scholarly journals Inducible Gene Expression from African Swine Fever Virus Recombinants: Analysis of the Major Capsid Protein p72

1998 ◽  
Vol 72 (4) ◽  
pp. 3185-3195 ◽  
Author(s):  
Ramón García-Escudero ◽  
Germán Andrés ◽  
Fernando Almazán ◽  
Eladio Viñuela
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
African Swine Fever Virus ◽  
Expression System ◽  
African Swine Fever ◽  
Firefly Luciferase ◽  
Fever Virus ◽  
Gene Encoding ◽  
Iptg Induction ◽  
Dense Membrane

ABSTRACT A method to study the function of individual African swine fever virus (ASFV) gene products utilizing the Escherichia coli lac repressor-operator system has been developed. Recombinant viruses containing both the lacI gene encoding thelac repressor and a strong virus late promoter modified by the insertion of one or two copies of the lac operator sequence at various positions were constructed. The ability of each modified promoter to regulate expression of the firefly luciferase gene was assayed in the presence and in the absence of the inducer isopropyl β-d-thiogalactoside (IPTG). Induction and repression of gene activity were dependent on the position(s) of the operator(s) with respect to the promoter and on the number of operators inserted. The ability of this system to regulate the expression of ASFV genes was analyzed by constructing a recombinant virus inducibly expressing the major capsid protein p72. Electron microscopy analysis revealed that under nonpermissive conditions, electron-dense membrane-like structures accumulated in the viral factories and capsid formation was inhibited. Induction of p72 expression allowed the progressive building of the capsid on these structures, leading to assembly of ASFV particles. The results of this report demonstrate that the transferred inducible expression system is a powerful tool for analyzing the function of ASFV genes.

Mapping and sequence of the gene coding for protein p72, the major capsid protein of african swine fever virus

Virology ◽  
1990 ◽  
Vol 175 (2) ◽  
pp. 477-484 ◽  
Author(s):  
C. López-Otín ◽  
J.M.P. Freije ◽  
F. Parra ◽  
E. Mendez ◽  
E. Viñuela
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Gene Coding

scholarly journals Structure of the African swine fever virus major capsid protein p72

Cell Research ◽  
2019 ◽  
Vol 29 (11) ◽  
pp. 953-955 ◽  
Author(s):  
Qi Liu ◽  
Bingting Ma ◽  
Nianchao Qian ◽  
Fan Zhang ◽  
Xu Tan ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus

scholarly journals A Virally Encoded Chaperone Specialized for Folding of the Major Capsid Protein of African Swine Fever Virus

2001 ◽  
Vol 75 (16) ◽  
pp. 7221-7229 ◽  
Author(s):  
C. Cobbold ◽  
M. Windsor ◽  
T. Wileman
Keyword(s):  
Protein Folding ◽  
Capsid Protein ◽  
Major Capsid Protein ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Capsid Proteins ◽  
Capsid Shell ◽  
Infected Cells ◽  
Virally Encoded

ABSTRACT It is generally believed that cellular chaperones facilitate the folding of virus capsid proteins, or that capsid proteins fold spontaneously. Here we show that p73, the major capsid protein ofAfrican swine fever virus (ASFV) failed to fold and aggregated when expressed alone in cells. This demonstrated that cellular chaperones were unable to aid the folding of p73 and suggested that ASFV may encode a chaperone. An 80-kDa protein encoded by ASFV, termed the capsid-associated protein (CAP) 80, bound to the newly synthesized capsid protein in infected cells. The 80-kDa protein was released following conformational maturation of p73 and dissociated before capsid assembly. Coexpression of the 80-kDa protein with p73 prevented aggregation and allowed the capsid protein to fold with kinetics identical to those seen in infected cells. CAP80 is, therefore, a virally encoded chaperone that facilitates capsid protein folding by masking domains exposed by the newly synthesized capsid protein, which are susceptible to aggregation, but cannot be accommodated by host chaperones. It is likely that these domains are ultimately buried when newly synthesized capsid proteins are added to the growing capsid shell.

scholarly journals Study on the Immunogenicity of Three Recombinant Lactic Acid Bacteria Expressing ASFV p14.5 Protein

2021 ◽  
Author(s):  
Quntao Huang ◽  
Tian-Ming Niu ◽  
Bo-Shi Zou ◽  
Jun-Hong Wang ◽  
Jun-Hong Xin ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Fusion Gene ◽  
African Swine Fever Virus ◽  
Expression System ◽  
African Swine Fever ◽  
Fever Virus ◽  
Control Group ◽  
Adjuvant Effect

Abstract The African Classical Swine Fever Virus (ASFV) has spread severely all over the world. The lack of vaccines has dealt a heavy blow to the pig industry.In this study, the p14.5 protein encoded by the African swine fever virus was used as the antigen, and the p14.5 protein gene was expressed in vitro using the Lactobacillus expression system. Three new functional recombinant Lactobacillus plantarum((L. plantarum) were constructed and the p14.5 was successfully detected using western technology.Protein, fusion gene p14.5-IL-33-mouse（P14.5-IL-33-Mus） protein and CTA1-p14.5-DD protein expression.After oral immunization of SPF mice with recombinant lactic acid bacteria, flow cytometry and ELISA were used to detect that the differentiation and maturity of T, B, and DC cells of the mice were higher than those of the control group, and specific antibodies were produced. In contrast, the immune effect of the adjuvant group was stronger than that of the single antigen group, and the IL-33 adjuvant effect was stronger than that of the CTA1-DD adjuvant. This study provides effective data support for the prevention of African swine fever virus infection with new lactic acid bacteria preparations, and has certain innovative significance.

scholarly journals The African Swine Fever Virus Nonstructural Protein pB602L Is Required for Formation of the Icosahedral Capsid of the Virus Particle

2006 ◽  
Vol 80 (24) ◽  
pp. 12260-12270 ◽  
Author(s):  
Carolina Epifano ◽  
Jacomine Krijnse-Locker ◽  
María L. Salas ◽  
Javier M. Rodríguez ◽  
José Salas
Keyword(s):  
Virus Particle ◽  
Capsid Protein ◽  
Recombinant Virus ◽  
Nonstructural Protein ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Viral Assembly ◽  
Fever Virus ◽  
Assembly Process ◽  
Icosahedral Capsid

ABSTRACT African swine fever virus (ASFV) protein pB602L has been described as a molecular chaperone for the correct folding of the major capsid protein p72. We have studied the function of protein pB602L during the viral assembly process by using a recombinant ASFV, vB602Li, which inducibly expresses the gene coding for this protein. We show that protein pB602L is a late nonstructural protein, which, in contrast with protein p72, is excluded from the viral factory. Repression of protein pB602L synthesis inhibits the proteolytic processing of the two viral polyproteins pp220 and pp62 and leads to a decrease in the levels of protein p72 and a delocalization of the capsid protein pE120R. As shown by electron microscopy analysis of cells infected with the recombinant virus vB602Li, the viral assembly process is severely altered in the absence of protein pB602L, with the generation of aberrant “zipper-like” structures instead of icosahedral virus particles. These “zipper-like” structures are similar to those found in cells infected under restrictive conditions with the recombinant virus vA72 inducibly expressing protein p72. Immunoelectron microscopy studies show that the abnormal forms generated in the absence of protein pB602L contain the inner envelope protein p17 and the two polyproteins but lack the capsid proteins p72 and pE120R. These findings indicate that protein pB602L is essential for the assembly of the icosahedral capsid of the virus particle.

scholarly journals Structural comparisons of host and African swine fever virus dUTPases reveal new clues for inhibitor development

2020 ◽  
pp. jbc.RA120.014005
Author(s):  
Rui Liang ◽  
Gang Wang ◽  
Ding Zhang ◽  
Gang Ye ◽  
Mengxia Li ◽  
...  
Keyword(s):  
Active Sites ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Structural Diversity ◽  
Fever Virus ◽  
Gene Encoding ◽  
Inhibitor Development ◽  
Ligand Free ◽  
The Difference ◽  
Swine Diseases

African swine fever, caused by the African swine fever virus (ASFV), is among the most significant swine diseases. There are currently no effective treatments against ASFV. ASFV contains a gene encoding a dUTPase (E165R), which is required for viral replication in swine macrophages, making it an attractive target for inhibitor development. However, the full structural details of the ASFV dUTPase and those of the comparable swine enzyme are not available, limiting further insights. Herein, we determine the crystal structures of ASFV dUTPase and swine dUTPase in both their ligand-free and ligand-bound forms. We observe that the swine enzyme employs a classical dUTPase architecture made up of three-subunit active sites, whereas the ASFV enzyme employs a novel two-subunit active site. We then performed a comparative analysis of all dUTPase structures uploaded in PDB, which showed classic and non-classical types were mainly determined by the C-terminal β-strand orientation, and the difference was mainly related to the four amino acids behind motif IV. Thus, our study not only explains the reason for the structural diversity of dUTPase but also reveals how to predict dUTPase type, which may have implications for the dUTPase family. Finally, we tested two dUTPase inhibitors developed for the Plasmodium falciparum dUTPase against the swine and ASFV enzymes. One of these compounds inhibited the ASFV dUTPase at low micromolar concentrations (Kd=15.6μM) and with some selectivity (~2x) over swine dUTPase. In conclusion, our study expands our understanding of the dUTPase family and may aid in the development of specific ASFV inhibitors.

scholarly journals Repression of African Swine Fever Virus Polyprotein pp220-Encoding Gene Leads to the Assembly of Icosahedral Core-Less Particles

2002 ◽  
Vol 76 (6) ◽  
pp. 2654-2666 ◽  
Author(s):  
Germán Andrés ◽  
Ramón García-Escudero ◽  
María L. Salas ◽  
Javier M. Rodríguez
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Proteolytic Processing ◽  
Viral Envelope ◽  
Fever Virus ◽  
Core Shell ◽  
The Core ◽  
Dense Membrane ◽  
Core Proteins ◽  
Membrane Anchoring

ABSTRACT African swine fever virus (ASFV) polyprotein pp220, encoded by the CP2475L gene, is an N-myristoylated precursor polypeptide that, after proteolytic processing, gives rise to the major structural proteins p150, p37, p34, and p14. These proteins localize at the core shell, a matrix-like virus domain placed between the DNA-containing nucleoid and the inner envelope. In this study, we have examined the role of polyprotein pp220 in virus morphogenesis by means of an ASFV recombinant, v220i, containing an inducible copy of the CP2475L gene regulated by the Escherichia coli repressor-operator system. Under conditions that repress pp220 expression, the virus yield of v220i was about 2.6 log units lower than that of the parental virus or of the recombinant grown under permissive conditions. Electron microscopy revealed that pp220 repression leads to the assembly of icosahedral particles virtually devoid of the core structure. Analysis of recombinant v220i by immunoelectron microscopy, immunoblotting, and DNA hybridization showed that mutant particles essentially lack, besides the pp220-derived products, a number of major core proteins as well as the viral DNA. On the other hand, transient expression of the CP2475L gene in COS cells showed that polyprotein pp220 assembles into electron-dense membrane-bound coats, whereas a mutant nonmyristoylated version of pp220 does not associate with cellular membranes but forms large cytoplasmic aggregates. Together, these findings indicate that polyprotein pp220 is essential for the core assembly and suggest that its myristoyl moiety may function as a membrane-anchoring signal to bind the developing core shell to the inner viral envelope.

scholarly journals Linear epitopes in African swine fever virus p72 recognized by monoclonal antibodies prepared against baculovirus-expressed antigen

2018 ◽  
Vol 30 (3) ◽  
pp. 406-412 ◽  
Author(s):  
Mallory E. Heimerman ◽  
Maria V. Murgia ◽  
Ping Wu ◽  
Andre D. Lowe ◽  
Wei Jia ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Major Capsid Protein ◽  
Vaccine Development ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
Fever Virus ◽  
E Coli ◽  
Important Target ◽  
Linear Epitopes

Protein p72 is the major capsid protein of African swine fever virus (ASFV) and is an important target for test and vaccine development. Monoclonal antibodies (mAbs) were prepared against a recombinant antigenic fragment, from amino acid (aa) 20–303, expressed in baculovirus. A total of 29 mAbs were recovered and tested by immunofluorescent antibody (IFA) staining on ASFV Lisbon-infected Vero cells. Six antibodies were IFA-positive and selected for further characterization. Epitope mapping was performed against overlapping polypeptides expressed in E. coli and oligopeptides. Based on oligopeptide recognition, the mAbs were divided into 4 groups: mAb 85 (aa 165–171); mAbs 65-3 and 6H9-1 (aa 265–280); mAbs 8F7-3 and 23 (aa 280–294); and mAb 4A4 (aa 290–303). All mAbs were located within a highly conserved region in p72. This panel of antibodies provides the opportunity to develop new assays for the detection of ASFV antibody and antigen.

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