Summary auto-generated
Borna disease virus (BDV) is an enveloped negative-strand RNA virus that uniquely replicates in the cell nucleus and is the prototypic member of the Bornaviridae family. This study identified the viral proteins required to generate infectious BDV-like particles (VLPs) using a reverse genetics approach. Researchers co-expressed individual BDV proteins in cells containing a BDV RNA mimic and monitored VLP formation and infectivity through a reporter gene assay. The results demonstrated that the BDV L, N, and P proteins are minimal requirements for RNA synthesis, while both the matrix protein (M) and surface glycoprotein (G) are necessary for efficient VLP assembly and release. The M protein appears essential for particle assembly, while G mediates receptor recognition and cell entry, as confirmed by neutralizing antibody blocking studies. Treatment with non-ionic detergent NP40 prevented VLP infectivity, confirming the enveloped nature of the particles. These findings establish a foundation for studying BDV molecular biology and provide the basis for future rescue of infectious BDV from cloned DNA.
Key findings
- BDV L, N, and P proteins constitute the minimal viral factors required for BDV RNA synthesis and replication
- Both M and G proteins are essential for formation of infectious BDV-like particles
- BDV glycoprotein (G) mediates infectivity through receptor recognition and cell entry, as demonstrated by neutralizing antibody inhibition
- M protein plays a critical role in particle assembly independent of its requirement for RNA replication
- The reverse genetics system enables investigation of viral protein interactions and provides a foundation for rescuing infectious BDV from plasmid DNA
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Abstract
Borna disease virus (BDV) is an enveloped virus with a non-segmented, negative-strand RNA genome that has an organization characteristic of Mononegavirales. However, based on its unique genetics and biological features BDV is considered to be the prototypic member of a new virus family, Bornaviridae. Here, the use of a reverse genetic approach to identify the viral proteins required for packaging of BDV RNA analogues (MG) into infectious virus-like particles (VLPs) was described. Plasmids encoding individual BDV proteins under the control of a RNA polymerase II promoter were co-transfected with a plasmid that allows for intracellular synthesis of a BDV MG mediated by the cellular RNA polymerase I. Clarified lysates from transfected cells were passaged onto fresh cells that were previously transfected with plasmids expressing the minimal BDV trans-acting factors L, N and P required for RNA synthesis mediated by the BDV polymerase. Reconstitution of BDV MG-specific packaging and passage of infectious VLP was monitored by expression of the chloramphenicol acetyl transferase reporter gene present in the BDV MG. BDV M and G, in addition to L, N and P, were sufficient for the passage of chloramphenicol acetyl transferase activity, which could be blocked by BDV neutralizing antibodies to G, indicating that VLP infectivity was fully mediated by BDV G. Passage of BDV MG was abrogated by omission of either M or G.