Characterization and Intracellular Trafficking of Epstein-Barr Virus BBLF1, a Protein Involved in Virion Maturation

Chiu, Ya-Fang; Sugden, Bill; Chang, Pey‐Jium; Chen, Lee-Wen; Lin, Ying-Ju; Lan, Yu Ching; Lai, Chih‐Ho; Liou, Jieh-Yuan; Liu, Shih‐Tung; Hung, Chien Hui

doi:10.1128/jvi.01126-12

Cited by 41 publications

(61 citation statements)

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“…Cells were fixed with 4% paraformaldehyde and processed for indirect immunofluorescence staining, which was conducted using a primary antibody against Flag (Sigma), BBLF1 (Chiu et al, 2012), or BGLF2, following incubation with fluorescently labeled secondary antibodies (Invitrogen). Mouse anti-BGLF2 monoclonal antibody was generated using the synthesized peptide 145 KTVEELQDITPS 155 (Abmart, China).…”

Section: Immunofluorescence Stainingmentioning

confidence: 99%

Interaction Between BGLF2 and BBLF1 Is Required for the Efficient Production of Infectious Epstein–Barr Virus Particles

et al. 2020

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BGLF2 is a tegument protein of the Epstein-Barr virus (EBV). This study finds that BGLF2 is expressed in the late stage of the EBV lytic cycle. Microscopic investigations reveal that BGLF2 is present in both the nucleus and the cytoplasm and colocalized with BBLF1 and gp350 at juxtanuclear regions in the cytoplasm. This study also finds that the basic KKK 69 motif of BGLF2 and acidic DYEE 31 motif of BBLF1 are crucial for the interaction between BGLF2 and BBLF1, which is required for the recruitment of BGLF2 to the BBLF1 that is anchored on the trans-Golgi-network (TGN). In addition, BGLF2 in a density gradient is co-sedimented with un-enveloped capsids, revealing that BGLF2 associates with the EBV capsid before the final envelopment. The knockout of BGLF2 expression is demonstrated to reduce the numbers of infectious virions that are released into the culture medium, but they do not affect the expression of lytic proteins and viral DNA replication. The production of infectious viral particles by a BGLF2-knockout mutant can be rescued by exogenously expressed BGLF2 but only partially rescued by BGLF2-3KA, which is a mutant with reduced ability to interact with BBLF1 but does not affect its ability to activate the MAPK pathway and the expression of the EBV lytic proteins, suggesting that the interaction of BGLF2 with BBLF1 is important to the efficient production of infectious viral particles during the maturation. The results of this study improve our understanding of how BGLF2 promotes EBV viral production.

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Section: Immunofluorescence Stainingmentioning

confidence: 99%

Interaction Between BGLF2 and BBLF1 Is Required for the Efficient Production of Infectious Epstein–Barr Virus Particles

et al. 2020

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“…This viral mimicry (see Davey et al, 2011) enables the viruses to assemble progeny, escape immune surveillance and prevent apoptosis. For example, PACS-1 binds to acidic clusters in the human cytomegalovirus (HCMV) envelope glycoprotein gB and the Epstein-Barr virus (EBV) tegument protein BBLF1 and localizes them to the TGN to support virus assembly Chiu et al, 2012) (see Fig. 3A).…”

Section: Introductionmentioning

confidence: 99%

Caught in the act – protein adaptation and the expanding roles of the PACS proteins in tissue homeostasis and disease

Thomas

Aslan

Thomas

et al. 2017

Journal of Cell Science

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Vertebrate proteins that fulfill multiple and seemingly disparate functions are increasingly recognized as vital solutions to maintaining homeostasis in the face of the complex cell and tissue physiology of higher metazoans. However, the molecular adaptations that underpin this increased functionality remain elusive. In this Commentary, we review the PACS proteins -which first appeared in lower metazoans as protein traffic modulators and evolved in vertebrates to integrate cytoplasmic protein traffic and interorganellar communication with nuclear gene expression -as examples of protein adaptation 'caught in the act'. Vertebrate PACS-1 and PACS-2 increased their functional density and roles as metabolic switches by acquiring phosphorylation sites and nuclear trafficking signals within disordered regions of the proteins. These findings illustrate one mechanism by which vertebrates accommodate their complex cell physiology with a limited set of proteins. We will also highlight how pathogenic viruses exploit the PACS sorting pathways as well as recent studies on PACS genes with mutations or altered expression that result in diverse diseases. These discoveries suggest that investigation of the evolving PACS protein family provides a rich opportunity for insight into vertebrate cell and organ homeostasis.

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“…The majority of functional data regarding the herpesvirus tegument comes primarily from research on alpha-and betaherpesviruses, which has shown that tegument proteins function in crucial roles in viral replication, including transcytosis of the herpesvirus capsid toward the nucleus during initial infection and egress from the nucleus toward the periphery during lytic replication (12,(36)(37)(38)(39). Additional functions of tegument proteins modulate the host cell environment during the immediateearly phase of infection (40), including shutoff of host gene expression (21,41,42), antagonism of the innate antiviral host response (43)(44)(45)(46)(47), and assembly and egress of herpesvirus virions (38,(48)(49)(50); reviewed in reference 51).…”

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Maturation and Vesicle-Mediated Egress of Primate Gammaherpesvirus Rhesus Monkey Rhadinovirus Require Inner Tegument Protein ORF52

Anderson

Loftus

Kedes

2014

J Virol

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The tegument layer of herpesviruses comprises a collection of proteins that is unique to each viral species. In rhesus monkey rhadinovirus (RRV), a close relative of the human oncogenic pathogen Kaposi's sarcoma-associated herpesvirus, ORF52 is a highly abundant tegument protein tightly associated with the capsid. We now report that ORF52 knockdown during RRV infection of rhesus fibroblasts led to a greater than 300-fold reduction in the viral titer by 48 h but had little effect on the number of released particles and caused only modest reductions in the levels of intracellular viral genomic DNA and no appreciable change in viral DNA packaging into capsids. These data suggested that the lack of ORF52 resulted in the production and release of defective particles. In support of this interpretation, transmission electron microscopy (TEM) revealed that without ORF52, capsidlike particles accumulated in the cytoplasm and were unable to enter egress vesicles, where final tegumentation and envelopment normally occur. TEM also demonstrated defective particles in the medium that closely resembled the accumulating intracellular particles, having neither a full tegument nor an envelope. The disruption in tegument formation from ORF52 suppression, therefore, prevented the incorporation of ORF45, restricting its subcellular localization to the nucleus and appearing, by confocal microscopy, to inhibit particle transport toward the periphery. Ectopic expression of small interfering RNA (siRNA)-resistant ORF52 was able to partially rescue all of these phenotypic changes. In sum, our results indicate that efficient egress of maturing virions and, in agreement with studies on murine gammaherpesvirus 68 (MHV-68), complete tegumentation and secondary envelopment are dependent on intact ORF52. IMPORTANCEThe tegument, or middle layer, of herpesviruses comprises both viral and cellular proteins that play key roles in the viral life cycle. A subset of these proteins is present only within members of one of the three subfamilies (alphaherpesviruses, betaherpesviruses, or gammaherpesviruses) of Herpesviridae. In this report, we show that the gammaherpesvirus-specific tegument protein ORF52 is critical for maturation of RRV, the closest relative of Kaposi's sarcoma-associated herpesvirus (KSHV) (a human cancer-causing pathogen) that has undergone this type of analysis. Without ORF52, the nascent subviral particles are essentially stuck in maturation limbo, unable to acquire the tegument or outer (envelope) layers. This greatly attenuates infectivity. Our data, together with earlier work on a murine homolog, as well as a more distantly related human homolog, provide a more complete understanding of how early protein interactions involving virus-encoded tegument proteins are critical for virus assembly and are also, therefore, potentially attractive therapeutic targets.

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Characterization and Intracellular Trafficking of Epstein-Barr Virus BBLF1, a Protein Involved in Virion Maturation

Cited by 41 publications

References 46 publications

Interaction Between BGLF2 and BBLF1 Is Required for the Efficient Production of Infectious Epstein–Barr Virus Particles

Interaction Between BGLF2 and BBLF1 Is Required for the Efficient Production of Infectious Epstein–Barr Virus Particles

Caught in the act – protein adaptation and the expanding roles of the PACS proteins in tissue homeostasis and disease

Maturation and Vesicle-Mediated Egress of Primate Gammaherpesvirus Rhesus Monkey Rhadinovirus Require Inner Tegument Protein ORF52

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