RESUMO
Viral infectivity factor (Vif) encoded by lentiviruses is essential for viral replication and escaping from antiviral activity of host defensive factors APOBEC3. Jembrana disease virus (JDV) causes an acute disease syndrome with approximately 20% case fatality rate in Bali cattle. However, the interplay mechanism between JDV Vif and Bos taurus APOBEC3 (btA3) is poorly understood. In this study, we determined that JDV Vif recruits ElonginB, ElonginC(ELOB/C), Cul2 and RBX1 but without the need of CBF-ß to form E3 ubiquitin ligase and induces the degradation of btA3 proteins. Further investigation identified BC-box (T149LQ151) motif required for ELOB/C binding, Cul2 box (Y167xxxxV/X172) and a zinc-binding motif (H95-C113-H115-C133) required for Cul2 binding in JDV Vif. The precise mechanism of JDV Vif overcoming the antiviral activity of btA3 proteins is helpful for the application of the broad spectrum antiviral drug targeting conserved functional domains of various species Vif proteins in the future.
Assuntos
Desaminases APOBEC/metabolismo , Produtos do Gene vif/metabolismo , Lentivirus Bovinos/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Elonguina/metabolismo , Produtos do Gene vif/química , Produtos do Gene vif/genética , Células HEK293 , Humanos , Lentivirus Bovinos/genética , Ligação Proteica , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
Jembrana disease virus (JDV) is an unusual bovine lentivirus that causes an acute disease syndrome with a 20% case fatality rate after a short incubation period in Bos javanicus (Bali cattle) in Indonesia. Analysis of tat mRNA transcription patterns has identified up to six differently spliced transcripts indicating that, in common with other lentiviruses, JDV uses a complex splicing pattern. RT-PCR analysis of mRNA transcripts produced during the acute phase of infection with JDV(TAB/87) revealed at least 12 differently spliced transcripts involving 9 different splice sites. A single unspliced gag/pol transcript, singly spliced vif and tmx specific transcripts and alternatively spliced env, tat and rev transcripts were identified. A 67 nucleotide putative non-coding exon was identified that shared the same splice acceptor (SA) as vif and was incorporated into alternative transcripts of tat, rev and env.
Assuntos
Doenças dos Bovinos/virologia , Infecções por Lentivirus/veterinária , Lentivirus Bovinos/genética , Lentivirus Bovinos/patogenicidade , RNA Mensageiro/metabolismo , Transcrição Gênica , Doença Aguda , Animais , Sequência de Bases , Bovinos , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Infecções por Lentivirus/virologia , Lentivirus Bovinos/classificação , Lentivirus Bovinos/metabolismo , Dados de Sequência Molecular , Sítios de Splice de RNA , Splicing de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Jembrana disease virus (JDV) is a lentivirus highly related to the bovine immunodeficiency virus (BIV). It causes an acute disease with high mortality rate within 1-2 weeks. JDV encodes the most potent Tat (JTat) of any of the lentiviruses. JTat can transactivate all LTRs and functionally substitute for HIV Tat in the viral genome and may function as a pivotal regulator in the acute pathogenesis of JDV. The goal of this paper is to study JTat internalization by cells, the mechanisms involved in internalization, and the effect of JTat on neighbouring cells. By quantification and fluorescence microscopy, we found that the internalization of extracellular EGFP-JTat fusion protein was both time and dose-dependent, but endocytosis and energy independent. We identified that arginines which were responsible for the internalization. Internalized JTat was distributed in both the nucleus and the cytoplasm, could transactivate JDV LTR and modulate cellular gene expression. Based on our findings, we propose that secretion and internalization of JTat may be a way for JDV to influence neighbouring cells and make the cellular environment more amenable to viral infection.
Assuntos
Produtos do Gene tat/fisiologia , Infecções por Lentivirus/virologia , Lentivirus Bovinos/fisiologia , Sequência de Aminoácidos , Animais , Arginina/fisiologia , Bovinos , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Endocitose , Corantes Fluorescentes , Produtos do Gene tat/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lentivirus Bovinos/metabolismo , Dados de Sequência Molecular , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Sequências Repetidas Terminais/fisiologia , Ativação TranscricionalRESUMO
The ability of BIV strain R29 to infect bovine cell lines in the presence or absence of a functional lentiviral Tat protein is described. Jembrana disease virus (JDV) Tat protein was stably expressed in MDBK cells. No viral replication could be detected in this cell line after infection with BIV R29. Transfection of MDBK cells and MDBK Tat expressing cells with BIV R29 proviral DNA established that BIV R29 could not replicate in MDBK cells. Whether viral entry into MDBK cells is also a block to BIV R29 infection of MDBK cells has yet to be established.