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

Rice stripe virus (RSV) transmitted by the small brown planthopper causes severe rice yield losses in Asian countries. Although viral nuclear entry promotes viral replication in host cells, whether this phenomenon occurs in vector cells remains unknown. Therefore, in this study, we systematically evaluated the presence and roles of RSV in the nuclei of vector insect cells. We observed that the nucleocapsid protein (NP) and viral genomic RNAs were partially transported into vector cell nuclei by utilizing the importin α nuclear transport system. When blocking NP nuclear localization, cytoplasmic RSV accumulation significantly increased. In the vector cell nuclei, NP bound the transcription factor YY1 and affected its positive regulation to FAIM. Subsequently, decreased FAIM expression triggered an antiviral caspase-dependent apoptotic reaction. Our results reveal that viral nuclear entry induces completely different immune effects in vector and host cells, providing new insights into the balance between viral load and the immunity pressure in vector insects.
Animals ; Cell Nucleus ; Hemiptera/metabolism* ; Insect Vectors/genetics* ; Insecta ; Nucleocapsid Proteins/metabolism* ; Oryza ; Plant Diseases ; Tenuivirus/metabolism* ; Virus Replication

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

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

Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.
Ebolavirus/physiology* ; HEK293 Cells ; HeLa Cells ; Humans ; Nucleocapsid Proteins/metabolism* ; RNA, Viral/metabolism* ; Viral Matrix Proteins/metabolism* ; Virion/metabolism* ; Virus Assembly

Severe fever with thrombocytopenia syndrome virus (SFTSV), a member of the Phlebovirus genus from the Bunyaviridae family endemic to China, is the causative agent of life-threatening severe fever with thrombocytopenia syndrome (SFTS), which features high fever and hemorrhage. Similar to other negative-sense RNA viruses, SFTSV encodes a nucleocapsid protein (NP) that is essential for viral replication. NP facilitates viral RNA encapsidation and is responsible for the formation of ribonucleoprotein complex. However, recent studies have indicated that NP from Phlebovirus members behaves in inhomogeneous oligomerization states. In the present study, we report the crystal structure of SFTSV NP at 2.8 Å resolution and demonstrate the mechanism by which it processes a ringshaped hexameric form to accomplish RNA encapsidation. Key residues essential for oligomerization are identified through mutational analysis and identified to have a significant impact on RNA binding, which suggests that correct formation of highly ordered oligomers is a critical step in RNA encapsidation. The findings of this work provide new insights into the discovery of new antiviral reagents for Phlebovirus infection.
Binding Sites ; Crystallography, X-Ray ; Mutation ; Nucleocapsid Proteins ; chemistry ; genetics ; metabolism ; Phlebovirus ; metabolism ; Protein Binding ; Protein Multimerization ; Protein Structure, Quaternary ; RNA, Viral ; metabolism ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

Ebola virus (EBOV) harbors an RNA genome encapsidated by nucleoprotein (NP) along with other viral proteins to form a nucleocapsid complex. Previous Cryo-eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451-739) alone is capable of forming a helical nucleocapsid-like complex (NLC). However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NPΔ451-739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensitive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro.
Ebolavirus ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Nucleocapsid ; chemistry ; genetics ; metabolism ; RNA, Viral ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Virus Assembly

BACKGROUNDTo sequence, analyze and express the nucleotide sequences of S and M segments of hantavirus strain 84Fli.

METHODSS and M segments of hantavirus 84Fli strain were amplified by RT-PCR, the PCR products were cloned into plasmid pCR2.1-TOPOr. Three clones of each segment which have been sequenced were randomly selected. The coding region of S and M segments were amplified by PCR, and cloned into expressing vector pcDNA3.0. Transient expression of nucleocapsid protein, G1 and G2 glycoproteins in COS7 cells were performed by Lipofectin transfection. The expression of NP, G1 and G2 have been conformed by using immunofluorescence, Western blot and immuno-precipitation methods.

RESULTSThe full length S and M segments cDNA have been amplified and cloned. The S and M segments of hantavirus strain 84Fli contained 1 688 and 3 616 nucleotides respectively.

CONCLUSIONSDeduced amino acid sequences of NP and glycoproteins revealed high homologue to other Hantaan viruses. NP, G1 and G2 proteins of 84Fli can be transiently expressed in COS7 cells.

Animals ; COS Cells ; metabolism ; Cloning, Molecular ; Gene Expression ; Glycoproteins ; biosynthesis ; Hantavirus ; genetics ; metabolism ; Nucleocapsid Proteins ; biosynthesis ; Sequence Analysis ; Viral Proteins ; biosynthesis

Production of Hepatitis E Virus capsid protein by high cell density culture in recombinant E. coli has been studied in 10L and 30L fermentors. The effects of different factors on growth and producing recombinant protein of E. coli have been studied by batch culture, such as different media, the ratio of phosphate and Magnesium sulfate. Comparison of fermentation performance for recombinant E. coli in different fed-methods culture has been investigated by fed-batch culture. The effects of inducing at different stages of growth and time of inducing on growth and producing recombinant protein, also obtained by fed-batch culture. At last, the solubility of inclusion body in different urea concentrations also has been obtained by fed-batch culture. The results show that the concentration of phosphate and Magnesium sulfate in the optimal media is 80mmol/L and 20mmol/L in batch culture respectively, that induction with 1.0mmol/L IPTG at mid log phase (about 45 OD at 600nm) is suitable for growth and recombinant protein expression, the cells were approaching stationary growth phase and the maximum cell OD at 600nm of 80 was achieved in 5h of fed-batch culture, and the expression level is 29.74%. The results also indicate that the solubility of inclusion body in 4mol/L urea solution induced at 37 degrees C reaches 14mg/mL, over 80% inclusion body was resolved. The culture process achieved in 10L fermentor could be successfully scaled up to 30L fenmentor with good reproducibility.
Bioreactors ; microbiology ; Colony Count, Microbial ; Escherichia coli ; genetics ; metabolism ; Hepatitis E virus ; genetics ; Nucleocapsid Proteins ; biosynthesis ; genetics ; Protein Engineering ; methods ; Recombinant Fusion Proteins ; biosynthesis ; genetics

Genes encoding nucleocaspid (N) and membrane (M) protein of SARS coronavirus were obtained by RT-PCR and were cloned into expression vector pET22b and pBV222. DNA sequencing showed that the genes cloned from a patient in Beijing were identical to the gene sequences from reported Toronto strain. The genes were over-expressed in E. coli either as inclusion body or as soluble form. The recombinant proteins were purified by ion-exchange, or ion-exchange followed by metal chelate affinity chromatography. The recombinant N protein was demonstrated highly antigenic and could be employed as antigen to detect SARS antibodies in ELISA system for SARS diagnosis.
Chromatography, Affinity ; Chromatography, Ion Exchange ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Nucleocapsid Proteins ; genetics ; isolation & purification ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; isolation & purification ; metabolism