Phase separation by the SARS-CoV-2 nucleocapsid protein: Consensus and open questions.

In response to the recent SARS-CoV-2 pandemic, a number of labs across the world have reallocated their time and resources to better our understanding of the virus. For some viruses, including SARS-CoV-2, viral proteins can undergo phase separation: a biophysical process often related to the partitioning of protein and RNA into membraneless organelles in vivo. In this review, we discuss emerging observations of phase separation by the SARS-CoV-2 nucleocapsid (N) protein-an essential viral protein required for viral replication-and the possible in vivo functions that have been proposed for N-protein phase separation, including viral replication, viral genomic RNA packaging, and modulation of host-cell response to infection. Additionally, since a relatively large number of studies examining SARS-CoV-2 N-protein phase separation have been published in a short span of time, we take advantage of this situation to compare results from similar experiments across studies. Our evaluation highlights potential strengths and pitfalls of drawing conclusions from a single set of experiments, as well as the value of publishing overlapping scientific observations performed simultaneously by multiple labs.

Aptamer/antibody sandwich method for digital detection of SARS-CoV2 nucleocapsid protein.

Nucleocapsid protein (N protein) is the most abundant protein in SARS-CoV2 and is highly conserved, and there are no homologous proteins in the human body, making it an ideal biomarker for the early diagnosis of SARS-CoV2. However, early detection of clinical specimens for SARS-CoV2 remains a challenge due to false-negative results with viral RNA and host antibodies based testing. In this manuscript, a microfluidic chip with femtoliter-sized wells was fabricated for the sensitive digital detection of N protein. Briefly, ß-galactosidase (ß-Gal)-linked antibody/N protein/aptamer immunocomplexes were formed on magnetic beads (MBs). Afterwards, the MBs and ß-Gal substrate fluorescein-di-ß-d-galactopyranoside (FDG) were injected into the chip together. Each well of the chip would only hold one MB as confined by the diameter of the wells. The MBs in the wells were sealed by fluorocarbon oil, which confines the fluorescent (FL) product generated from the reaction between ß-Gal and FDG in the individual femtoliter-sized well and creates a locally high concentration of the FL product. The FL images of the wells were acquired using a conventional inverted FL microscope. The number of FL wells with MBs (FL wells number) and the number of wells with MBs (MBs wells number) were counted, respectively. The percentage of FL wells was calculated by dividing (FL wells number) by (MBs wells number). The higher the percentage of FL wells, the higher the N protein concentration. The detection limit of this digital method for N protein was 33.28 pg/mL, which was 300 times lower than traditional double-antibody sandwich based enzyme-linked immunosorbent assay (ELISA).

A novel approach for the purification of aggregation prone proteins.

The protein aggregation is one of the major challenges of the biotechnological industry, especially in the areas of development and commercialization of successful protein-based drug products. The inherent high aggregation tendency of proteins during various manufacturing processes, storage, and administration has significant impact upon the product quality, safety and efficacy. We have developed an interesting protein purification approach that separates the functionally active protein from inactive aggregates using a detergent concentration gradient. The C-terminally His tagged nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV) has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the new purification approach reported here, the freshly purified protein by NiNTA chromatography was further processed using a detergent gradient. In this new purification approach the active protein is retained in the low detergent concentration zone while the inactive aggregates are promptly removed by their rapid migration to the high detergent concentration zone. The method prevented further aggregation and retained the RNA binding activity in the native protein despite numerous freeze thaw cycles. This simple approach prevents protein aggregation by rapidly separating the preformed early aggregates and creating the appropriate microenvironment for correctly folded proteins to retain their biological activity. It will be of potential importance to the biotechnological industry and other fields of protein biochemistry that routinely face the challenges of protein aggregation.

Molecular Targets for the Testing of COVID-19.

The pandemic outbreaks of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread all over the world in a short period of time. Efficient identification of the infection by SARS-CoV-2 has been one of the most important tasks to facilitate all the following counter measurements in dealing with the infectious disease. In Taiwan, a COVID-19 Open Science Platform adheres to the spirit of open science: sharing sources, data, and methods to promote progress in academic research while corroborating findings from various disciplines has established in mid-February 2020, for collaborative research in support of the development of detection methods, therapeutics, and a vaccine for COVID-19. Research priorities include infection control, epidemiology, clinical characterization and management, detection methods (including viral RNA detection, viral antigen detection, and serum antibody detection), therapeutics (neutralizing antibody and small molecule drugs), vaccines, and SARS-CoV-2 pathogenesis. In addition, research on social ethics and the law are included to take full account of the impact of the COVID-19 virus.

Production of Recombinant N Protein of Infectious Bronchitis Virus Using the Baculovirus Expression System and Its Assessment as a Diagnostic Antigen.

The avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is recognized as an important avian pathogen, and new viral variants are a continuous threat to the poultry industry worldwide. Sensitive diagnostics and efficacious vaccines are necessary to combat IBV infections in chickens. The aim of this study was to produce recombinant N protein of IBV in the baculovirus system to use in ELISA diagnostic tests in order to enable the assessment of the sero-prevalence and risk of IBV infections in chickens in Turkey. For this, the gene encoding the N protein of the Beaudette strain of IBV was expressed using a recombinant baculovirus expression system. The recombinant N protein was purified using Ni-NTA affinity chromatography. An estimated 50-kDa recombinant protein corresponding to the expected molecular weight of IBV N including the 6xHis tag was detected using an anti-His monoclonal antibody. Specific immunoreactivity of the recombinant protein was confirmed by Western blot using antiserum obtained from vaccinated and naturally infected chicken from Turkey as well as using a monoclonal antibody raised against the N protein of the IBV Massachusetts strain. The results obtained with the in-house ELISA had high agreement with a commercial ELISA. Immunoreactivity analysis using antisera in Western blotting and the in-house ELISA suggests that the recombinant IBV N protein could be broadly cross-reactive with antisera produced against different IBV strains. We conclude that the recombinant baculovirus expressed IBV N protein could serve as a useful diagnostic antigen for detection of IBV infections in chickens by ELISA.

A method for analyzing the composition of viral nucleoprotein complexes, produced by heterologous expression in bacteria.

Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the total protein and nucleic acid content of particle preparations is a recurrent biochemical problem. We describe a method for resolving this problem, developed when characterizing particles resembling the Menangle Virus nucleocapsid. The protein content was quantified using the biuret assay, which is largely independent of amino acid composition. Bound nucleic acids were quantified by determining the phosphorus content, using inductively coupled plasma mass spectrometry. Estimates for the amount of RNA packaged within the particles were consistent with the structurally-characterized packaging mechanism. For a bacterially-produced nucleoprotein complex, phosphorus usually provides a unique elemental marker of bound nucleic acids, hence this method of analysis should be routinely applicable.

Baculovirus expression and purification of peste-des-petits-ruminants virus nucleocapsid protein and its application in diagnostic assay.

Peste-des-petits-ruminants (PPR) is a contagious and highly devastating disease of small ruminants. For control of endemic PPR, adequate supply of affordable and reliable diagnostics is critical for effective surveillance, along with the use of highly efficacious live vaccines that are currently available. The nucleocapsid (N) protein of PPR virus (PPRV) is an important candidate antigen for developing specific diagnostic, as it is a major viral protein being highly immunogenic and conserved among the structural proteins. In the present study, we expressed the N protein of PPRV (Sungri/96 strain), in baculovirus expression system and purified using affinity column chromatography. The recombinant protein reacted well with PPRV anti-N monoclonal antibodies and PPRV-specific polyclonal antiserum, suggesting that the expressed protein was authentic and in native form. The recombinant protein was evaluated as antigen in the diagnostic ELISA as reference positive control in place of whole virus antigen. The utility of recombinant PPRV N protein circumvents the need to use live PPRV antigen in the routinely used diagnostics targeting 'N' protein of PPRV, thus allowing large-scale field application of the test.

Serine 105 and 120 are important phosphorylation sites for porcine reproductive and respiratory syndrome virus N protein function.

The nucleocapsid (N) protein is the most abundant protein of porcine reproductive and respiratory syndrome virus (PRRSV). It has been shown to be multiphosphorylated. However, the phosphorylation sites are still unknown. In this study, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyze the phosphorylation sites of N protein expressed in Sf9 cells. The results showed that N protein contains two phosphorylation sites. Since N protein can regulate IL-10, which may facilitate PRRSV replication, we constructed four plasmids (pCA-XH-GD, pCA-A105, pCA-A120 and pCA-A105-120) and transfected them into Pig alveolar macrophages (PAMs,3D4/2). The qPCR results showed that mutations at residues 105 and 120 were associated with down-regulation of the IL-10 mRNA level, potentially decreasing the viral growth ability. Then, we mutated the phosphorylation sites (S105A and S120A) and rescued three mutated viruses, namely, A105, A120 and A105-120. Compared with wild-type virus titers, the titers of the mutated viruses at 48 hpi were significantly decreased. The Nsp(non-structural protein) 9 qPCR results were consistent with the multistep growth kinetics results. The infected PAMs (primary PAMs) results were similar with Marc-145.The findings indicated that the mutations impaired the viral replication ability. The confocal microscopy results suggested that mutations to residues 105 and 120 did not affect N protein distribution. Whether the mutations affected other functions of N protein and what the underlying mechanisms are need further investigation. In conclusion, our results show that residues 105 and 120 are phosphorylation sites and are important for N protein function and for viral replication ability.

An enhanced immunochromatographic strip test using colloidal gold nanoparticle-labeled dual-type N proteins for detection of antibodies to PRRS virus.

Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most important infectious diseases causing serious economic loss in the swine industry worldwide. Due to its increasing genetic diversity, a rapid and accurate diagnosis is critical for PRRS control. The immunochromatographic strip test (ICST) is a rapid and convenient type of immunoassay. In this study, an on-site immunochromatographic assay-based diagnostic method was developed for detection of PRRS virus (PRRSV)-specific antibodies. The method utilized colloidal gold nanoparticle-labeled dual-type nucleocapsid proteins encoded by open reading frame 7. We evaluated 991 field samples from pig farms and 66 serum samples from experimentally PRRSV-inoculated pigs. Based on true PRRSV-specific antibody-positive or -negative sera determined by immunofluorescence assay and IgM enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of the ICST were 97.5% and 91.1%, respectively, similar to those of a commercial ELISA (IDEXX PRRS X3 Ab). More importantly, the ICST was completed within 15 min and could detect the PRRSV-specific antibody at an earlier stage of infection (3-7 days) than that of ELISA (7+ days). The results demonstrate that the developed ICST has great potential as an on-farm diagnostic method, providing excellent diagnostic performance in a quick and convenient manner.

Sin Nombre hantavirus nucleocapsid protein exhibits a metal-dependent DNA-specific endonucleolytic activity.

We demonstrate that the nucleocapsid protein of Sin Nombre hantavirus (SNV-N) has a DNA-specific endonuclease activity. Upon incubation of SNV-N with DNA in the presence of magnesium or manganese, we observed DNA digestion in sequence-unspecific manner. In contrast, RNA was not affected under the same conditions. Moreover, pre-treatment of SNV-N with RNase before DNA cleavage increased the endonucleolytic activity. Structure-based protein fold prediction using known structures from the PDB database revealed that Asp residues in positions 88 and 103 of SNV-N show sequence similarity with the active site of the restriction endonuclease HindIII. Crystal structure of HindIII predicts that residues Asp93 and Asp108 are essential for coordination of the metal ions required for HindIII DNA cleavage. Therefore, we hypothesized that homologous residues in SNV-N, Asp88 and Asp103, may have a similar function. Replacing Asp88 and Asp103 by alanine led to an SNV-N protein almost completely abrogated for endonuclease activity.

A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus.

Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-react with antisera against porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine group A rotavirus, classical swine fever virus, porcine circovirus-2, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus; the receiver operating characteristic (ROC) curve analysis revealed 100% sensitivity and 90.4% specificity of the rPDCoV-N-ELISA based on samples of known status (n=62). Analyses of field samples (n=319) using the rPDCoV-N-ELISA indicated that 11.59% of samples were positive for antibodies against PDCoV. These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province, northeast China.

The crystal structure of the Hazara virus nucleocapsid protein.

BACKGROUND: Hazara virus (HAZV) is a member of the Bunyaviridae family of segmented negative stranded RNA viruses, and shares the same serogroup as Crimean-Congo haemorrhagic fever virus (CCHFV). CCHFV is responsible for fatal human disease with a mortality rate approaching 30 %, which has an increased recent incidence within southern Europe. There are no preventative or therapeutic treatments for CCHFV-mediated disease, and thus CCHFV is classified as a hazard group 4 pathogen. In contrast HAZV is not associated with serious human disease, although infection of interferon receptor knockout mice with either CCHFV or HAZV results in similar disease progression. To characterise further similarities between HAZV and CCHFV, and support the use of HAZV as a model for CCHFV infection, we investigated the structure of the HAZV nucleocapsid protein (N) and compared it to CCHFV N. N performs an essential role in the viral life cycle by encapsidating the viral RNA genome, and thus, N represents a potential therapeutic target. RESULTS: We present the purification, crystallisation and crystal structure of HAZV N at 2.7 Å resolution. HAZV N was expressed as an N-terminal glutathione S-transferase (GST) fusion protein then purified using glutathione affinity chromatography followed by ion-exchange chromatography. HAZV N crystallised in the P212121 space group with unit cell parameters a = 64.99, b = 76.10, and c = 449.28 Å. HAZV N consists of a globular domain formed mostly of alpha helices derived from both the N- and C-termini, and an arm domain comprising two long alpha helices. HAZV N has a similar overall structure to CCHFV N, with their globular domains superposing with an RMSD = 0.70 Å, over 368 alpha carbons that share 59 % sequence identity. Four HAZV N monomers crystallised in the asymmetric unit, and their head-to-tail assembly reveals a potential interaction site between monomers. CONCLUSIONS: The crystal structure of HAZV N reveals a close similarity to CCHFV N, supporting the use of HAZV as a model for CCHFV. Structural similarity between the N proteins should facilitate study of the CCHFV and HAZV replication cycles without the necessity of working under containment level 4 (CL-4) conditions.

Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that was first reported in China in 2011. It is caused by SFTS virus (SFTSV) which is a member of the Phlebovirus genus in the Bunyaviridae family. SFTSV has been classified as a BSL3 pathogen. There is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients. METHODS: The full length nucleocapsid (N) gene of SFTSV Yamaguchi strain was amplified by RT-PCR and cloned to an expression vector pQE30. The recombinant (r) SFTSV-N protein was expressed by using Escherichia coli (E. coli) expression system and purified under native conditions. rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. One hundred fifteen serum samples from clinically suspected-SFTS patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich ELISA system. RESULTS: The native form of recombinant (r) SFTSV-N protein was expressed and purified. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. The rSFTSV-N protein based indirect IgM ELISA missed 8 positive samples that were detected by the total antibody sandwich ELISA. The sensitivity and specificity of rSFTSV-N-IgM capture ELISA were 90.59 and 100 %, respectively. CONCLUSIONS: The rSFTSV-N protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. Its preparation is simpler in comparison with that used for the total antibody sandwich system. Our rSFTSV-N protein-based IgG and IgM ELISA systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. They are safe to use for diagnosis of SFTS virus infection and especially fit in large-scale epidemiological investigations.

Preparation and characterization of a stable BHK-21 cell line constitutively expressing the Schmallenberg virus nucleocapsid protein.

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that predominantly infects livestock such as cattle, sheep, and goats. Its nucleocapsid (N) protein is an ideal target antigen for SBV diagnosis. In this study, a stable BHK-21 cell line, BHK-21-EGFP-SBV-N, constitutively expressing the SBV N protein was obtained using a lentivector-mediated gene transfer system combined with puromycin selection. To facilitate the purification of recombinant SBV N protein, the coding sequence for a hexa-histidine tag was introduced into the C-terminus of the SBV N gene during construction of the recombinant lentivirus vector pLV-EGFP-SBV-N. The BHK-21-EGFP-SBV-N cell line was demonstrated to spontaneously emit strong enhanced green fluorescent protein (EGFP) signals that exhibited a discrete punctate distribution throughout the cytoplasm. SBV N mRNA and protein expression in this cell line were detected by real-time RT-PCR and western blot, respectively. The expressed recombinant SBV N protein carried an N-terminal EGFP tag, and was successfully purified using Ni-NTA agarose by means of its C-terminal His tag. The purified SBV N protein could be recognized by SBV antisera and an anti-SBV monoclonal antibody (mAb) 2C8 in an indirect enzyme-linked immunosorbent assay and western blot analyses. Indirect immunofluorescence assays further demonstrated that the stable cell line reacts with SBV antisera and mAb 2C8. These results suggest that the generated cell line has the potential to be used in the serological diagnosis of SBV.

Development of an immunochromatography strip test based on truncated nucleocapsid antigens of three representative hantaviruses.

BACKGROUND: Hantaviruses are causative agents of hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica (NE) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. There is a need for time-saving diagnostic methods. In the present study, recombinant N antigens were used as antigens in an immunochromatography strip (ICG) test to detect specific IgG antibodies. METHODS: The N-terminal 103 amino acids (aa) of Hantaan virus (HTNV), Puumala virus (PUUV) and Andes virus (ANDV) nucleocapsid (N) protein were expressed in E. coli as representative antigens of three groups (HFRS, NE and HPS-causing viruses) of hantavirus. Five different types of ICG test strips, one antigen line on one strip for each of the three selected hantaviruses (HTNV, PUUV and ANDV), three antigen lines on one strip and a mixed antigen line on one strip, were developed and sensitivities were compared. RESULTS: A total of 87 convalescent-phase patient sera, including sera from 35 HFRS patients, 36 NE patients and 16 HPS patients, and 25 sera from healthy seronegative people as negative controls were used to evaluate the ICG test. Sensitivities of the three-line strip and mixed-line strip were similar to those of the single antigen strip (97.2 to 100%). On the other hand, all of the ICG test strips showed high specificities to healthy donors. CONCLUSION: These results indicated that the ICG test with the three representative antigens is an effective serodiagnostic tool for screening and typing of hantavirus infection in humans.

Expression, purification, crystallization and preliminary X-ray crystallographic study of the nucleocapsid protein of tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV), which causes severe damage to various agricultural crops such as tomato, pepper, lettuce and peanut, is a negative-stranded RNA virus belonging to the Tospovirus genus of the Bunyaviridae family. Viral genomic RNA molecules are packaged in the form of ribonucleoprotein complexes, each of which contains one viral RNA molecule that is coated with many nucleocapsid (N) proteins. Here, the expression and crystallization of TSWV N protein are reported. Native and selenomethionine-substituted crystals of N protein belonged to the same space group P2(1). Their unit-cell parameters were a = 66.8, b = 97.2, c = 72.0 Å, ß = 112.8° and a = 66.5, b = 96.3, c = 72.1 Å, ß = 113.4°, respectively.

[Methods for the Crimean-Congo hemorrhage fever diagnosis].

Several distinct methods currently used for the Crimean-Congo Hemorrhage Fever diagnosis (CCHF) were suggested in this work. We demonstrated that the ELISA-based diagnostic kits, which are based on CCHFV recombinant antigens produced in E. coli cells, still possessed a few substantial shortcomings, which are yet to be addressed. In this work we presented the development of the unique CCHFV detection system fully based on reverse transcription--nested two-step polymerase chain reaction (RT-PCR). Our RT-PCR-based diagnostic kit for the CCHFV detection is now commercially available. We also developed a simple screening method for the samples, potentially containing CCHFV, which is based on restriction fragment length polymorphism (RFLP) amplicons analysis and allows for preliminary genotyping of the CCHFV isolates.

[Detection and antigenic characteristics of the recombinant nucleocapsid proteins of Lassa and Marburg viruses].

Two plasmid vectors, which allow the recombinant polypeptides of Lassa and Marburg viruses to be expressed in prokaryotic cells E. coli strain BL21 (DE3), were produced. The two recombinant polypeptides are able to bind specific antibodies. This provides an opportunity to use them as antigenic components of immunoassay diagnostic test kits.