Proteolytic processing in African swine fever virus: evidence for a new structural polyprotein, pp62.

ABSTRACT African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV.

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African Swine Fever Virus Protein p17 Is Essential for the Progression of Viral Membrane Precursors toward Icosahedral Intermediates

Journal of Virology ◽

10.1128/jvi.00600-10 ◽

2010 ◽

Vol 84 (15) ◽

pp. 7484-7499 ◽

Cited By ~ 20

Author(s):

Cristina Suárez ◽

Javier Gutiérrez-Berzal ◽

Germán Andrés ◽

María L. Salas ◽

Javier M. Rodríguez

Keyword(s):

Early Stage ◽

African Swine Fever Virus ◽

Transmembrane Protein ◽

African Swine Fever ◽

Integral Membrane Protein ◽

Proteolytic Processing ◽

Fever Virus ◽

Morphological Evidence ◽

Virus Protein

ABSTRACT The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that protein p54, a viral structural integral membrane protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of protein p17, an abundant transmembrane protein localized at the viral internal envelope, in these processes. Using an inducible virus for this protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyproteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.

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African Swine Fever Virus Polyprotein pp62 Is Essential for Viral Core Development

Journal of Virology ◽

10.1128/jvi.01858-09 ◽

2009 ◽

Vol 84 (1) ◽

pp. 176-187 ◽

Cited By ~ 16

Author(s):

Cristina Suárez ◽

María L. Salas ◽

Javier M. Rodríguez

Keyword(s):

Gene Expression ◽

Viral Particle ◽

Virus Assembly ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Proteolytic Processing ◽

Fever Virus ◽

Clear Correlation ◽

Late Gene Expression ◽

The Core

ABSTRACT One of the most characteristic features of African swine fever virus gene expression is its use of two polyproteins, pp220 and pp62, to produce several structural proteins that account for approximately 32% of the total protein virion mass. Equimolecular amounts of these proteins are the major components of the core shell, a thick protein layer that lies beneath the inner envelope, surrounding the viral nucleoid. Polyprotein pp220, which is located immediately underneath the internal envelope, is essential for the encapsidation of the core of the viral particle. In its absence, the infection produces essentially coreless particles. In this study we analyzed, by means of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible virus, the role of polyprotein pp62 in virus assembly. Polyprotein pp62 is indispensable for viral replication. The repression of polyprotein pp62 expression does not alter late gene expression or the proteolytic processing of the polyprotein pp220. However, it has a profound impact on the subcellular localization of polyprotein pp220. Electron microscopy studies revealed that polyprotein pp62 is necessary for the correct assembly and maturation of the core of the viral particle. Its repression leads to the appearance of a significant fraction of empty particles, to an increase in the number of immature-like particles, and to the accumulation of defective particles. Immunoelectron microscopy analysis showed a clear correlation between the amount of polyprotein pp62, the quantity of polyprotein pp220, and the state of development of the core, suggesting that the complete absence of polyprotein pp62 during morphogenesis would produce a homogenous population of empty particles.

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African Swine Fever Virus Proteinase Is Essential for Core Maturation and Infectivity

Journal of Virology ◽

10.1128/jvi.77.10.5571-5577.2003 ◽

2003 ◽

Vol 77 (10) ◽

pp. 5571-5577 ◽

Cited By ~ 23

Author(s):

Alí Alejo ◽

Germán Andrés ◽

María L. Salas

Keyword(s):

African Swine Fever Virus ◽

Sequence Similarity ◽

Cysteine Proteinase ◽

African Swine Fever ◽

Virus Production ◽

Electron Microscopic Examination ◽

Proteolytic Processing ◽

Fever Virus ◽

Core Shell ◽

Electron Microscopic

ABSTRACT African swine fever virus (ASFV) encodes two polyprotein precursors named pp220 and pp62 that are sequentially processed during viral infection, giving rise to six major structural proteins. These reside at the core shell, a matrix domain located between the endoplasmic reticulum-derived inner envelope and the DNA-containing nucleoid. Proteolytic processing of the polyprotein precursors is catalyzed by the viral proteinase pS273R, a cysteine proteinase that shares sequence similarity with the SUMO1-processing peptidases. We describe here the construction and characterization of an ASFV recombinant, vS273Ri, that inducibly expresses the ASFV proteinase. Using vS273Ri, we show that repression of proteinase expression inhibits polyprotein processing and strongly impairs infective virus production. Electron microscopic examination of vS273Ri-infected cells showed that inhibition of proteolytic processing leads to the assembly of defective icosahedral particles containing a noncentered electron-dense nucleoid surrounded by an abnormal core shell of irregular thickness. The analysis of purified extracellular defective particles revealed that they contain the unprocessed pp220 and pp62 precursors, as well as the major DNA-binding nucleoid proteins p10 and pA104R. Altogether, these results indicate that the proteolytic processing of the polyproteins is not required for their incorporation into the assembling particles nor for the incorporation of the DNA-containing nucleoid. Instead, the ASFV proteinase is involved in a late maturational step that is essential for proper core assembly and infectivity.

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

Journal of Virology ◽

10.1128/jvi.76.6.2654-2666.2002 ◽

2002 ◽

Vol 76 (6) ◽

pp. 2654-2666 ◽

Cited By ~ 42

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.

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African Swine Fever Virus Polyproteins pp220 and pp62 Assemble into the Core Shell

Journal of Virology ◽

10.1128/jvi.76.24.12473-12482.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 12473-12482 ◽

Cited By ~ 37

Author(s):

Germán Andrés ◽

Alí Alejo ◽

José Salas ◽

María L. Salas

Keyword(s):

Virus Particle ◽

Virus Assembly ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Proteolytic Processing ◽

Fever Virus ◽

Core Shell ◽

Virion Protein ◽

Protein Mass ◽

The Core

ABSTRACT African swine fever virus (ASFV), a complex enveloped DNA virus, expresses two polyprotein precursors, pp220 and pp62, which after proteolytic processing give rise to several major components of the virus particle. We have analyzed the structural role of polyprotein pp62, the precursor form of mature products p35 and p15, in virus morphogenesis. Densitometric analysis of one- and two-dimensional gels of purified virions showed that proteins p35 and p15, as well as the pp220-derived products, are present in equimolecular amounts in the virus particle. Immunoelectron microscopy revealed that the pp62-derived products localize at the core shell, a matrix-like domain placed between the DNA-containing nucleoid and the inner envelope, where the pp220-derived products are also localized. Pulse-chase experiments indicated that the processing of both polyprotein precursors is concomitant with virus assembly. Furthermore, using inducible ASFV recombinants, we show that pp62 processing requires the expression of the pp220 core precursor, whereas the processing of both precursors pp220 and pp62 is dependent on expression of the major capsid protein p72. Interestingly, when p72 expression is blocked, unprocessed pp220 and pp62 polyproteins assemble into aberrant zipper-like elements consisting of an elongated membrane-bound protein structure reminiscent of the core shell. Moreover, the two polyproteins, when coexpressed in COS cells, interact with each other to form zipper-like structures. Together, these findings indicate that the mature products derived from both polyproteins, which collectively account for about 30% of the virion protein mass, are the basic components of the core shell and that polyprotein processing represents a maturational process related to ASFV morphogenesis.

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African Swine Fever Virus IAP Homologue Inhibits Caspase Activation and Promotes Cell Survival in Mammalian Cells

Journal of Virology ◽

10.1128/jvi.75.6.2535-2543.2001 ◽

2001 ◽

Vol 75 (6) ◽

pp. 2535-2543 ◽

Cited By ~ 70

Author(s):

Marı́a L. Nogal ◽

Gonzalo González de Buitrago ◽

Clara Rodrı́guez ◽

Beatriz Cubelos ◽

Angel L. Carrascosa ◽

...

Keyword(s):

Cell Death ◽

Caspase 3 ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Vero Cells ◽

Proteolytic Processing ◽

Fever Virus ◽

Caspase Activation ◽

Apoptosis Protein ◽

Infected Cells

ABSTRACT African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.

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