The Scripps Research Institute, Department of Neuropharmacology IMM-6, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Correspondence
Juan Carlos de la Torre
juanct{at}scripps.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
BDV is an enveloped virus with a non-segmented, negative-strand (NNS) RNA genome. Its genome (ca 8·9 kb), the smallest among known NNS RNA viruses, has an organization similar to that of other mononegaviruses (de la Torre, 1994; Schneemann et al., 1995
). Six major open reading frames (ORFs) are found in the BDV genome sequence (de la Torre, 1994
; Schneemann et al., 1995
). Based on their positions in the viral genome (3'-N-p10/P-M-G-L-5'), together with their biochemical and sequence features, as well as recent functional studies using reverse genetic approaches (Kawaoka, 2004
) these polypeptides are the counterparts of the nucleoprotein (N), phosphoprotein (P) transcriptional activator, matrix (M) protein, surface glycoprotein (G) and L polymerase, respectively, found in other NNS RNA viruses (Conzelmann, 2004
; Tordo et al., 1992
). The p10 ORF starts 49 nt upstream from P within the same mRNA and p10 overlaps, but in a different frame, with the 71 N-terminal amino acids of P. Notably, BDV has the property, unique among known animal NNS RNA viruses, of a nuclear site for the replication and transcription of its genome (Briese et al., 1992
; Cubitt & de la Torre, 1994
). In addition, BDV uses a remarkable diversity of strategies, including RNA splicing, for the regulation of its genome expression (Cubitt et al., 2001
; de la Torre, 1994
; Jehle et al., 2000
; Schneemann et al., 1995
; Tomonaga et al., 2000
). Based on its distinct genetic and biological features among known NNS RNA viruses, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales.
As with other negative-strand RNA viruses, the template of the BDV polymerase is exclusively a nucleocapsid (NC) consisting of the genomic RNA tightly encapsidated by the virus N protein. This NC associated with the viral polypeptides of the polymerase complex forms a ribonucleoprotein (RNP) complex active in transcription and replication, which is also the minimum unit of infectivity (Conzelmann, 1998, 2004
; Garcia-Sastre & Palese, 1993
; Tordo et al., 1992
). Thus, generation of biologically active synthetic virus from cDNA will require trans-complementation by all viral proteins involved in virus replication and transcription. For a variety of negative-strand RNA viruses, systems have been developed which permit the encapsidation, transcription, replication and packaging of synthetic genomic RNA analogues into virus-like particles (VLPs) in cells expressing all the required viral polypeptides from plasmid (Kawaoka, 2004
). These VLPs are budded into the extracellular space and can infect new cells, where they will replicate if the required trans-acting viral proteins are also expressed. These systems have facilitated the investigation of the viral cis-acting sequences and proteins required for genome packaging, as well as maturation and budding of VLPs. Moreover, it has allowed the generation and rescue of infectious viruses entirely from cloned cDNAs for members of several different families of negative-strand RNA viruses (Conzelmann, 1998
; Garcia-Sastre & Palese, 1993
; Kawaoka, 2004
; Neumann et al., 2002
).
Recently, we (Perez et al., 2003b) and others (Schneider et al., 2003
) have documented the establishment of a reverse genetic system for intracellular reconstitution of BDV replication and transcription. Similarly to other NNS RNA viruses examined (Conzelmann, 1998
; Garcia-Sastre & Palese, 1993
; Neumann et al., 2002
), BDV L, N and P constituted the minimal viral trans-acting factors required for MG expression (Perez et al., 2003b
; Schneider et al., 2003
). Notably, of the two isoforms of the BDV N (Np40 and Np38) found in BDV-infected cells, only Np40 was competent in promoting BDV MG replication and expression (Perez et al., 2003b
; Schneider et al., 2003
). The polypetide of 10 kDa encoded by the p10 ORF and present in BDV-infected cells (Wehner et al., 1997
) was not required for RNA synthesis mediated by the BDV polymerase, but rather it exhibited a strong inhibitory effect in both RNA replication and transcription of the BDV MG (Perez et al., 2003b
; Schneider et al., 2003
). Several negative-strand RNA viruses code accessory proteins that are not strictly required for RNA synthesis mediated by the virus polymerase, but they contribute to the regulation of a variety of steps in the virus life cycle (Neumann et al., 2002
). Interestingly, the accessory C proteins of the rhabdovirus vesicular stomatitis virus (VSV) and several paramyxoviruses are encoded, as p10, by the P gene and are expressed via RNA editing or from additional ORFs overlapping the P gene (Nagai, 1999
). These C proteins have been implicated in different activities including virus assembly, virulence and viral countermeasures of the interferon induced antiviral stage, as well as regulation of RNA synthesis by the virus polymerase (Nagai, 1999
; Neumann et al., 2002
). Whether BDV p10 might have similar kind of functions remains to be determined.
Here, we have examined the requirements of viral proteins for production of BDV infectious VLPs. For most negative-strand RNA viruses, this process is assumed to depend on the interaction between the RNP core and the virus-encoded transmembrane glycoproteins (G) (Lyles et al., 1992; Mebatsion et al., 1999
). The M protein is thought to play an essential role in this interaction. Moreover, budding of rabies virus and VSV does not require strictly the presence of G, suggesting an intrinsic budding activity of the M protein (Justice et al., 1995
; Mebatsion et al., 1999
; Takada et al., 1997
). Nevertheless, G can significantly enhance budding (Mebatsion et al., 1996
; Robison & Whitt, 2000
).
Using a pseudotype approach based on a recombinant VSV in which the gene for green fluorescent protein is substituted for the VSV G protein gene (VSVG*) (Takada et al., 1997
), we have shown that BDV G is sufficient to mediate receptor recognition and cell entry (Perez et al., 2001
). Based on this observation and the evidence accumulated with other NNS RNA viruses, we hypothesized that also for BDV, the viral M and G proteins were sufficient, to direct the assembly of VLPs. To test this hypothesis, we transfected 293T cells with the minimal viral trans-acting factors (N, P and L) together with plasmids expressing M and G, as well as the BDV MG, and examined whether G-containing BDV infectious VLPs could be generated. For these studies, we used a plasmid (hPol I-MG) that allowed for intracellular synthesis of a BDV MG RNA directed by the human polymerase I present in 293T cells. To generate this construct, the previously described BDV MG (pol I-MG) (Perez et al., 2003b
) was subcloned into a plasmid containing the human RNA polymerase I promoter (Fodor et al., 1999
). We first determined transfection conditions that allowed for co-expression of M and G without significantly affecting levels of BDV MG expression. For this we transfected 293T cells (1x106/M6 well) with hPol I-MG, together with the indicated combination of plasmids expressing N (pC-N), P (pC-P), L (pC-L), p10 (pC-p10), and various amounts of plasmids expressing M and G. The pC-P construct used for these experiments only contains the P ORF and hence it cannot direct expression of p10. Sixty hours later cell extracts (CE) were prepared and assayed for CAT activity as described previously (Perez & de la Torre, 2003
). Previously, we have shown that levels of MG-derived CAT activity correlates well with levels of RNA synthesis mediated by the virus polymerase (Perez et al., 2003b
). As previously reported BDV L, N and P were sufficient for efficient BDV MG replication and expression (Fig. 1
, lane 1). Moreover, we observed that in cells co-transfected also with 0·2 µg of each plasmid expressing M and G, levels of BDV MG expression remained unaffected (Fig. 1
, lane 2). Previously, we (Perez et al., 2003b
) and others (Schneider et al., 2003
) have shown that low amounts of pC-p10 (60 ng plasmid per 1x106 cells) completely inhibited BDV MG expression. Consistent with this we observed that addition of 100 ng pC-p10 to the transfection mix caused a very strong inhibitory effect on BDV MG expression (Fig. 1
, lane 3). Notably, this inhibitory effect was released in cells co-transfected also with M and G (Fig. 1
, lane 4). The reason for this finding remains to be determined, but it would suggest that in the presence of M and G additional interactions among viral, or viral and cellular, proteins take place and prevent p10 from exerting its inhibitory effect on RNA synthesis mediated by the virus polymerase. It is worth noting that we have observed a similar situation with the arenavirus small RING finger Z protein (Lee et al., 2002
). Thus, the very powerful inhibitory of the arenavirus Z on RNA synthesis mediated by the virus polymerase was dramatically diminished in the presence of the virus surface G (Perez et al., 2003a
).
|
|
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Briese, T., de la Torre, J. C., Lewis, A., Ludwig, H. & Lipkin, W. I. (1992). Borna disease virus, a negative-strand RNA virus, transcribes in the nucleus of infected cells. Proc Natl Acad Sci U S A 89, 1148611489.
Carbone, K. M. (2001). Borna disease virus and human disease. Clin Microbiol Rev 14, 513527.
Conzelmann, K. K. (1998). Nonsegmented negative-strand RNA viruses: genetics and manipulation of viral genomes. Annu Rev Genet 32, 123162.[CrossRef][Medline]
Conzelmann, K. K. (2004). Reverse genetics of mononegavirales. Curr Top Microbiol Immunol 283, 141.[Medline]
Cubitt, B. & de la Torre, J. C. (1994). Borna disease virus (BDV), a nonsegmented RNA virus, replicates in the nuclei of infected cells where infectious BDV ribonucleoproteins are present. J Virol 68, 13711381.[Abstract]
Cubitt, B., Ly, C. & de la Torre, J. C. (2001). Identification and characterization of a new intron in Borna disease virus. J Gen Virol 82, 641646.
Danner, K. & Mayr, A. (1979). In vitro studies on Borna virus. II. Properties of the virus. Arch Virol 61, 261271.[CrossRef][Medline]
Danner, K., Heubeck, D. & Mayr, A. (1978). In vitro studies on Borna virus. I. The use of cell cultures for the demonstration, titration and production of Borna virus. Arch Virol 57, 6375.[CrossRef][Medline]
de la Torre, J. C. (1994). Molecular biology of Borna disease virus: prototype of a new group of animal viruses. J Virol 68, 76697675.[Medline]
Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G. & Garcia-Sastre, A. (1999). Rescue of influenza A virus from recombinant DNA. J Virol 73, 96799682.
Garcia-Sastre, A. & Palese, P. (1993). Genetic manipulation of negative-strand RNA virus genomes. Annu Rev Microbiol 47, 765790.[CrossRef][Medline]
Hatalski, C. G., Lewis, A. J. & Lipkin, W. I. (1997). Borna disease. Emerg Infect Dis 3, 129135.[Medline]
Ikuta, K., Hagiwara, H., Taniyama, H. & Nowotny, N. (2002). Epidemiology and infection of natural animal hosts. In Borna Disease Virus and its Role in Neurobehavioral Disease, pp. 87124. Edited by K. M. Carbone. Washington, DC: ASM Press.
Jehle, C., Lipkin, W. I., Staeheli, P., Marion, R. M. & Schwemmle, M. (2000). Authentic Borna disease virus transcripts are spliced less efficiently than cDNA-derived viral RNAs. J Gen Virol 81, 19471954.
Justice, P. A., Sun, W., Li, Y., Ye, Z., Grigera, P. R. & Wagner, R. R. (1995). Membrane vesiculation function and exocytosis of wild-type and mutant matrix proteins of vesicular stomatitis virus. J Virol 69, 31563160.[Abstract]
Kawaoka, Y. (2004). Biology of negative strand RNA viruses: the power of reverse genetics. In Current Topics in Microbiology and Immunology, 1st edn, vol. 283. Berlin: Springer.
Lee, K. J., Perez, M., Pinschewer, D. D. & de la Torre, J. C. (2002). Identification of the lymphocytic choriomeningitis virus (LCMV) proteins required to rescue LCMV RNA analogs into LCMV-like particles. J Virol 76, 63936397.
Lefrancois, L. & Lyles, D. S. (1982). The interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus. II. Monoclonal antibodies of nonneutralizing and cross-reactive epitopes of Indiana and New Jersey serotypes. Virology 121, 168174.[CrossRef][Medline]
Lyles, D. S., McKenzie, M. & Parce, J. W. (1992). Subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein. J Virol 66, 349358.[Abstract]
Mebatsion, T., Konig, M. & Conzelmann, K. K. (1996). Budding of rabies virus particles in the absence of the spike glycoprotein. Cell 84, 941951.[CrossRef][Medline]
Mebatsion, T., Weiland, F. & Conzelmann, K. K. (1999). Matrix protein of rabies virus is responsible for the assembly and budding of bullet-shaped particles and interacts with the transmembrane spike glycoprotein G. J Virol 73, 242250.
Nagai, Y. (1999). Paramyxovirus replication and pathogenesis. Reverse genetics transforms understanding. Rev Med Virol 9, 8399.[CrossRef][Medline]
Neumann, G., Whitt, M. A. & Kawaoka, Y. (2002). A decade after the generation of a negative-sense RNA virus from cloned cDNA what have we learned? J Gen Virol 83, 26352662.
Perez, M. & de la Torre, J. C. (2003). Characterization of the genomic promoter of the prototypic arenavirus lymphocytic choriomeningitis virus. J Virol 77, 11841194.[CrossRef][Medline]
Perez, M., Watanabe, M., Whitt, M. A. & de la Torre, J. C. (2001). N-terminal domain of Borna disease virus G (p56) protein is sufficient for virus receptor recognition and cell entry. J Virol 75, 70787085.
Perez, M., Craven, R. C. & de la Torre, J. C. (2003a). The small RING finger protein Z drives arenavirus budding: implications for antiviral strategies. Proc Natl Acad Sci U S A 100, 1297812983.
Perez, M., Sanchez, A., Cubitt, B., Rosario, D. & de la Torre, J. C. (2003b). A reverse genetics system for Borna disease virus. J Gen Virol 84, 30993104.
Planz, O., Bechter, K. & Schwemmle, M. (2002). Human Borna disease virus infection. In Borna Disease Virus and its Role in Neurobehavioral Disease, pp. 179226. Edited by K. M. Carbone. Washington, DC: ASM Press.
Pletnikov, M., Gonzalez-Dunia, D. & Stitz, L. (2002). Experimental infection: Pathogenesis of neurobehavioral disease. In Borna Disease Virus and its Role in Neurobehavioral Disease, pp. 125178. Edited by K. M. Carbone. Washington, DC: ASM Press.
Richt, J. A. & Rott, R. (2001). Borna disease virus: a mystery as an emerging zoonotic pathogen. Vet J 161, 2440.[CrossRef][Medline]
Richt, J. A., Pfeuffer, I., Christ, M., Frese, K., Bechter, K. & Herzog, S. (1997). Borna disease virus infection in animals and humans. Emerg Infect Dis 3, 343352.[Medline]
Robison, C. S. & Whitt, M. A. (2000). The membrane-proximal stem region of vesicular stomatitis virus G protein confers efficient virus assembly. J Virol 74, 22392246.
Rott, R. & Becht, H. (1995). Natural and experimental Borna disease in animals. In Borna Disease, pp. 1730. Edited by H. Koprowski & W. I. Lipkin. Springer.
Schneemann, A., Schneider, P. A., Lamb, R. A. & Lipkin, W. I. (1995). The remarkable coding strategy of borna disease virus: a new member of the nonsegmented negative strand RNA viruses. Virology 95, 18.[CrossRef]
Schneider, U., Naegele, M., Staeheli, P. & Schwemmle, M. (2003). Active borna disease virus polymerase complex requires a distinct nucleoprotein-to-phosphoprotein ratio but no viral X protein. J Virol 77, 1178111789.
Staeheli, P., Sauder, C., Hausmann, J., Ehrensperger, F. & Schwemmle, M. (2000). Epidemiology of Borna disease virus. J Gen Virol 81, 21232135.
Takada, A., Robison, C., Goto, H., Sanchez, A., Murti, K. G., Whitt, M. A. & Kawaoka, Y. (1997). A system for functional analysis of Ebola virus glycoprotein. Proc Natl Acad Sci U S A 94, 1476414769.
Tomonaga, K., Kobayashi, T., Lee, B.-J., Watanabe, M., Kamitani, W. & Ikuta, K. (2000). Identification of alternative splicing and negative splicing activity of a nonsegmented negative-strand RNA virus, Borna disease virus. Proc Natl Acad Sci U S A 97, 1278812793.
Tordo, N., DeHaan, P., Goldbach, R. & Poch, O. (1992). Evolution of negative-stranded RNA genomes. Semin Virol 3, 341357.
Wehner, T., Ruppert, A., Herden, C., Frese, K., Becht, H. & Richt, J. A. (1997). Detection of a novel Borna disease virus-encoded 10 kDa protein in infected cells and tissues. J Gen Virol 78, 24592466.[Abstract]
Received 29 January 2005;
accepted 10 March 2005.