Summary
1. N-protein: construction and composition
The nucleocapsid (N) protein is encoded by the ninth ORF of SARS-CoV. The identical ORF additionally codes for one more distinctive accent protein known as ORF9b, although in a special studying body, whose operate is but to be outlined. The N-protein is a 46 kDa protein composed of 422 amino acids (Rota et al., 2003). Its N-terminal area consists principally of positively charged amino acids, which is liable for RNA binding. A lysine wealthy area is current between 373 and 390 amino acids on the C-terminus, which is predicted to be the nuclear localization sign. Moreover that, a SR-rich motif is current within the center area encompassing 177–207 amino acids. Biophysical research accomplished by Chang et al. (2006) have urged that this protein consists of two impartial structural domains and a linker area. The primary area is current on the N-terminus, contained in the putative RNA binding area. The second area consists of the C-terminal area that’s able to self-association. Between these two structural domains, there lies a extremely disordered area, which serves as a linker. This area has been reported to work together with the membrane (M) protein and human mobile hnRNPA1 protein (Fang et al., 2006, Luo et al., 2005a, Luo et al., 2005b). Moreover, this area can also be predicted to be a scorching spot for phosphorylation. Therefore, in abstract, the N-protein may be categorized into three distinct areas (Fig. 1
), which can serve fully completely different features throughout completely different levels of the viral life cycle. An identical mode of group has been reported for different coronavirus nucleocapsid proteins.
2. Stability of the N-protein
In vitro thermodynamic research accomplished by C. Luo et al. (2004) and H. Luo et al. (2004) utilizing purified recombinant N-protein have proven it to be steady between pH 7 and 10, with most conformational stability close to pH 9. Additional, it was noticed to bear irreversible thermal-induced denaturation. It begins to unfold at 35 °C and is totally denatured at 55 °C (Wang et al., 2004). Nonetheless, chemical compounds corresponding to urea or guanidium chloride-induced denaturation of the N-protein is a reversible course of.
3. Put up-translational modification
As in different coronavirus N-proteins, SARS-CoV N-protein has been predicted and later experimentally confirmed to bear varied post-translational modifications corresponding to acetylation, phosphorylation, and sumoylation.
Acetylation is the primary modification of the N-protein to be experimentally confirmed. By mass spectrometric evaluation of convalescent sera from a number of SARS sufferers, it has been proven that the N-terminal methionine of N is eliminated in addition to all different methionines are oxidised and the ensuing N-terminal serine is acetylated. Nonetheless, the purposeful relevance of this modification, if any, stays to be elucidated (Krokhin et al., 2003).
One other distinctive modification of the N-protein is its capacity to get sumoylated. Research accomplished by Y.H. Li et al. (2005) and F.Q. Li et al. (2005) have clearly established that heterologously expressed N in mammalian cells is sumoylated. Utilizing a site-directed mutagenesis method, the sumoylation motif has been mapped to the lysine residue 62, which is current in a putative sumo-modification area (GK62EE). Their information additional means that sumoylation might play a key position in modulating homo-oligomerization, nucleolar translocation and cell-cycle deregulatory property of the N-protein. Additional experimental help concerning sumoylation of N-protein got here from one other impartial research carried out by Fan et al. (2006) whereby they’ve demonstrated an affiliation between the N-protein and Hubc9, which is a ubiquitin conjugating enzyme of the sumoylation system. They’ve additionally mapped the interplay area to the SR-rich motif, which is in settlement with the sooner report. Nonetheless, they did not detect the involvement of the GKEE motif in mediating this interplay (Fan et al., 2006).
Initially, the SARS-CoV N-protein was predicted to be closely phosphorylated. Afterward, from outcomes obtained in our laboratory in addition to by different researchers, it’s now clear that the N-protein is a substrate of a number of mobile kinases. First experimental proof for the phosphorylation standing of the N-protein got here from the research accomplished by Zakhartchouk et al. (2005) whereby by 32P orthophosphate labelling, they have been in a position to observe phosphorylation of adenovirus vector expressed N-protein in 293T cells. Additional research accomplished in our laboratory clearly confirmed this commentary. Majority of the N-protein was discovered to be phosphorylated at its serine residues (though the involvement of threonine and tyrosine residues couldn’t be detected, they might be occurring in vivo). As well as, utilizing a wide range of biochemical assays, it was proved that, no less than in vitro, the N-protein may get phosphorylated by mitogen activated protein kinase (MAP kinase), cyclin-dependent kinase (CDK), glycogen synthase kinase 3 (GSK3), and casein kinase 2 (CK2). Additionally, this information gives preliminary indication concerning phosphorylation-dependent nucleo-cytoplasmic shuttling of the N-protein (Surjit et al., 2005). Whether or not these occasions truly bear any purposeful significance in vivo, stays but to be established.
4. Localization of the N-protein – “n protein”
In distinction to the N-protein of many different coronaviruses, the SARS-CoV N-protein is predominantly distributed within the cytoplasm, when expressed heterologously or in contaminated cells (Surjit et al., 2005, You et al., 2005, Rowland et al., 2005). In contaminated cells, a number of cells exhibited nucleolar localization (You et al., 2005). As reported by You et al. (2005) the N-protein incorporates pat4, pat7, and bipartite-type nuclear localization indicators. It has additionally been predicted to own a possible CRM-1 dependent nuclear export sign. Nonetheless, no clear experimental proof could possibly be obtained concerning the involvement of those signature sequences in regulating the localization of the N-protein. Curiously, research accomplished in our laboratory revealed that almost all of N-protein localized to the nucleus in serum starved cells. This phenomenon could possibly be reproducibly noticed each in biochemical fractionation in addition to immunofluorescence research. As well as, therapy of cells with particular inhibitors of various mobile kinases corresponding to CK2 inhibitor and CDK inhibitor, resulted in retention of a fraction of the N-protein within the nucleus, whereas GSK3 and MAPK inhibitor had little or no impact. Additional, N was discovered to be effectively phosphorylated by the cyclin–CDK complicated, which is understood to be lively solely within the nucleus. The N-protein was additionally discovered to affiliate with 14-3-3 protein in a phospho-specific method and inhibition of the 14-3-3θ protein stage by siRNA resulted in nuclear accumulation of the N-protein. Though these experiments are too preliminary to conclusively present any reply concerning the intra-cellular localization of N-protein, nonetheless they do present substantial clues concerning the bodily presence of the N-protein within the nucleus, below sure circumstances, which can be a really dynamic phenomenon. One other research accomplished by Timani et al. (2005) utilizing completely different deletion mutants of the N-protein fused to EGFP, confirmed that the N-terminal of N, which incorporates the NLS 1 (aa 38–44) localizes to the nucleus, whereas the C-terminal area containing each NLS 2 (aa 257–265) and NLS 3 (aa 369–390) localizes to the cytoplasm and nucleolus. Utilizing a mixture of various deletion mutants, they concluded that the N-protein might act as a shuttle protein between cytoplasm–nucleus and nucleolus. Taken collectively, all these outcomes additional counsel that the N-protein per se has the bodily capacity to localize to the nucleus. Whether or not this localization is regulated via phosphorylation mediated activation of a possible NLS or piggy-backing by affiliation with one other mobile nuclear protein or via every other mechanism stays to be established.
5. Genome encapsidation: main operate of a viral capsid protein
Being the capsid protein, the first operate of the N-protein is to package deal the genomic RNA in a protecting overlaying. With the intention to obtain this construction, the N-protein have to be outfitted with two completely different attribute properties corresponding to: (i) with the ability to acknowledge the genomic RNA and affiliate with it; (ii) self-associate into an oligomer to type the capsid. The N-protein of SARS-CoV has been experimentally confirmed to own these properties in vitro, as mentioned under.
6. Perturbation of host mobile course of by the N-protein
Moreover being the capsid protein of the virus, the N-protein of many coronaviruses is understood to double up as a regulatory protein. The N-protein of the SARS-CoV too has been proven to modulate the host mobile equipment in vitro, thereby indicating its potential regulatory position throughout its viral life cycle. Among the main mobile processes perturbed by heterologous expression of the N-protein are mentioned under.
“n protein”