RESUMO
Epstein-Barr virus (EBV) and rhesus lymphocryptovirus (rLCV) are closely related gammaherpesviruses in the lymphocryptovirus subgroup that express viral microRNAs (miRNAs) during latent infection. In addition to many host mRNAs, EBV miRNAs are known to target latent viral transcripts, specifically those encoding LMP1, BHRF1, and EBNA2. The mRNA targets of rLCV miRNAs have not been investigated. Using luciferase reporter assays, photoactivatable cross-linking and immunoprecipitation (PAR-CLIP), and deep sequencing, we demonstrate that posttranscriptional regulation of LMP1 expression is a conserved function of lymphocryptovirus miRNAs. Furthermore, the mRNAs encoding the rLCV EBNA2 and BHRF1 homologs are regulated by miRNAs in rLCV-infected B cells. Homologous to sites in the EBV LMP1 and BHRF1 3'-untranslated regions (UTRs), we also identified evolutionarily conserved binding sites for the cellular miR-17/20/106 family in the LMP1 and BHRF1 3'UTRs of several primate LCVs. Finally, we investigated the functional consequences of LMP1 targeting by individual EBV BART miRNAs and show that select viral miRNAs play a role in the previously observed modulation of NF-κB activation.
Assuntos
Infecções por Vírus Epstein-Barr/virologia , Evolução Molecular , Regulação Viral da Expressão Gênica , Lymphocryptovirus/genética , MicroRNAs/genética , Doenças dos Primatas/virologia , RNA Viral/genética , Proteínas Virais/genética , Animais , Sequência de Bases , Herpesvirus Humano 4/química , Herpesvirus Humano 4/classificação , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Lymphocryptovirus/química , Lymphocryptovirus/classificação , Lymphocryptovirus/metabolismo , Macaca mulatta , MicroRNAs/química , MicroRNAs/metabolismo , Dados de Sequência Molecular , Primatas , RNA Viral/química , RNA Viral/metabolismo , Alinhamento de Sequência , Proteínas Virais/metabolismoRESUMO
Acute Epstein-Barr virus (EBV) infection is the most common cause of Infectious Mononucleosis. Nearly all adult humans harbor life-long, persistent EBV infection which can lead to development of cancers including Hodgkin Lymphoma, Burkitt Lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and lymphomas in immunosuppressed patients. BARF1 is an EBV replication-associated, secreted protein that blocks Colony Stimulating Factor 1 (CSF-1) signaling, an innate immunity pathway not targeted by any other virus species. To evaluate effects of BARF1 in acute and persistent infection, we mutated the BARF1 homologue in the EBV-related herpesvirus, or lymphocryptovirus (LCV), naturally infecting rhesus macaques to create a recombinant rhLCV incapable of blocking CSF-1 (ΔrhBARF1). Rhesus macaques orally challenged with ΔrhBARF1 had decreased viral load indicating that CSF-1 is important for acute virus infection. Surprisingly, ΔrhBARF1 was also associated with dramatically lower virus setpoints during persistent infection. Normal acute viral load and normal viral setpoints during persistent rhLCV infection could be restored by Simian/Human Immunodeficiency Virus-induced immunosuppression prior to oral inoculation with ΔrhBARF1 or infection of immunocompetent animals with a recombinant rhLCV where the rhBARF1 was repaired. These results indicate that BARF1 blockade of CSF-1 signaling is an important immune evasion strategy for efficient acute EBV infection and a significant determinant for virus setpoint during persistent EBV infection.
Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Infecções por Herpesviridae/imunologia , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Fator Estimulador de Colônias de Macrófagos/metabolismo , Proteínas Virais/metabolismo , Animais , Vírus Defeituosos/genética , Vírus Defeituosos/patogenicidade , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/virologia , Técnicas de Inativação de Genes , Infecções por Herpesviridae/virologia , Herpesvirus Humano 4/metabolismo , Imunidade Inata , Lymphocryptovirus/genética , Lymphocryptovirus/imunologia , Lymphocryptovirus/metabolismo , Macaca mulatta/metabolismo , Macaca mulatta/virologia , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologia , Carga Viral/genética , Proteínas Virais/genética , Replicação ViralRESUMO
Glycoproteins gB and gH/gL are required for entry of Epstein-Barr virus (EBV) into cells, but the role of each glycoprotein and how they function together to mediate fusion is unclear. Analysis of the functional homology of gB from the closely related primate gammaherpesvirus, rhesus lymphocryptovirus (Rh-LCV), showed that EBV gB could not complement Rh gB due to a species-specific dependence between gB and gL. To map domains of gB required for this interaction, we constructed a panel of EBV/Rh gB chimeric proteins. Analysis showed that insertion of Rh gB from residues 456 to 807 restored fusion function of EBV gB with Rh gH/gL, suggesting this region of gB is important for interaction with gH/gL. Split YFP bimolecular complementation (BiFC) provided evidence of an interaction between EBV gB and gH/gL. Together, our results suggest the importance of a gB-gH/gL interaction in EBV-mediated fusion with B cells requiring the region of EBV gB from 456 to 807.
Assuntos
Herpesvirus Humano 4/fisiologia , Lymphocryptovirus/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/química , Herpesvirus Humano 4/genética , Lymphocryptovirus/química , Lymphocryptovirus/fisiologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas do Envelope Viral/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is a large transcriptional regulator essential for EBV-mediated immortalization of B lymphocytes. We previously identified interactions between EBNA-3C and two cellular transcription factors, J kappa and Spi proteins, through which EBNA-3C regulates transcription. To better understand the contribution of these interactions to EBNA-3C function and EBV latency, we examined whether they are conserved in the homologous proteins of nonhuman primate lymphocryptoviruses (LCVs), which bear a strong genetic and biological similarity to EBV. The homologue of EBNA-3C encoded by the LCV that infects baboons (BaLCV) was found to be only 35% identical in sequence to its EBV counterpart. Of particular significance, this homology localized predominantly to the N-terminal half of the molecule, which encompasses the domains in EBNA-3C that interact with J kappa and Spi proteins. Like EBNA-3C, both BaLCV and rhesus macaque LCV (RhLCV) 3C proteins bound to J kappa and repressed transcription mediated by EBNA-2 through its interaction with J kappa. Both nonhuman primate 3C proteins were also able to activate transcription mediated by the Spi proteins in the presence of EBNA-2. Like EBNA-3C, a domain encompassing the putative basic leucine zipper motif of the BaLCV-3C protein directly interacted with both Spi-1 and Spi-B. Surprisingly, a recently identified motif in EBNA-3C that mediates repression was not identifiable in the BaLCV-3C protein. Finally, although the C terminus of BaLCV-3C bears minimal homology to EBNA-3C, it nonetheless contains a C-terminal domain rich in glutamine and proline that was able to function as a potent transcriptional activation domain, as does the C terminus of EBNA-3C. The conservation of these functional motifs despite poor overall homology among the LCV 3C proteins strongly suggests that the interactions of EBNA-3C with J kappa and Spi do indeed play significant roles in the life cycle of EBV.
