridae: the viruses and their replication. In: D.M. Knipe and P.M. Howley, Editors, Fields Virology, Lippincott Williams and Wilkins, Philadelphia (2001), pp. 1163–1185.

Thiel V, Ivanov KA, Putics A, Hertzig T, Schelle B, Bayer S, Weissbrich B, Snijder EJ, Rabenau H, Doerr HW, Gorbalenya AE, Ziebuhr J. Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol. 84(9), 2305-2315 (2003).

Pedersen KW, van der Meer Y, Roos N, Snijder EJ. Open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex. J Virol. 1999 Mar;73(3):2016-26.