Assembly of African swine fever virus: role of polyprotein pp220

Abstract

Polyprotein processing is a common strategy of gene expression in many positive-strand RNA viruses and retroviruses but not in DNA viruses. African swine fever virus (ASFV) is an exception because it encodes a polyprotein, named pp220, to produce several major components of the virus particle, proteins p150, p37, p34, and p14. In this study, we analyzed the assembly pathway of ASFV and the contribution of the polyprotein products to the virus structure. Electron microscopic studies revealed that virions assemble from membranous structures present in the viral factories. Viral membranes became polyhedral immature virions after capsid formation on their convex surface. Beneath the lipid envelope, two distinct domains appeared to assemble consecutively: first a thick protein layer that we refer to as core shell and then an electron-dense nucleoid, which was identified as the DNA-containing domain. Immunofluorescence studies showed that polyprotein pp220 is localized in the viral factories. At the electron microscopic level, antibodies to pp220 labeled all identifiable forms of the virus from the precursor viral membranes onward, thus indicating an early role of the polyprotein pp220 in ASFV assembly. The subviral localization of the polyprotein products, examined on purified virions, was found to be the core shell. In addition, quantitative studies showed that the polyprotein products are present in equimolar amounts in the virus particle and account for about one-fourth of its total protein content. Taken together, these results suggest that polyprotein pp220 may function as an internal protein scaffold which would mediate the interaction between the nucleoid and the outer layers similarly to the matrix proteins of other viruses. MATERIALS AND METHODS Cells and viruses. Vero cells (ATCC CCL81) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, which was reduced to 2% during viral infection. Swine alveolar macrophages, obtained by bronchoalveolar lavage, were maintained and infected in Dulbecco's modified Eagle's medium with 10% newborn calf serum. ASFV strain BA71V, adapted to grow in Vero cells, has been previously described (14). Highly purified ASFV was obtained by Percoll equilibrium centrifugation (7). Antibodies. Monoclonal antibody 18H.H7, against proteins pp220 and p150, as well as rabbit polyclonal anti-p150, anti-p37/p14, and anti-p34 sera, which also recognize the precursor form pp220, have been characterized previously (28, 33). Monoclonal antibody 19B.A2, against protein p72, has been described before (28). The anti-DNA immunoglobulin M (Ac 30-10

African swine fever virus-cell interactions: From virus entry to cell survival

“…FPP and GGPP are formed in the reaction catalyzed by the viral enzyme. This enzyme has the unique characteristic that it is associated with precursor viral membranes derived from the ER at the viral assembly sites (Alejo et al, 1999;Andres et al, 1997). GGPP synthesized by B318L product serves as a substrate for protein prenylation, required during virus replication and morphogenesis.…”

A Guide to Viral Inclusions, Membrane Rearrangements, Factories, and Viroplasm Produced During Virus Replication

“…Appearance and contents of viral origin a Viral membranes, assembling and complete particles, electron dense condensations, viral DNA, A224L IAP apoptosis inhibitor, A104R (5AR) DNA binding histone like, A137R p11.5, B119L Erv1p homologue, B438L p49, B646L p73 major capsid protein, CP2475L pp220 precursor to p150; p37; p34 and p14 CP530R pp62 precursor to p35 and p15, O61R p12 attachment, D117L (i1L) transmembrane, S273R (i6R) cysteine protease, H108R (j5R) membrane, E183L p54 (j13L) dynein interacting, E199L (j18L) membrane, E120R (k3R) p14.5 DNA binding necessary for viral exit from factory Alcamí et al, 1993;Alonso et al, 2001;Andrés et al, 1997Andrés et al, , 2001Borca et al, 1996;Brookes et al, 1998a,b;Carrascosa et al, 1986;Chacó n et al, 1995;Cobbold et al, 1996;Galindo et al, 2000;García-Beato et al, 1992;Heath et al, 2001;Hingamp et al, 1992;Jouvenet and Wileman, 2005;Jouvenet et al, 2004;Martinez-Pomares et al, 1997;Moura Nunes et al, 1975;Rodríguez et al, 2006;Rouiller et al, 1998;Sanz et al, 1985;Simón-Mateo et al, 1997;Sun et al, 1996;Vigário et al, 1967 Contents of cellular origin Ubiquitin, hsp70 chaperone, g-tubulin, Pericentrin, p21, mdm1 Surrounded by: ER membranes, vimentin, p230 Golgin, mitochondria, and tubulin. Granja et al, 2004;Heath et al, 2001;Hingamp et al, 1992;Jouvenet and Wileman, 2005;Netherton et al, 2004Netherton et al, , 2006Rojo et al, 1998;Rouiller et al, 1998;Stefanovic et al, 2005 Poxviridae, Chordopoxvirinae, Orthopoxvirus Vaccinia virus Cytoplasmic A-type inclusion…”

Visualization of the African swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera

“…The 170-kb genome of ASFV encodes some 150 open reading frames, and as many as 50 viral proteins are incorporated to the viral particle (Esteves et al, 1986). Approximately 35% of the mass of the virion is provided by p72, the major capsid protein, while the structural proteins p150, p37, p34 and p14, all of them derived from polyprotein p220, provides another 25% of the virion mass (Andres et al, 1997). ASFV particles assemble within cytoplasmic viral factories from endoplasmic reticulum-derived viral membranes (Andres et al, 1997Rouiller et al, 1998) at perinuclear sites (Nunes et al, 1975) that contain fully assembled virions seen as 200-nm-diameter hexagons in cross-section, and a series of one to six-sided assembly intermediates (Rouiller et al, 1998).…”

“…Biochemical data also suggest that protein p72 is assembled in a time-dependent fashion into large membrane-bound complexes that may correspond to capsid-like structures (Cobbold and Wileman, 1998). Concomitantly, the core shell is formed underneath the viral envelope, and subsequently the viral DNA and nucleoproteins are packaged and condensed to form the electrondense nucleoid (Andres et al, 1997(Andres et al, , 2002Brookes et al, 1998). Intracellular mature virions made at the assembly sites are infectious (Andres et al, 2001).…”