VP1, the Putative RNA-Dependent RNA Polymerase of Infectious Bursal Disease Virus, Forms Complexes with the Capsid Protein VP3, Leading to Efficient Encapsidation into Virus-Like Particles

Lombardo, Eleuterio; Maraver, Antonio; Castón, José R.; Rivera, José; Fernández-Arias, Armando; Serrano, Antonio; Carrascosa, José L.; Rodrı́guez, José F.

doi:10.1128/jvi.73.8.6973-6983.1999

Cited by 123 publications

(55 citation statements)

References 38 publications

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“…Cells, viruses, and antibodies. The IBDV Soroa strain, a virulent serotype 1 virus, was propagated in QM7 quail muscle cells (ATCC CRL-1962) as previously described (20). Human epithelial HeLa cells and avian fibroblast QM7 cells were grown at 37°C in an atmosphere of 95% air and 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Argentina) containing 10% fetal calf serum (FCS; Internegocios, Argentina).…”

Section: Methodsmentioning

confidence: 99%

The Endosomal Pathway and the Golgi Complex Are Involved in the Infectious Bursal Disease Virus Life Cycle

Delgui

Rodrı́guez

Colombo

2013

J Virol

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Infectious bursal disease virus (IBDV), a double-stranded RNA virus belonging to the Birnaviridae family, causes immunosuppression in chickens. In this study, we defined the localization of IBDV replication complexes based on colocalization analysis of VP3, the major protein component of IBDV ribonucleoproteins (RNPs). Our results indicate that VP3 localizes to vesicular structures bearing features of early and late endocytic compartments located in the juxtanuclear region. Interfering with the endocytic pathway with a dominant negative version of Rab5 after the internalization step leads to a reduction in virus titer. Triple-immunostaining studies between VP3, the viral RNA-dependent RNA polymerase VP1, and viral double-stranded RNA (dsRNA) showed a well-defined colocalization, indicating that the three critical components of the RNPs colocalize in the same structure, likely representing replication complexes. Interestingly, recombinant expressed VP3 also localizes to endosomes. Employing Golgi markers, we found that VP3-containing vesicles were closely associated with this organelle. Depolymerization of microtubules with nocodazole caused a profound change in VP3 localization, showing a punctate distribution scattered throughout the cytoplasm. However, these VP3-positive structures remained associated with Golgi ministacks. Similarly, brefeldin A (BFA) treatment led to a punctate distribution of VP3, scattered throughout the cytoplasm of infected cells. In addition, analysis of intra-and extracellular viral infective particles after BFA treatment of avian cells suggested a role for the Golgi complex in viral assembly. These results constitute the first study elucidating the localization of IBDV replication complexes (i.e., in endocytic compartments) and establishing a role for the Golgi apparatus in the assembly step of a birnavirus.

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Section: Methodsmentioning

confidence: 99%

The Endosomal Pathway and the Golgi Complex Are Involved in the Infectious Bursal Disease Virus Life Cycle

Delgui

Rodrı́guez

Colombo

2013

J Virol

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“…VP1 was considered to be involved in the generation of the cap structure in the genome 5= ends (15), while Dobos reported that VP1 lacks the enzymatic activities for generating a cap structure (16). VP3 interacts with both VP1 and the viral genomic dsRNA to assemble into ribonucleoprotein (RNP) complexes (17,18) in which VP3 significantly stimulates the RdRp activity of VP1 (19). Further, VP3 is an antiapoptotic protein that prevents the activation of the cellular dsRNA-dependent protein kinase (PKR) (20) and a competitor of MDA5 to bind IBDV genomic dsRNA to prevent the induction of beta interferon (IFN-␤) (21).…”

Section: Importancementioning

confidence: 99%

VP1 and VP3 Are Required and Sufficient for Translation Initiation of Uncapped Infectious Bursal Disease Virus Genomic Double-Stranded RNA

Wang

Zhang

et al. 2018

J Virol

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Infectious bursal disease virus (IBDV) is a bi-segmented double-strand RNA (dsRNA) virus of the family. While IBDV genomic dsRNA lacks a 5' cap, the means by which the uncapped IBDV genomic RNA is translated effectively is unknown. In this study, we describe a cap-independent pathway of translation initiation of IBDV uncapped RNA that relies on VP1 and VP3. We show that neither purified IBDV genomic dsRNA nor the uncapped viral plus-sense RNA transcripts was directly translated and rescued into infectious viruses in host cells. This defect in translation of the uncapped IBDV genomic dsRNA was rescued by-supplementation of the viral proteins VP1 and VP3, which was dependent on both the intact polymerase activity of VP1 and the dsRNA binding activity of VP3. Deletion analysis showed that both 5' - and 3' -UTRs of IBDV dsRNA were essential for the VP1/VP3-dependent translation initiation. Significantly, VP1 and VP3 could also mediate the recovery of infectious IBDV from the authentic minus-sense strand of IBDV dsRNA. Moreover, down-regulation or inhibition of the cap-binding protein eIF4E did not decrease, but rather enhanced the VP1/VP3-mediated translation of the uncapped IBDV RNA. Collectively, our findings for the first time reveal that VP1 and VP3 compensate for the deficiency of 5' cap and replace eIF4E to confer upon the uncapped IBDV RNA the ability to be translated and rescued into infectious viruses.A key point of control for virus replication is the viral translation initiation. The current study shows that the uncapped IBDV RNA cannot be translated into viral proteins directly by host translation machinery, and is thus noninfectious. Our results constitute the first direct experimental evidence that the VP1 and VP3 are required and sufficient to initiate translation of uncapped IBDV genomic RNA by acting as a substitute of cap and replacing the cap-binding protein eIF4E. Significantly, the VP1/VP3 mediate the recovery of infectious IBDV not only from the plus-sense but also from the minus-sense strand of the IBDV dsRNA. These findings provide not only new insights into the molecular mechanisms of the life cycle of IBDV, but also a new tool for an alternative strategy for the recovery of IBDV from both the plus- and the minus-sense strand of the viral genomic dsRNA.

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“…The IBDV Soroa strain, a virulent serotype 1 virus, was propagated in QM7 avian myoblasts cells (ATCC, CRL-1962) as previously described (Lombardo et al, 1999). Human epithelial HeLa and QM7 cells were grown at 37°C in an atmosphere of 95% air and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Buenos Aires, Argentina) containing 10% FCS (PAA Laboratories, Ciudad Autónoma de Buenos Aires, Argentina).…”

Section: Cells and Virusmentioning

confidence: 99%

Infectious bursal disease virusuptake involves macropinocytosis and trafficking to early endosomes in a Rab5-dependent manner

et al. 2015

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SummaryInfectious bursal disease virus (IBDV) internalization is sparsely known in terms of molecular components of the pathway involved. To describe the cell biological features of IBDV endocytosis, we employed perturbants of endocytic pathways such as pharmacological inhibitors and overexpression of dominant-negative mutants. Internalization analysis was performed quantifying infected cells by immunofluorescence and Western blot detection of the viral protein VP3 at 12 h post-infection reinforced by the analysis of the capsid protein VP2 localization after virus uptake at 1 h post-infection. We compared IBDV infection to the internalization of well-established ligands with defined endocytic pathways: transferrin, cholera-toxin subunit B and dextran. To describe virus endocytosis at the morphological level, we performed ultrastructural studies of viral internalization kinetics in control and actin dynamics-blocked cells. Our results indicate that IBDV endocytic internalization was clathrin-and dynamin-independent, and that IBDV uses macropinocytosis as the primary entry mechanism.After uptake, virus traffics to early endosomes and requires exposure to the low endocytic pH as well as a functional endocytic pathway to complete its replication cycle. Moreover, our results indicate that the GTPase Rab5 is crucial for IBDV entry supporting the participation of the early endosomal pathway in IBDV internalization and infection of susceptible cells.

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VP1, the Putative RNA-Dependent RNA Polymerase of Infectious Bursal Disease Virus, Forms Complexes with the Capsid Protein VP3, Leading to Efficient Encapsidation into Virus-Like Particles

Cited by 123 publications

References 38 publications

The Endosomal Pathway and the Golgi Complex Are Involved in the Infectious Bursal Disease Virus Life Cycle

The Endosomal Pathway and the Golgi Complex Are Involved in the Infectious Bursal Disease Virus Life Cycle

VP1 and VP3 Are Required and Sufficient for Translation Initiation of Uncapped Infectious Bursal Disease Virus Genomic Double-Stranded RNA

Infectious bursal disease virusuptake involves macropinocytosis and trafficking to early endosomes in a Rab5-dependent manner

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