RESUMEN
During its lytic cycle, Epstein-Barr virus (EBV) expresses Rta, a factor encoded by BRLF1 that activates the transcription of viral lytic genes. We found that upstream stimulating factor (USF) binds to E1, one of the five E boxes located at - 79 in the BRLF1 promoter (Rp), to activate BRLF1 transcription. Furthermore, Rta was shown to interact with USF1 in coimmunoprecipitation and glutathione S-transferase (GST)-pulldown assays, and confocal laser-scanning microscopy further confirmed that these two proteins colocalize in the nucleus. Rta was also found to bind with the E1 sequence in a biotin-labelled E1 probe, but only in the presence of USF1, suggesting that these two proteins likely form a complex on E1. We subsequently constructed p188mSZ, a reporter plasmid that contained the sequence from - 188 to +5 in Rp, within which the Sp1 site and Zta response element were mutated. In EBV-negative Akata cells cotransfected with p188mSZ and plasmids expressing USF1 and Rta, synergistic activation of Rp transcription was observed. However, after mutating the E1 sequence in p188mSZ, USF1 and Rta were no longer able to transactivate Rp, indicating that Rta autoregulates BRLF1 transcription via its interaction with USF1 on E1. This study showed that pUSF1 transfection after EBV lytic induction in P3HR1 cells increases Rta expression, indicating that USF1 activates Rta expression after the virus enters the lytic cycle. Together, these results reveal a novel mechanism by which USF interacts with Rta to promote viral lytic development, and provide additional insight into the viral-host interactions of EBV.
Asunto(s)
Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Proteínas Inmediatas-Precoces/genética , Transactivadores/genética , Transactivadores/metabolismo , Activación Transcripcional , Factores Estimuladores hacia 5'/metabolismo , Secuencia de Bases , Sitios de Unión , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/química , Herpesvirus Humano 4/metabolismo , Interacciones Huésped-Patógeno , Humanos , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Transactivadores/química , Factores Estimuladores hacia 5'/genéticaRESUMEN
The Epstein-Barr virus (EBV) capsid contains a major capsid protein, VCA; two minor capsid proteins, BDLF1 and BORF1; and a small capsid protein, BFRF3. During the lytic cycle, these capsid proteins are synthesized and imported into the host nucleus for capsid assembly. This study finds that EBV capsid proteins colocalize with promyelocytic leukemia (PML) nuclear bodies (NBs) in P3HR1 cells during the viral lytic cycle, appearing as nuclear speckles under a confocal laser scanning microscope. In a glutathione S-transferase pulldown study, we show that BORF1 interacts with PML-NBs in vitro. BORF1 also colocalizes with PML-NBs in EBV-negative Akata cells after transfection and is responsible for bringing VCA and the VCA-BFRF3 complex from the cytoplasm to PML-NBs in the nucleus. Furthermore, BDLF1 is dispersed throughout the cell when expressed alone but colocalizes with PML-NBs when BORF1 is also present in the cell. In addition, this study finds that knockdown of PML expression by short hairpin RNA does not influence the intracellular levels of capsid proteins but reduces the number of viral particles produced by P3HR1 cells. Together, these results demonstrate that BORF1 plays a critical role in bringing capsid proteins to PML-NBs, which may likely be the assembly sites of EBV capsids. The mechanisms elucidated in this study are critical to understanding the process of EBV capsid assembly. IMPORTANCE Capsid assembly is an important event during the Epstein-Barr virus (EBV) lytic cycle, as this process is required for the production of virions. In this study, confocal microscopy revealed that the EBV capsid protein BORF1 interacts with promyelocytic leukemia (PML) nuclear bodies (NBs) in the host nucleus and is responsible for transporting the other EBV capsid proteins, including VCA, BDLF1, and BFRF3, to these subnuclear locations prior to initiation of capsid assembly. This study also found that knockdown of PML expression by short hairpin RNA significantly reduces EBV capsid assembly capabilities. This enhanced understanding of capsid assembly offers potential for the development of novel antiviral strategies and therapies that can prevent the propagation and spread of EBV.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Antígenos Virales/metabolismo , Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas de Neoplasias/metabolismo , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Línea Celular Tumoral , Células HEK293 , Herpesvirus Humano 4/genética , Humanos , Leucemia Promielocítica Aguda/virología , Microscopía Confocal , Proteínas Nucleares/metabolismo , Transporte de Proteínas/genética , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
Epstein-Barr virus (EBV) expresses two immediate-early proteins, Rta and Zta, which are key transcription factors that can form a complex with MCAF1 at Zta-responsive elements (ZREs) to synergistically activate several viral lytic genes. Our previous research indicated that RanBPM interacts with Rta and enhances Rta sumoylation. Here we showed that RanBPM binds to Zta in vitro and in vivo, and acts as an intermediary protein in Rta-Zta complex formation. The Rta-RanBPM-Zta complex was observed to bind with ZREs in the transcriptional activation of key viral genes, such as BHLF1 and BHRF1, while the introduction of RanBPM short hairpin RNA (shRNA) subsequently reduced the synergistic activity of Zta and Rta. RanBPM was found to enhance Zta-dependent transcriptional activity via the inhibition of Zta sumoylation. Interestingly, Z-K12R, a sumoylation-defective mutant of Zta, demonstrated transcriptional activation capabilities that were stronger than those of Zta and apparently unaffected by RanBPM modulation. Finally, RanBPM silencing inhibited the expression of lytic proteins. Taken together, these results shed light on the mechanisms by which RanBPM regulates Zta-mediated transcriptional activation, and point to an important role for RanBPM in EBV lytic progression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Citoesqueleto/genética , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Regulación Viral de la Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Nucleares/genética , Unión Proteica , Transactivadores/genéticaRESUMEN
Autophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of Epstein-Barr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, including LC3A, LC3B, and ATG9B genes, as well as those that are involved in the regulation of autophagy, including the genes TNF, IRGM, and TRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of extracellular signal-regulated kinase (ERK) signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 small interfering RNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development.