Prompt detection is essential to limit the spread of pathogens. The availability of the complete genomic sequence of SARS CoV-2 has facilitated the development of a variety of diagnostic tests for SARS-CoV-2. Reverse transcription polymerase chain reaction (RT-PCR) has been used as a rapid diagnostic test during the epidemic [52]. However, the sensitivity of viral RNA testing varies depending on the timing of testing relative to exposure, which could lead to false-negative results [53]. Thus, more and more laboratories pay attention to serological tests.

Among the four CoVs structural proteins, the S and N proteins are the main immunogens [54]. There have been several serological tests showing that S and N induced a strong antibody response in hosts [55,56]. During the detection process, it was found that the detection rate of N protein was higher than that of S protein in PCR-positive patients [57]. Hence, it is a feasible method to use N proteins for serological tests or combined N and S proteins as capture antigens to increase the sensitivity of this assay. However, one question raised is that the test for specific antibodies against SARS-COV-2 in the serum will appear positive only about 7 days after infection or later, making it difficult to detect the infection at an early stage. Given this situation, it is necessary to explore the diagnostic value of SARS-CoV-2 proteins in the early stages of SARS-COV-2 infection. Several studies have detected serum N protein level in SARS-COV-2 infected patients and analyzed the correlation with serum N protein antibody level using the commercial kit. Based on the CUT-OFF value determined from the receiver operating characteristic (ROC) curve, the specificity of the SARS-COV-2 serum N protein detection was 96.84%, and the sensitivity was 92% before the appearance of antibodies, suggesting that the detection of SARS-COV-2 serum N protein has a high diagnostic value for infected patients before the appearance of antibodies, and shortens the window of serological diagnosis [58]. Meanwhile, several laboratories have tried to identify the immunodominant epitopes of N protein and develop specific monoclonal antibodies that can be used in ELISA. Amrun et al. identified four immunodominant epitopes: S14P5, S20P2, S21P2, and N4P5, on the S and N viral proteins. IgG responses to all identified epitopes displayed a strong detection profile, with N4P5 achieving the highest level of specificity (100%) and sensitivity (>96%) against SARS-CoV-2, suggesting the feasibility of developing mAbs to these epitopes alone or in combination used in ELISA to detect SARS-CoV-2 [59].

Taken together, all these data support the notion that the N-protein could be used as an efficient diagnostic tool for detection of SARS-CoV-2 infection and the specific detection methods of N protein should further be validated in more patient samples.

Despite extensive research on COVID-19, there is currently no effective treatment available for clinical use. Based on the conservation of CoVs N protein in evolution and its key role in viral replication, it is a promising target for drug discovery. Firstly, since the RNA binding activity of N protein is pivotal to viral RNP formation and genome replication, blocking the RNA binding of N-NTD has been proven to be a considering strategy. To date, there have been some small compounds targeting other CoVs considered as candidate inhibitors for SARS-CoV-2 by virtual screening. For example, the compounds PJ34 and H3, which targeted the RNA binding site of N-NTD, can inhibit HCoV-OC43 replication [14]. Notably, the key residues that are involved in the RNA binding interactions, including S51, F53, R107, Y109, Y111, and R149 (in SARS-CoV-2 N-NTD numbering), are conserved, suggesting potential development possibility (Figure 1) [60,61].

In addition, blocking normal N protein oligomerization or triggering abnormal RNP formation is also an attractive inhibitory strategy. More recently, Lin et al. identified 5-benzyloxygramine (P3) is a novel inhibitor for MERS-CoV by virtual screening. This compound could mediate MERS-CoV N-NTD non-native dimerization and induce N protein aggregation. The structure-based study showed that P3 targets the non-native interface of N-NTD dimers and simultaneously interacts with the hydrophobic pockets in both N-NTD protomers. It was demonstrated that P3 was able to replace the vector-fusion residues of promoter 2 to occupy its binding cavity in promoter 1 under the legend free condition, which, in turn, stabilized the dimeric status by triggering massive hydrophobic interactions [14,62]. By comparing the binding sites of P3 in the hydrophobic cavity, it was found that almost all of the residues of the N-NTD involved in the interactions are conserved, except F135 in MERS-CoV, which is replaced by I146 in SARS-CoV-2(Figure 3). Although both residues are nonpolar amino acids, the effect on SARS-CoV-2 replication needs to be further verified. For other viruses, such as the human immunodeficiency virus and influenza virus, the researchers proposed a strategy to inhibit viral N protein oligomerization by developing competing peptides [63,64]. For CoVs, it has been shown that the excessive peptide based on the C-terminal tail sequence can interfere with CTD oligomerization of HCoV-229E N protein and decrease the viral titer, providing a reference for relevant studies on SARS COV-2 N protein [65]. Notably, the LLPS of N protein induced by viral genomic RNA is also a potential target [35]. Slowing viral infection by increasing or decreasing the N protein LLPS is a strategy that could be considered. 1,6-hexanediol, lipoic acid, and aminoglycoside kanamycin, each of which potentially alters LLPS by a representative and distinct mechanism. In terms of SARS-CoV-2, further experiments showed that the formation or the size of condensates could be reduced after treatment with these small molecules [34]. Meanwhile, high-throughput virtual screening is underway, several potential drug candidates have been proposed, and the next focus is on rigorous experimental validation, such as (−)-catechin gallate and (−)-gallocatechin gallate [66] (Table 1).