Virology Group, ICGEB, 10504, Aruna Asaf Ali Highway, New Delhi, 110067 India
Summary
Introduction
By definition, nucleocapsid is a viral protein coat that surrounds the genome (both DNA or RNA). Nucleocapsid protein is the foremost constituent of a viral nucleocapsid. It’s able to associating with itself and with the genome, thus packaging the genome inside a closed cavity. In some viruses, nucleocapsid protein might also be assisted by different viral cofactors to type the capsid. Nonetheless, in coronaviruses (together with SARS-CoV), the nucleocapsid protein alone is able to forming the capsid. The first benefit of the virus for encoding the nucleocapsid protein is that the latter encloses and protects the viral genome from coming into direct contact with the cruel atmosphere within the host. In actual fact, in some easy viruses like hepatitis E virus and polio virus, the nucleocapsid protein is the one coat that protects the genome from the surface world. Nonetheless, in advanced viruses, like hepatitis B virus and coronaviruses (together with SARS-CoV), the nucleocapsid is roofed by a further coat composed of different viral proteins (spike protein is a serious element of this coat). Moreover this property, nucleocapsid proteins of a number of viruses have been demonstrated to play a number of regulatory roles throughout viral pathogenesis. They’re outfitted with particular structural motifs and/or signature sequences, by which they affiliate with different viral/ host elements and skew the host mobile equipment in such a fashion that it turns into extra favorable for the survival of the virus. Nucleocapsid protein can be probably the most abundantly expressed viral proteins and it’s the main antigen acknowledged by convalescent antisera. Therefore, it’s tempting to judge its potential as a candidate diagnostic device or vaccine in opposition to the virus.
Subsequently, understanding the properties of the nucleocapsid protein is of utmost significance to any virologist with a view to perceive the biology of the virus and develop efficient instruments to regulate the an infection. For the reason that identification and isolation of SARS-CoV in 2003, a number of laboratories all over the world have focussed their analysis on characterization of varied properties of the nucleocapsid protein. An oblique measure of the curiosity amongst SARS-CoV researchers to check the nucleocapsid protein is revealed from the truth that in PubMed the variety of SARS-CoV analysis publications focussed on nucleocapsid protein is second solely to these on spike protein. Proof gathered from these articles has helped us achieve substantial understanding of the properties of this protein. On this article, we’ll present a complete description of all of the completely different properties of the nucleocapsid protein, as established by impartial staff from a number of laboratories. We are going to conclude this text with the dialogue of among the remaining challenges on this subject that should be addressed in future.
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 referred to as ORF9b, although in a distinct studying body, whose perform 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 largely of positively charged amino acids, that are liable for RNA binding. A lysine-rich area is current between amino acids 373 and 390 on the C-terminus, which is predicted to be the nuclear localization sign. Moreover these, an SR-rich motif is current within the center area encompassing amino acids 177–207. Biophysical research performed by Chang et al. (2006) have steered 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, and 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. 2005). Moreover, this area can be predicted to be a scorching spot for phosphorylation. Therefore, in abstract, the N-protein may be categorised into three distinct areas (Fig. 9.1), which can serve fully completely different features throughout completely different levels of the viral life-cycle. An analogous mode of group has been reported for different coronavirus nucleocapsid proteins.
Stability of the N-Protein
In-vitro thermodynamic research performed by Luo et al. (2004b) utilizing purified recombinant N-protein have proven it to be secure 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, denaturation of the N-protein induced by chemical substances resembling urea or guanidium chloride is a reversible course of.
Posttranslational Modification – “n protein definition”
As in different coronavirus N-proteins, SARS-CoV N-protein has been predicted and later experimentally confirmed to bear numerous posttranslational modifications resembling 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 and all different methionines are oxidized 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 means to turn out to be sumoylated. Research performed by Li et al. (2005a) 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 62nd lysine residue, which is current in a putative sumo-modification area (GK62EE). Their knowledge 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. In a while, 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 performed by Zakhartchouk et al. (2005) wherein, utilizing [32P]orthophosphate labelling, they have been in a position to observe phosphorylation of adenovirus-vector-expressed N-protein in 293T cells. Additional research performed in our laboratory clearly confirmed this statement. The vast 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 could be occurring in vivo). As well as, utilizing a wide range of biochemical assays, it was proved that, at the very least in vitro, the N-protein may turn out to be phosphorylated by mitogen-activated protein kinase (MAP kinase), cyclin-dependent kinase (CDK), glycogen synthase kinase 3 (GSK3), and casein kinase 2 (CK2). Additionally, this knowledge offered preliminary indication concerning phosphorylation-dependent nucleo-cytoplasmic shuttling of the N-protein (Surjit et al. 2005). A latest report printed by Wu et al. (2008) has additional confirmed that N-protein is a substrate of GSK3 enzyme, each in vitro and in vivo. Utilizing a wide range of biochemical and genetic assays, it was clearly demonstrated that serine 177 residue of N-protein was phosphorylated by GSK3. An antibody particular to phospho 177 residue of the N-protein may effectively detect the phospho N-protein each in vitro and in SARS-CoV contaminated cells. Curiously, biochemically mediated inhibition of GSK3 exercise in SARS-CoV contaminated cells additionally results in round 80% discount in viral titer and subsequent induction of a virus-induced cytopathic impact. The authors proposed that GSK3 could also be a serious regulator of SARS-CoV replication, presumably by advantage of its means to phosphorylate the N-protein. Nonetheless, phosphorylation of different viral and/or host proteins by GSK3 might also be a determinant of the noticed cytopathic impact.
Localization of the 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 couple 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 might be obtained concerning the involvement of those signature sequences in regulating the localization of the N-protein. Curiously, research performed in our laboratory revealed that almost all of N-protein localized to the nucleus in serum-starved cells. This phenomenon might be reproducibly noticed each in biochemical fractionation in addition to immunofluorescence research. As well as, remedy of cells with particular inhibitors of various mobile kinases resembling 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-protein was discovered to be effectively phosphorylated by the cyclin–CDK advanced, which is thought to be energetic 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 degree by siRNA resulted in nuclear accumulation of the N-protein. Though these experiments are too preliminary to conclusively present any reply concerning the intracellular localization of N-protein, nonetheless they do present substantial clues concerning the bodily presence of the N-protein within the nucleus, beneath sure circumstances, which can be a really dynamic phenomenon. One other research performed by Timani et al. (2005) utilizing completely different deletion mutants of the N-protein fused to EGFP confirmed that the N-terminal of N-protein, 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 recommend that the N-protein per se has the bodily means to localize to the nucleus. Whether or not this localization is regulated by means of phosphorylation-mediated activation of a possible NLS or piggy-backing by affiliation with one other mobile nuclear protein or by means of another mechanism stays to be established.
Genome Encapsidation: Main Operate of a Viral Capsid Protein
Being the capsid protein, the first perform 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 should be outfitted with two completely different attribute properties; resembling (1) having the ability to acknowledge the genomic RNA and affiliate with it, and (2) 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.
“n protein definition”