Nucleocapsid protein (NPs) of negative-sense single-stranded RNA (-ssRNA) viruses function in different stages of viral replication, transcription, and maturation. Structural investigations show that -ssRNA viruses that encode NPs preliminarily serve as structural building blocks that encapsidate and protect the viral genomic RNA and mediate the interaction between genomic RNA and RNA-dependent RNA polymerase. However, recent structural results have revealed other biological functions of -ssRNA viruses that extend our understanding of the versatile roles of virally encoded NPs.
Animals ; Capsid ; metabolism ; Humans ; Lassa virus ; chemistry ; physiology ; Nucleocapsid Proteins ; chemistry ; metabolism ; Orthobunyavirus ; chemistry ; physiology ; RNA Viruses ; chemistry ; physiology

BACKGROUNDConstruction of replication-deficient recombinant adenovirus expressing gag-pol and env genes of human immunodeficiency virus (HIV) in mice.

METHODSgag-polDelta and gp140TM genes were cloned into shuttle vector pAdTrack-CMV respectively, and then the plasmids containing gag-polDelta or gp140TM gene were cotransformed with the backbone of adenovirus into E.coli BJ5183. Transfections of the recombinants were performed to obtain recombinant adenoviruses. Its immunogenicity was evaluated by testing antibody levels of mice primed with DNA vaccines and boosted with recombinant adenoviruses.

RESULTSThe replication-deficient recombinant adenovirus could express Gp140TM, Gag P55 and P24 proteins correctly. The mice primed with DNA vaccines and boosted with recombinant adenoviruses elicited high titer of HIV-1-specific antibody compared with that inoculated with DNA vaccines only.

CONCLUSIONReplication-deficient recombinant adenovirus expressing gag-polDelta and gp140TM can elicit high titer HIV-1-specific antibodies.

AIDS Vaccines ; immunology ; Adenoviridae ; genetics ; Animals ; Female ; Fusion Proteins, gag-pol ; biosynthesis ; genetics ; Gene Products, env ; biosynthesis ; genetics ; HIV-1 ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Recombination, Genetic ; Transfection ; Vaccines, DNA ; immunology ; env Gene Products, Human Immunodeficiency Virus

COVID-19 ; Coronavirus Nucleocapsid Proteins ; Humans ; Nucleocapsid Proteins ; Phosphoproteins ; SARS-CoV-2

Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).
Anti-HIV Agents ; pharmacology ; Benzoxazines ; pharmacology ; Bioluminescence Resonance Energy Transfer Techniques ; methods ; Fusion Proteins, gag-pol ; genetics ; metabolism ; HEK293 Cells ; HIV Protease ; metabolism ; physiology ; HIV-1 ; enzymology ; High-Throughput Screening Assays ; methods ; Humans ; Plasmids ; genetics ; Protein Precursors ; metabolism ; physiology ; Pyridazines ; pharmacology ; Transfection ; Virion ; growth & development ; Virus Assembly ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism

The genome of Hantaan virus, the prototype of the hantavirus genus, is composed of three segmented, single stranded negative sense RNA genome. The 5' and 3' termini of the Hantaan virus RNA genome contain noncoding regions (NCRs) that are highly conserved and complementary to form panhandle stuctures. There are some reports that these NCRs seems to control gene expression and viral replication in influenza virus and vesicular stomatitis virus. In this study, we examined whether NCRs in Hantaan virus play a role in expression of the viral nucleocapsid protein (Np) and foreign (luciferase) gene. The 5' and/or 3' NCR-deleted mutants were constructed and analysed. The Np expression of 5' NCR-deleted clone, it showed 40% reduction. To investigate the role of NCR in foreign gene expression, the clones which are replaced ORF of Hantaan viral Np gene with that of luciferase gene were constructed. The results were similar to those of the experiments using Np gene. These results suggest that 3' NCR is more important than 5' NCR in protein expression. To find out a critical region of 3' NCR in more important than 5' NCR in protein expression. To find out a critical region of 3' NCR in protein expression, several clones with a deleted part of 3' NCR were constructed and analyzed. The deletion of the conserved region in 3' NCR showed 20~30% decrease in Np expression. However there were no change in luciferase activities between clones with or without non-conserved region of 3' NCR. These results suggest that the 3' NCR of Hantaan virus S genome, especially conserved region in 3' NCR, plays and important role in the expression of Hantaan viral Np and foreign genes.
Animals ; Clone Cells ; Ecthyma, Contagious ; Gene Expression ; Genome* ; Hantaan virus ; Hantavirus ; Luciferases ; Nucleocapsid Proteins* ; Nucleocapsid* ; Orthomyxoviridae ; RNA ; Vesicular Stomatitis

Apoptosis, as a part of the natural defense mechanisms that protect against viral infection, plays a vital role in the pathogenic mechanisms. It also plays a key role in the pathogenesis of diseases including many viral diseases. Mechanisms of virus-induced apoptosis are not completely understood because of the complexity of the underlying biochemical cascades and all of the participating host factors. Mumps virus belongs to the genus Rubulavirus in the family Paramyxoviridae. It contains single stranded RNA genome with negative polarity. It was observed that mumps virus induced apoptosis in VeroE6 cells, and adsorption and penetration of mumps virus to cell membrane alone were not sufficient for the induction of cell death. When mumps virus was superinfected onto nucleocapsid protein (NP) expressing VeroE6 cells, cell viability and facterial titer were maintained until 13 and 12 day, respectively. The levels of p53, Bax, and Bcl-2 were increased in NP-expressing VeroE6 cells, and the increase in Bax, and Bcl-2 was outstanding. It was observed that NP protein did not directly affect the efficiency of the infection of mumps virus in NP-expressing VeroE6 cells. The levels of p53, and Bax were decreased in both mock-infected VeroE6 cells and NP-expressing VeroE6 cells infected with mumps virus. However, the Bcl-2 level was little affected by the virus infection.
Adsorption ; Apoptosis* ; Cell Death ; Cell Membrane ; Cell Survival ; Defense Mechanisms ; Genome ; Humans ; Mumps virus* ; Mumps* ; Nucleocapsid Proteins* ; Nucleocapsid* ; Paramyxoviridae ; RNA ; Rubulavirus ; Virus Diseases

OBJECTIVEThe purpose of this study is to express partial S gene of Hantavirus Z10.

METHODSThe 300 bp S gene of Z10 strain was synthesized by using a successive PCR method for the optimal expression in Pichia pastoris and subcloned into pMD19-T. The SP300 gene was constructed into pPICZaA and sequenced. The recombinant pPICZaA-SP300 and pPICZaA-S300 was transformed into Pichia with LiCI.

RESULTSThe recombination Pichia were cultivate, and expressed the SP300 or S300 gene induced in Pichia by methanol.

CONCLUSIONThe nucleocapsid secreted from the Pichia can be detected by ELISA and WesternBlot.

Gene Expression ; Hantavirus ; genetics ; metabolism ; Nucleocapsid Proteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism

To develop the large scale serological assay for severe fever with thrombocytopenia syndrome virus (SFTSV) infection, we evaluated two different enzyme-linked immunosorbent assay (ELISA) methods using nucleocapsid protein (NP) and Gn proteins of CB1 (genotype B) SFTSV strains. The NP-based ELISA tests showed more sensitive with broad cross-reactivity between two different genotype A and B strains compared with those of Gn-based ELISA tests. However, Gn-based ELISA showed more genotype specificity and specificity. These result suggested that NP-based ELISA test could be applicable for general sero-prevalence studies of SFTSV infections, while Gn-based ELISA could be applicable for a certain specific genotype sero-prevalence study.
Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Fever ; Genotype ; Nucleocapsid Proteins ; Sensitivity and Specificity ; Thrombocytopenia

Since the outbreak of corona virus disease 2019 (COVID-19), viral strains have mutated and evolved. Vaccine research is the most direct and effective way to control COVID-19. According to different production mechanisms, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines included inactivated virus vaccine, live attenuated vaccine, mRNA vaccine, DNA vaccine, viral vector vaccine, virus-like particle vaccine and protein subunit vaccine. Among them, viral protein subunit vaccine has a wide application prospect due to its high safety and effectiveness. Viral nucleocapsid protein has high immunogenicity and low variability which could be a new direction for vaccine production. We summarized the current development of vaccine research by reviewing the current progress, vaccine safety and vaccine immune efficiency. It is hoped that the proposed possible development strategies could provide a reference for epidemic prevention work in future.
Humans ; SARS-CoV-2/genetics* ; COVID-19/prevention & control* ; Protein Subunits ; Vaccines, DNA ; Nucleocapsid Proteins

The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.
Cloning, Molecular ; Nucleocapsid Proteins ; biosynthesis ; genetics ; immunology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; SARS Virus ; genetics