The structural basis of African swine fever virus core shell protein p15 binding to DNA

ABSTRACT Programmed cell death is a tightly controlled process critical for the removal of damaged or infected cells. Pro- and antiapoptotic proteins of the Bcl-2 family are pivotal mediators of this process. African swine fever virus (ASFV) is a large DNA virus, the only member of the Asfarviridae family, and harbors A179L, a putative Bcl-2 like protein. A179L has been shown to bind to several proapoptotic Bcl-2 proteins; however, the hierarchy of binding and the structural basis for apoptosis inhibition are currently not understood. We systematically evaluated the ability of A179L to bind proapoptotic Bcl-2 family members and show that A179L is the first antiapoptotic Bcl-2 protein to bind to all major death-inducing mammalian Bcl-2 proteins. We then defined the structural basis for apoptosis inhibition of A179L by determining the crystal structures of A179L bound to both Bid and Bax BH3 motifs. Our findings provide a mechanistic understanding for the potent antiapoptotic activity of A179L by identifying it as the first panprodeath Bcl-2 binder and serve as a platform for more-detailed investigations into the role of A179L during ASFV infection. IMPORTANCE Numerous viruses have acquired strategies to subvert apoptosis by encoding proteins capable of sequestering proapoptotic host proteins. African swine fever virus (ASFV), a large DNA virus and the only member of the Asfarviridae family, encodes the protein A179L, which functions to prevent apoptosis. We show that A179L is unusual among antiapoptotic Bcl-2 proteins in being able to physically bind to all core death-inducing mammalian Bcl-2 proteins. Currently, little is known regarding the molecular interactions between A179L and the proapoptotic Bcl-2 members. Using the crystal structures of A179L bound to two of the identified proapoptotic Bcl-2 proteins, Bid and Bax, we now provide a three-dimensional (3D) view of how A179L sequesters host proapoptotic proteins, which is crucial for subverting premature host cell apoptosis.

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Crystal structure of the African swine fever virus structural protein p35 reveals its role for core shell assembly

Protein & Cell ◽

10.1007/s13238-020-00730-w ◽

2020 ◽

Vol 11 (8) ◽

pp. 600-605

Author(s):

Guobang Li ◽

Dan Fu ◽

Guangshun Zhang ◽

Dongming Zhao ◽

Mingyu Li ◽

...

Keyword(s):

Crystal Structure ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Core Shell ◽

Structural Protein

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The structural basis of African swine fever virus pA104R binding to DNA and its inhibition by stilbene derivatives

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922523117 ◽

2020 ◽

Vol 117 (20) ◽

pp. 11000-11009 ◽

Cited By ~ 4

Author(s):

Ruili Liu ◽

Yeping Sun ◽

Yan Chai ◽

Su Li ◽

Shihua Li ◽

...

Keyword(s):

Dna Binding ◽

African Swine Fever Virus ◽

Dna Bending ◽

Complex Structure ◽

African Swine Fever ◽

Fever Virus ◽

Structural Basis ◽

Bending Angle ◽

Stilbene Derivatives ◽

Dna Complex

African swine fever virus (ASFV) is a highly contagious nucleocytoplasmic large DNA virus (NCLDV) that causes nearly 100% mortality in swine. The development of effective vaccines and drugs against this virus is urgently needed. pA104R, an ASFV-derived histone-like protein, shares sequence and functional similarity with bacterial HU/IHF family members and is essential for viral replication. Herein, we solved the crystal structures of pA104R in its apo state as well as in complex with DNA. Apo-pA104R forms a homodimer and folds into an architecture conserved in bacterial heat-unstable nucleoid proteins/integration host factors (HUs/IHFs). The pA104R-DNA complex structure, however, uncovers that pA104R has a DNA binding pattern distinct from its bacterial homologs, that is, the β-ribbon arms of pA104R stabilize DNA binding by contacting the major groove instead of the minor groove. Mutations of the basic residues at the base region of the β-strand DNA binding region (BDR), rather than those in the β-ribbon arms, completely abolished DNA binding, highlighting the major role of the BDR base in DNA binding. An overall DNA bending angle of 93.8° is observed in crystal packing of the pA104R-DNA complex structure, which is close to the DNA bending angle in the HU-DNA complex. Stilbene derivatives SD1 and SD4 were shown to disrupt the binding between pA104R and DNA and inhibit the replication of ASFV in primary porcine alveolar macrophages. Collectively, these results reveal the structural basis of pA104R binding to DNA highlighting the importance of the pA104R-DNA interaction in the ASFV replication cycle and provide inhibitor leads for ASFV chemotherapy.

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Structural insight into molecular inhibitory mechanism of InsP6 on African Swine Fever Virus mRNA-decapping enzyme g5Rp

10.1101/2021.06.23.449648 ◽

2021 ◽

Author(s):

Yang Yan ◽

Changhui Zhang ◽

Li Li ◽

Xuehui Li ◽

Xin Yang ◽

...

Keyword(s):

Rna Binding ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Structural Basis ◽

Viral Gene ◽

Inhibitory Mechanism ◽

Mrna Decapping ◽

Decapping Enzyme ◽

Rna Interaction

Removal of 5' cap on cellular mRNAs by the African Swine Fever Virus (ASFV) decapping enzyme g5R protein (g5Rp) is beneficial to viral gene expression during the early stages of infection. As the only nucleoside diphosphate linked moiety X (Nudix) decapping enzyme encoded in the ASFV genome, g5Rp works in both the degradation of cellular mRNA and hydrolyzation of the diphosphoinositol polyphosphates. Here, we report the structures of dimeric g5Rp and its complex with inositol hexakisphosphate (InsP6). The two g5Rp protomers interact head to head to form a dimer, and the dimeric interface is formed by extensive polar and nonpolar interactions. Each protomer composed a unique N terminal helical domain and C terminal classic Nudix domain. As a mRNA decapping enzyme, we identified key residues, including K8, K94, K95, K98, K175, R221, and K243 located on the substrate RNA binding interfaces of g5Rp, are important to RNA binding and decapping enzyme activity. Furthermore, we identified that the g5Rp mediated mRNA decapping was inhibited by the InsP6. The g5Rp/InsP6 complex structure showed that the InsP6 molecules occupy the same regions that primarily mediate g5Rp-RNA interaction, elucidating the roles of InsP6 in the regulation of the viral decapping activity of g5Rp in mRNA degradation. Collectively, these results provide the structural basis of interaction between RNA and g5Rp and highlight the inhibitory mechanism of InsP6 on mRNA decapping by g5Rp.

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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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