Showing papers by "Javier M. Rodríguez published in 2006"

African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

Abstract: Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.

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

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.

Antigenic properties and diagnostic potential of African swine fever virus protein pp62 expressed in insect cells.

Abstract: African swine fever (ASF) is an infectious and economically important disease of domestic pigs. The absence of vaccine renders the diagnostic test the only tool that can be used for the control of new outbreaks of the disease. At present, the enzyme-linked immunosorbent assay (ELISA) test is the most useful method for large-scale ASF serological studies, although false positives have been detected, mainly on poorly preserved sera. In order to improve the current diagnostic test available for ASF, we have studied the antigenic properties of the ASF virus polyprotein pp62 and its suitability for use in a novel ELISA. Two well-known antigenic proteins of ASF virus, p32 and p54, were also included in this study. These proteins were expressed in the baculovirus expression system and used as antigens in ASF serological tests. Our results indicate that the use of these recombinant proteins as antigens in the ELISAs improves the sensitivity and specificity obtained with the conventional diagnosis test used to detect antibodies against ASF virus. Furthermore, the use of polyprotein pp62 in an ELISA for testing poorly preserved sera allows performance of the diagnosis of ASF without the need to confirm the results by the immunoblot test. These features make pp62 one of the most interesting viral proteins to be used for serological ASF diagnosis.

Generation of Filamentous Instead of Icosahedral Particles by Repression of African Swine Fever Virus Structural Protein pB438L

Abstract: The mechanisms involved in the construction of the icosahedral capsid of the African swine fever virus (ASFV) particle are not well understood at present. Capsid formation requires protein p72, the major capsid component, but other viral proteins are likely to play also a role in this process. We have examined the function of the ASFV structural protein pB438L, encoded by gene B438L, in virus morphogenesis. We show that protein pB438L associates with membranes during the infection, behaving as an integral membrane protein. Using a recombinant ASFV that inducibly expresses protein pB438L, we have determined that this structural protein is essential for the formation of infectious virus particles. In the absence of the protein, the virus assembly sites contain, instead of icosahedral particles, large aberrant tubular structures of viral origin as well as bilobulate forms that present morphological similarities with the tubules. The filamentous particles, which possess an aberrant core shell domain and an inner envelope, are covered by a capsid-like layer that, although containing the major capsid protein p72, does not acquire icosahedral morphology. This capsid, however, is to some extent functional, as the filamentous particles can move from the virus assembly sites to the plasma membrane and exit the cell by budding. The finding that, in the absence of protein pB438L, the viral particles formed have a tubular structure in which the icosahedral symmetry is lost supports a role for this protein in the construction or stabilization of the icosahedral vertices of the virus particle.