Showing papers by "Javier M. Rodríguez published in 2001"

African Swine Fever Virus Structural Protein pE120R Is Essential for Virus Transport from Assembly Sites to Plasma Membrane but Not for Infectivity

Abstract: African swine fever virus (ASFV), the only member of the new family Asfarviridae, is a complex enveloped deoxyvirus responsible for a severe disease of domestic pigs (19, 23, 39, 49). ASFV infects soft ticks of the Ornithodoros genus and different species of suids, being the only known arbovirus that contains DNA, ASFV is unique among DNA viruses in that it resembles the poxviruses in its genome structure and gene expression strategy but morphologically is similar to the iridoviruses (39). The viral genome is a double-stranded DNA molecule of 170 to 190 kbp with terminal inverted repetitions and terminal cross-links (29, 47). The genome of the ASFV strain BA71V encodes more than 150 polypeptides, including structural proteins; a variety of enzymes involved in DNA replication and repair, gene transcription, and protein modification, and proteins potentially involved in the modulation of the virus-host interaction (39, 50). The virus particle possesses a complex structure composed by several concentric domains with an overall icosahedral shape and an average diameter of 200 nm (4, 5, 14). The viral core consists of a DNA-containing nucleoid covered by a thick protein coat, the core shell. The core is surrounded by an inner lipid envelope and an icosahedral protein capsid. Extracellular particles possess an additional envelope derived from the plasma membrane (9). ASFV particles assemble within cytoplasmic viral factories (4, 9, 10, 31) from precursor membranous structures that probably represent collapsed cisternae derived from the endoplasmic reticulum (5, 18, 38). These membranes give rise to the inner viral envelope, which becomes an icosahedral structure by the progressive assembly of the capsid layer (5, 27). Envelopment and capsid formation depend on calcium gradients and ATP (18). The core is formed beneath the inner envelope through the consecutive assembly of the core shell domain and the electron-dense DNA-containing nucleoid (4, 11). The intracellular particles associate with microtubules to reach the plasma membrane (3, 16) and are finally released from the cell by budding (9). The mature virion contains about 50 proteins (25), some of which are produced by the proteolytic processing of two viral polyproteins by the viral cysteine proteinase pS273R (6). The core shell proteins p150, p37, p34, and p14, which represent about 25% of the total protein mass of the virus particle, are derived from polyprotein pp220 (4, 43). Similarly, the structural proteins p35 and p15 are derived from polyprotein pp62 (44). At least three major structural proteins have DNA-binding properties (8, 30, 32); one of them (protein 5AR) has been located within the nucleoid and is similar to bacterial histone-like proteins (8). Among the 26 putative membrane proteins encoded by the ASFV genome (36), the structural proteins p12 and pE183L have been involved in the virus attachment to the host cell (1, 13, 28, 34). The icosahedral capsid is mainly composed by protein p72, which represents about one-third of the virus protein mass and probably forms the hexagonal capsomers (4, 27). Despite the emerging information about the ASFV structural components, little is known on their particular role in virus morphogenesis. To facilitate this study, our laboratory recently adapted to ASFV an inducible expression system based on the Escherichia coli lac operon (27). By using an ASFV recombinant with an inducible copy of protein p72 gene, the role of the major capsid protein in virus assembly and also the origin of the inner viral envelope were analyzed (5, 27). In this report, we have employed the same strategy to investigate the role of protein pE120R in ASFV replication. Protein pE120R, also called p14.5, has been previously characterized as a structural protein expressed as different molecular weight forms late after infection (30). In vitro assays revealed that protein pE120R has DNA-binding properties and that it interacts with a late virus-induced protein of 72 kDa. Data presented here show that protein pE120R is a capsid component which associates with the major capsid protein p72. Furthermore, protein pE120R was found to be essential for virus dissemination but not for infectivity.

African swine fever virus-induced polypeptides in porcine alveolar macrophages and in Vero cells: two-dimensional gel analysis.

Abstract: High-resolution two-dimensional electrophoresis followed by computer analysis has been used to study quantitatively the patterns of protein synthesis produced in porcine alveolar macrophages and in Vero cells infected with African swine fever virus (ASFV). Initially, a protein database for each cell type was constructed. The porcine alveolar macrophage database includes 995 polypeptides (818 acidic, isoelectric focusing (IEF) and 177 basic, nonequilibrium pH gradient electrophoresis (NEPHGE)) whereas the Vero database contains 1,398 polypeptides (1,127 acidic, IEF and 271 basic, NEPHGE). Taking these databases as reference, ASFV highly virulent strain E70 induces 57 acid and 43 basic polypeptides in porcine alveolar macrophages, which account for most of the information content of the virus DNA. The kinetics of synthesis of the virus-induced polypeptides showed the existence of three classes of proteins: one whose synthesis starts early after infection, continues for a period and then switches off; another whose synthesis also starts early but continues for prolonged periods; and a third which requires DNA replication. The attenuated, cell adapted, strain BA71V induces 92 acidic and 37 basic proteins in Vero cells. Significant differences were observed when comparing the patterns of polypeptides induced by the two viral strains. In both cell systems studied, ASFV infection produces a general shutoff of protein synthesis that affects up to 65% of the cellular proteins. Interestingly, 28 proteins of porcine alveolar macrophages and 48 proteins of Vero cells are stimulated at least two times by ASFV infection.