Adenoviruses are icosahedral virions containing double-stranded linen DNA. They are 70 nm to 90 nm in diameter and capsid is composed of 252 capsomeres. Several members of this group, including types commonly associated with respiratory disease in man, are capable of producing malignant tumors in young hamsters and a few types have been shown to be oncogenic in young rat. Previous report involving effect of caffein on transformation induced by Adenovirus type 12 [9] has been carried out. The present report represents a continuation of previous study. To obtain evidence concerning the of(tract of MNNG (N-methyl-N'-nitro-N-nitroguanidine) on transformation, investigation of adenovirus type 12 of this group was undertaken. For practical consideration it was desirable to investigate the effect of MNNG on the adenovirus type 12-induced transformation in L cell. Results were as following 1. Adeno virus type 12 induced transformation was enhanced in the presence of MNNG. 2. Yields of adeno type 12 virus in L cell were slightly inhibited by treatment of MNNG.
Adenoviridae* ; Animals ; Bedding and Linens ; Capsid ; Cricetinae ; DNA ; Methylnitronitrosoguanidine ; Rats ; Virion

Turnip yellow mosaic virus (TYMV) is a non-enveloped icosahedral virus composed of 20 kDa single coat proteins. In this study, we modified the TYMV coat protein (CP) ORF by inserting an oligonucleotide linker corresponding to T7, HSV, Tat, (Arg)9, or (RxR)4 peptide at the 5'-end of the CP ORF and examined its effect on replication, RNA packaging, and virion assembly. The results showed that the constructs containing (Arg)9 and (RxR)4 sequences were barely capable of replication. The TYMV constructs containing T7 and Tat peptide produced virions that co-migrated with wild-type virions. However, the insertion of T7 and Tat sequences impaired genomic RNA (gRNA) accumulation and packaging, respectively. When only the CP gene was expressed, CPs with (Arg)9 or (RxR)4 successfully produced virus-like particles whose mobility was comparable to that of wild type. In the case of CP having a HSV tag, the virion band was not detected, although a sufficient amount of CP was produced. This indicates that CP with the HSV tag failed to assemble into virions. Overall, the results suggest that TYMV replication, RNA packaging and virion assembly are strongly influenced by the insertion sequence.
Animals ; Brassica napus* ; Capsid Proteins ; Ecthyma, Contagious ; Product Packaging* ; RNA* ; Tymovirus* ; Virion*

For the purpose of morphologic analysis of human cytomegalovirus (HCMV) antigens reacting with specific monoclonal antibodies, we observed HCMV particles after immunogold staining. HCMV was cultured in human fetal lung fibroblasts to be concentrated by polyethylene glycol 6,000. The HCMV stock was dropped onto Formva-coated grids and was fixed by 2% glutaraldehyde. The grids were reacted with MCMVA57, 93, 135 or with SCMVM1, 6, 14, 49 monoclonal antibodies (MoAbs) follwed by gold (10 nm)-conjugated goat anti-mouse IgG. Then the grids were stained with 2.5% uranyl acetate to be observed under Hitachi 500 or Jeol 1,200 electron microscope. When HCMV was reacted with SCMVM14 and SCMVM49 MoAbs, gold particles were adsorbed to virion envelopes, suggesting that the reactive antigens were envelope proteins. In cases of MCMVA135 and SCMVM6 MoAbs, gold particles were adsorbed to dense bodies as well as to virion envelope. These results, together with the previous results of immunologic and genetic characterization, suggested that the reactive antigens of MCMVA135 and SCMVM6 MoAbs were gB homologue and structural protein, respectively. In case of SCMVM1 MoAb, gold particles were adsorbed to capsids, envelopes, and dense bodies, suggesting that the reactive antigen was structural protein. In case of SCMVM8 MoAb, gold particles were observed between the envelopes and capsids, which space was supposed to be the tegument, suggesting that the reactive antigen was carbohydrate moiety of glycoprotein or its polymer. In cases of MCMVA57 and MCMVA93 MoAbs, gold particles were adsorbed to only dense bodies, suggesting that the reactive antigens were precursors of structural proteins.
Antibodies, Monoclonal* ; Capsid ; Cytomegalovirus* ; Fibroblasts ; Glutaral ; Glycoproteins ; Goats ; Humans* ; Immunoglobulin G ; Lung ; Polyethylene Glycols ; Polymers ; Virion

The HIV-1 capsid protein (CA) plays an essential role in viral core assembly and maturation. Proteolytic cleavage at the MA-CA junction of the retroviral gag polyprotein refolds the amino-terminal end of capsid into a beta-helix structure that is stabilized by a salt bridge between the protein's processed amino-terminus and a conserved acidic residue. The refolded capsid aminoterminus then creates a new CA-CA interface, allowing assembly of the mature capsid core. Recently, researches focus on assembly of CA in vitro and development of CA vaccine. CA vaccine will provide widely immune protection because CA is comparatively conserved. Experiments demonstrate that fusing as few as four matrix residues onto the amino-terminus of capsid redirects protein assembly from cylinder to spheres in vitro. Evaluation of immunogenicity showed that immunization with virus-like particles induced both cellular and neutralizing antibody responses. Furthermore, mucosal administration of virus-like particles effectively induced both mucosal and systemic immune responses. These results indicate that virus-like particles consisting of HIV structural proteins are an attractive vaccine platform for eliciting anti-viral immune responses, especially neutralizing antibody responses. The production of antigens for vaccines in plants indicates that plant-based transgenic expression represents a viable means of producing CA vaccine for the development of HIV vaccine and for use in HIV diagnostic procedures and it has the potential as a safe and cost-effective alternative to traditional production systems.
AIDS Vaccines ; immunology ; Capsid ; immunology ; metabolism ; Capsid Proteins ; genetics ; immunology ; metabolism ; HIV-1 ; genetics ; immunology ; metabolism ; Virion ; genetics ; immunology ; metabolism

OBJECTIVETo express the L1 protein of human papillomavirus type 16 (HPV16) in insect cell suspension culture system.

METHODSOptimized the conditions of suspension culture, recombinant virus amplification and protein expression. Determined the virus tilter by plague analysis and detected the target protein by SDS-PAGE and Western blot; The formation of VLPs by HPV16 L1 protein was observed with TEM.

RESULTSThe Sf9 cells could grow better in suspension culture with seeding density of 5 x 10-5 cell/mL and the maximum expression quantity was obtained by infection of cells with rBacV/HPV16L1 (MOI =10) and harvesting after 72-84 h. HPV16L1 protein could assemble into VLPs in Sf9 cells observed with TEM.

CONCLUSIONThe conditions of cell culture, virus amplification and protein expression were optimized. HPV16 L1 protein could assemble into VLPs in Sf9 cells, which would provide a foundation for further study of the vaccine and diagnosis kits.

Animals ; Capsid Proteins ; biosynthesis ; Cell Proliferation ; Oncogene Proteins, Viral ; biosynthesis ; Recombinant Proteins ; biosynthesis ; Spodoptera ; Suspensions ; Virion ; ultrastructure

Genital HPV infection is the most common sexually transmitted infection, but the majority of infections are self-limited. However, persistent infection with high-risk types can cause cervical cancer in women, which is the most common female genital cancer in Korea. In addition, HPV infection is the cause of genital warts and is associated with other anogenital cancers. The HPV vaccine is composed of the HPV L1 protein, the major capsid protein of HPV. Expression of the L1 protein in yeast using recombinant DNA technology produces noninfectious virus-like particles (VLP) that resemble HPV virions. The quadrivalent HPV vaccine is a mixture of four HPV type-specific VLPs prepared from the L1 proteins of HPV 6, 11, 16, and 18 combined with an aluminum adjuvant. Clinical trials indicate that the vaccine has high efficacy in preventing persistent HPV infection, cervical cancer precursor lesions, vaginal and vulvar cancer precursor lesions, and genital warts caused by HPV types 6, 11, 16, or 18 among females who have not already been infected with the respective HPV type. The recommended age for primary vaccination of Korean females is 15-17 years, considering sexual debut and duration of protection of the vaccine. Vaccine can be administered as young as age 9 years. Catch-up vaccination is recommended for females aged 18-26 years who have not been previously vaccinated. Vaccination is not a substitute for routine cervical cancer screening, and vaccinated females should have cervical cancer screening as recommended.
Aluminum ; Capsid Proteins ; Colposcopy* ; Condylomata Acuminata ; DNA, Recombinant ; Female ; Human papillomavirus 6 ; Humans* ; Korea ; Mass Screening ; Uterine Cervical Neoplasms ; Vaccination ; Virion ; Vulvar Neoplasms ; Yeasts

Recombinant adeno-associated virus (rAAV)-based vectors that can stably express therapeutic genes in vivo without detectable side-effect have shown great promise for human gene therapy. A major challenge for translation of promising research to clinical development is how to establish clinically compatible purification methods in separating rAAV from potentially pathogenic impurities, especially rAAV vector-related impurities, a class of impurities corresponding to AAV particles that closely resemble bona fide vectors and are difficult to remove. In this review we summarize the assembly process of rAAV vector-related impurities and their characteristics differed with rAAV vectors, and evaluate several current technologies to prevent their formation or separate them from rAAV stocks.
Capsid Proteins ; isolation & purification ; Dependovirus ; genetics ; isolation & purification ; physiology ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; isolation & purification ; Recombination, Genetic ; Virion ; isolation & purification ; Virus Assembly ; genetics ; Virus Replication ; genetics

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

Here, we presented a method to bacterially express the major structural protein L1 of Human Papillomavirus type 18 (HPV18) as soluble form. We found that the purified L1 could self-assemble to virus-like particles (VLPs). Further, we investigated the immunogenicity and the induced level of neutralizing antibody using these VLPs. First, the genome of HPV18 was cloned from a patient in Xiamen. It was used as template for PCR amplification of HPV18 L1 gene. The resultant DNA fragment was inserted into expression vector pTrxFus and expressed in Escherichia coli GI724. Second, L1 protein was purified by ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography; and the purified L1 was subjected to self-assembly to form VLPs with the removal of premixed reductant DTT. Finally, the size and morphology of these VLPs was investigated by Dynamic Light Scattering and Transmission Electronic Microscopy as 29.34 nm in hydrated radius and globular particles similar with native HPV18. The half effective dosage (ED50) and maximum level of neutralizing antibody elicitation were measured by vaccinations on mice, rabbit and goat using pseudovirus neutralization cell model. The results showed that the ED50 of HPV18 VLPs is 0.006 microg in mice, and the maximum titer of neutralizing antibody elicited in rabbit and goat is up to 10(7). As a conclusion, we can provide HPV18 VLPs with highly immunogenicity from prokaryote expression system, which may pave a new way for research and development of prophylactic vaccine for HPV18.
Animals ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Goats ; Human papillomavirus 18 ; immunology ; isolation & purification ; Mice ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Virion ; genetics ; immunology

The baculovirus expression system was employed to prepare the virus-like particles (VLPs) of human parvovirus B19. The synthesized VP2 gene of B19 was inserted into the multi-cloning site (MCS) of pFastBac1 vector; the resulting plasmid was transferred to the Escherichia coli DH10Bac competent cells, which contain a baculovirus shuttle vector (Bacmid), to generate Bacmid-VP2 by site-specific transposition. Recombinant baculovirus carrying VP2 gene (rBac-VP2) was then rescued from Bacmid-VP2-transfected Sf9 cells. Indirect immunofluorescence and Western blotting were used to identify the VP2 protein in rBac-VP2-infected Sf9 cells, and the VLPs were observed under transmission electron microscope after being enriched by ultracentrifugation. The B19 VLPs were successfully produced in insect cells with baculovirus expression system, which will facilitate the development of diagnostic reagents to detect the antibody against B19 virus in human serum.
Animals ; Antibodies, Viral ; blood ; Baculoviridae ; genetics ; metabolism ; Capsid Proteins ; biosynthesis ; genetics ; Cell Line ; Cloning, Molecular ; Genetic Vectors ; genetics ; Parvovirus B19, Human ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Virion ; genetics ; metabolism