Generation and Characterization of a Recombinant Vesicular Stomatitis Virus Expressing the Glycoprotein of Borna Disease Virus

Borna disease virus (BDV) causes central nervous system (CNS) disease in a variety of vertebrate species, frequently manifested by behavioral abnormalities (31). BDV was originally identified as the causative agent of Borna disease, an often fatal immune-mediated neurological disease naturally occurring mainly in horses and sheep within regions of endemicity in central Europe (31). Current epidemiological data, however, indicate that the natural host range, prevalence, and geographic distribution of BDV are broader than originally thought (8, 16, 20, 29). Experimentally, BDV has a wide host range, from birds to rodents and nonhuman primates (8, 10, 16, 20, 29). Both host and viral factors contribute to a variable period of incubation and significant heterogeneity in the symptoms and pathology associated with infection (8, 10, 16, 20, 29). BDV is an enveloped virus with a nonsegmented negative-strand RNA genome with a characteristic mononegavirales organization (4, 34, 35). However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. The BDV genome (ca 8.9 kb) contains six major open reading frames (ORFs) in the order 3′-N-p10/P-M-G-L-5′ (4, 34, 35). These ORFs code for the virus nucleoprotein (N), phosphoprotein (P) transcriptional activator, matrix (M), surface glycoprotein (G), and RNA-dependent RNA polymerase (L) found in other mononegavirales. The p10 open reading frame (ORF) encodes a nonstructural polypeptide of 10 kDa (p10) that is present in BDV-infected cells (40). BDV is noncytolytic and highly neurotropic (10, 15, 16). Although viral RNA and proteins are readily detectable in BDV-infected cells, production of cell-free virus is conspicuously absent and cell-associated infectivity is extremely low (0.05 to 0.1 focus-forming unit [FFU]/cell). Notably, BDV propagates rapidly within the CNS and cultured cells in the absence of detectable assembled virions. These findings led to the view that BDV dissemination involves an initial cell penetration mediated by specific cellular receptors, followed by cell-to-cell spread. BDV provides an important model for the investigation of viral persistence in the CNS and associated disorders (19, 25-27, 32). Studies on BDV are contributing to the elucidation of immune-mediated pathological events involved in virally induced neurological disease, as well as the mechanisms whereby viruses induce neurodevelopmental and behavioral disturbances in the absence of the hallmarks of cytolysis and inflammation. The elucidation of the mechanisms underlying the bases of BDV persistence and its effects on brain cell functions will ultimately require a detailed understanding of the mechanisms governing BDV cell tropism within the CNS, as well as signaling pathways triggered at the cell surface through binding of the virus to the receptor. BDV G has been implicated both in cell entry by receptor-mediated endocytosis (13, 14) and in cell-to-cell propagation (1). The BDV G gene directs the synthesis of a polypeptide with a predicted molecular mass of 56 kDa, but due to its extensive glycosylation it migrates with an Mr of 84,000 to 94,000 (GPC). GPC is posttranslationally cleaved by the cellular subtilisin-like protease furin (14, 28). The C-terminus product (GP2) is readily detected in virally infected cells and migrates with an Mr of 43,000. In contrast, detection of the predicted N-terminal product (GP1) of G has been difficult due to its high content of N glycans, which shield antigenic sites recognized by antibodies (18). GPC accumulates in the endoplasmic reticulum and perinuclear region, but it is typically not detected on the plasma membrane, whereas GP2 accumulates at the cell surface (13). Intriguingly, both GPC and GP2 are associated with infectious BDV particles (13). Studies aimed at identifying BDV candidate cellular receptors have been jeopardized by the inability to grow cell-free infectious BDV to high titers. We have described the use of a pseudotype approach based on the prototypic rhabdovirus vesicular stomatitis virus (VSV) to examine the role of BDV G in virus cell entry (23). Pseudotyped viruses are competent for one single round of infection, and therefore new stocks need to be produced by trans complementation, via transfection, with the glycoprotein of interest. Replication-competent recombinant VSV (rVSV) expressing heterologous glycoproteins has been documented (17, 21) and has proved to be a far more powerful tool for investigating the role of viral glycoproteins in infection in both cell culture and whole organisms. Here we describe the generation and characterization of an rVSV (rVSVΔG*/BDVG) in which the VSV G gene was replaced by the coding region for green fluorescent protein (GFP) and the BDV G gene was incorporated as an additional ORF between the M and GFP ORFs of rVSVΔG*/BDVG. We present evidence that rVSVΔG*/BDVG recreates the tropism and mechanism of cell entry exhibited by the bona fide BDV. We also show that rVSVΔG*/BDVG is highly attenuated both in cell culture and in vivo. We discuss the implications of these findings for the identification of cellular receptors of BDV and the investigation of the effects of BDV G on the biology of virally infected cells.