The nucleolus is the site of Borna disease virus RNA transcription and replication.

Borna disease virus (BDV) is a neurotropic nonsegmented negative-strand RNA virus with limited homology to rhabdoviruses and paramyxoviruses. A distinguishing feature of BDV is that it replicates in the nucleus of infected cells. Strand-specific probes used for in situ hybridization of infected rat brain showed that there was differential localization of positive- and negative-strand RNAs within the nucleus of neurons. Within nuclei, sense-strand RNAs were preferentially localized within nucleolar regions while genomic-sense RNAs were found in both nucleolar and nonnucleolar regions. These results suggested a role for the nucleolus in BDV replication. Nucleoli isolated from persistently infected neuroblastoma cells contained both genomic and antigenomic BDV RNA species as well as an enrichment of the 39/38-kDa and gp18 BDV proteins. Since the nucleolus is the site of rRNA transcription, we examined BDV transcription in the presence of inhibitors of RNA polymerase I. Inhibition of RNA polymerase I did not affect levels of BDV transcription.

Molecular basis for the differential subcellular localization of the 38- and 39-kilodalton structural proteins of Borna disease virus.

Borna disease virus (BDV) is a nonsegmented negative-strand (NNS) RNA virus that is unusual because it replicates in the nucleus. The most abundant viral protein in infected cells is a 38/39-kDa doublet that is presumed to represent the nucleocapsid. Infectious particles also contain high levels of this protein, accounting for at least 50% of the viral proteins. The two forms of the protein differ by an additional 13 amino acids that are present at the amino terminus of the 39-kDa form and missing from the 38-kDa form. To examine whether this difference in amino acid content affects the localization of this protein in cells, the 39- and 38-kDa proteins were expressed in transfected cells. The 39-kDa form was concentrated in the nucleus, whereas the 38-kDa form was found in both the nucleus and cytoplasm. Inspection of the extra 13 amino acids present in the 39-kDa form revealed a sequence (Pro-Lys-Arg-Arg) that is very similar to the nuclear localization signals (in both sequence homology and amino-terminal location) of the VP1 proteins of simian virus 40 and polyomavirus. Primer extension analysis of total RNA from infected cells suggests that there are two mRNA species encoding the two forms of the nucleocapsid protein. In infected cells, the 39-kDa form is expressed at about twofold-higher levels than the 38-kDa form at both the RNA and protein levels. The novel nuclear localization of the 39-kDa nucleocapsid-like protein suggests that this form of the protein is targeted to the nucleus, the site for viral RNA replication, and that it may associate with genomic RNA.

Induction of protection against Borna disease by inoculation with high-dose-attenuated Borna disease virus.

Borna disease is a chronic neurological disease caused by an enveloped negative-strand RNA virus (BDV). Experimental disease can be reproduced in rats with brain homogenates derived from infected animals or with virus derived from infected cells in culture. The virus replicates in cultured cells without evidence of cytopathic effect or production of significant levels of cell-free virus. Borna disease is caused by an immunopathological response to viral infection of neural cells. To further investigate the pathogenesis of Borna disease, rats were inoculated with different doses of BDV attenuated by culture in MDCK cells. Low doses of attenuated BDV (10(2)-10(4) TCID50) resulted in typical clinical disease and severe encephalitis; however, the lag period between inoculation and disease was considerably longer than that with virulent BDV. In contrast, animals inoculated with a high dose of attenuated BDV (10(5)-10(6) TCID50) did not develop clinical disease, although a mild encephalitic response was present that did not progress beyond the mild encephalitis. Animals inoculated with a high dose of BDV developed high titers of anti-BDV antibody and were protected against virulent challenge. Protection was correlated with the rapid induction of an immune response in the animals and the lack of any biologically detectable virus in the CNS.

Partial purification and characterization of Borna disease virions released from infected neuroblastoma cells.

Borna disease is a rare but severe neurological disease of horses and sheep. Borna disease virus (BDV) has not been fully characterized because cell-free virus has not been isolated. Homogenates of infected brain are infectious both for animals and for some cell lines in culture. We report here the partial purification and characterization of cell-free BDV from the tissue culture supernatant of infected human neuroblastoma SKNSH cells. A single negative strand 10-kb RNA was detected in purified virions. Immunoprecipitation analysis of the BDV proteins in purified virions shows the presence of the 60-, 38-, 24-, and 14-kDa proteins previously identified as BDV-specific proteins in infected cells.

Molecular characterization of Borna virus RNAs.

Borna disease virus is cell-associated in infected animals. Antibodies in animals are directed against BDV proteins of 38/39, 24, and 14.5 kD. cDNA clones that encode these proteins hybridize to five mRNAs of 10.5, 3.6, 2.1, 1.4, and 0.85 kb. The 10.5, 3.6, 2.1, and 0.85 kb RNAs are 3' co-terminal; the 1.4 kb RNA is contained within the 10.5, 3.6, and 2.1 kb species but is not 3' co-terminal. A negative strand 10 kb RNA is also present in infected cells. To determine which of the large 10 kb species represents the genomic RNA, strand-specific probes were used for Northern analyses of RNA from infectious particles isolated by Freon extraction of BDV-infected rat brain. RNA purified from these particles contained both positive and negative sense 10 kb species. Treatment of particles with RNaseA before isolation of RNA resulted in detection of only negative strand species, suggesting that BDV is a negative strand RNA virus. However, the genomic organization of BDV is unlike any known negative strand RNA virus.

Genomic organization of the structural proteins of borna disease virus revealed by a cDNA clone encoding the 38-kDa protein.

Borna disease is a rare neurological disease of sheep and horses. The etiological agent, borna disease virus (BDV), has been shown to be an RNA virus but has not been characterized sufficiently to assign it to a virus family. Previous studies have shown that three BDV-specific proteins of 14, 24 and 38 to 39 kDa are found in infected animals and cell culture (Ludwig et al., 1988, Prog. Med. Virol. 35, 107-151). cDNA clones have been isolated that encode the 14- and 24-kDa proteins; using the nucleotide sequences from these clones additional cDNAs were isolated that contained a large open reading frame (ORF) corresponding to the 38-kDa protein. Monoclonal antibodies against the BDV 38- to 39-kDa protein recognized the protein product of the large ORF. The relative gene order of the three BDV proteins (5' 38, 14, and 24 kDa 3') can be deduced from cDNAs which include portions of both the 24- and 38-kDa ORFs. The abundance of these proteins in BDV-infected animals and cultured cells suggests that these proteins are structural components of the virus. Previously all BDV-specific mRNAs (10.5, 3.6, 2.1, and 0.85 kb) were thought to be organized as overlapping 3' coterminal RNAs. Oligonucleotide probes made to the nucleotide sequence of the cDNA that encodes the 38-kDa protein identified an additional BDV-specific mRNA of 1.4 kb. This 1.4-kb mRNA species partially overlaps with the 2.1-kb RNA but is not 3' coterminal.

Identification of a novel neuronal C-SRC exon expressed in human brain.

Neuronal cells are known to express at least two different forms of the C-SRC proto-oncogene as a consequence of alternative splicing events which add an 18-nucleotide exon (the NI exon) between C-SRC exons 3 and 4. Here we report that a second neuronal exon of C-SRC is also present between C-SRC exons 3 and 4. This neuronal exon (the NII exon) of C-SRC was isolated from human adult and fetal brain-derived cDNAs and contains 33 nucleotides capable of encoding 11 amino acids (Gln-Thr-Trp-Phe-Thr-Phe-Arg-Trp-Leu-Gln-Arg). The human NI exon was located approximately 390 nucleotides from the end of C-SRC exon 3, whereas the NII exon was approximately 1,000 nucleotides from the beginning of C-SRC exon 4. Analysis of human brain RNA revealed that the NII exon is utilized primarily in conjunction with the NI exon to yield transcripts capable of encoding C-SRC products possessing 17 additional amino acids. These splicing events, which occur between the NI and NII exons, are predicted to alter the sixth amino acid encoded by the NI exon from an arginine to a serine residue, producing a potentially novel phosphorylation site. Analysis of the different C-SRC RNA transcripts revealed that the level of C-SRC RNA containing both NI and NII exons is similar in adult and fetal brain tissue, whereas the level of C-SRC RNA containing only the NI exon or the nonneuronal form of C-SRC RNAs is significantly higher in fetal brain tissues. These results indicate that the expression and splicing pattern of the C-SRC gene are developmentally regulated in the human brain.

Neuron-specific splicing of C-SRC RNA in human brain.

The C-SRC, C-YES, and FYN genes encode three closely related tyrosine protein kinases that are expressed in human neural tissues. A unique form of the C-SRC gene has been demonstrated to be expressed in avian and murine brain tissues as the result of alternative splicing between the third and fourth exons. This neuronal-specific splicing event adds to the C-SRC mRNA an 18 base pair exon capable of encoding the same six amino acids in both avian and murine neural tissues. The C-YES and FYN genes share with C-SRC similar exon-intron boundaries and a high degree of amino acid sequence homology in the 3/4 exon coding region. However, potential alternative splicing of the C-YES and FYN genes in this region has not been previously investigated. In this study we have compared the expression of C-SRC, C-YES, and FYN RNAs in human lung, liver, brain, and placenta tissues and prepared cDNA clones spanning exons 3 and 4 for each of these genes from the different tissues. Sequence analysis of these cDNA clones revealed that the splicing patterns for the FYN and C-YES genes were the same among the various tissues, whereas C-SRC cDNAs isolated from brain contained 18 additional bases with the capacity to code for the same six amino acids present in the neural-specific forms of avian and murine pp60c-src.

Hyaluronidase enhances cell fusion and synthesis of viral DNA during infection with caprine arthritis encephalitis virus.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus which infects goats and causes chronic progressive arthritis after a prolonged incubation period. CAEV replicates productively in cultures of goat synovial membrane cells and causes cytopathic effects characterized by multinucleated giant cell formation. The enzyme hyaluronidase was found to accelerate this virus induced fusion of GSM cells. Hyaluronidase treatment also resulted in synthesis of increased levels of unintegrated viral DNA early after infection. However, there was no significant increase in viral RNA in the infected cells or in the amount of virus produced. These studies suggest that hyaluronidase facilitates the interaction of CAEV with the target cells. Further it suggests that only a few copies of viral DNA are required to achieve maximal levels of virus replication. Additional copies of viral DNA appear to be redundant not contributing to viral specific transcription or increased production of virus.

Nucleotide sequence and transcriptional activity of the caprine arthritis-encephalitis virus long terminal repeat.

Caprine arthritis-encephalitis virus (CAEV) and visna virus are pathogenic lentiviruses of goats and sheep which share morphologic features and sequence homology with human T-cell lymphotropic virus type III (HTLV-III), the etiologic agent of the acquired immune deficiency syndrome. The nucleotide sequence of the CAEV long terminal repeat (LTR) was determined, and it was found to be 450 base pairs long, with U3, R, and U5 regions of 287, 85, and 78 base pairs, respectively. Portions of the CAEV LTR are closely homologous to analogous regions of visna virus. The CAEV LTR is not significantly homologous with the HTLV-III LTR; however, like HTLV-III, visna virus, and equine infectious anemia virus, CAEV uses tRNA lysine as a primer for reverse transcription. The transcriptional activity of the CAEV and visna virus LTRs was measured by a chloramphenicol acetyltransferase assay, and the activity of the visna virus LTR was generally higher in a variety of uninfected cell types. Infection of cells with visna virus markedly increased gene expression directed by either the CAEV or visna virus LTR, but in contrast, infection of cells with CAEV had little effect on the activity of either LTR. The lack of trans-activation by CAEV, a virus which causes debilitating arthritis and encephalitis in goats, suggests that trans-activation may not be a general property of pathogenic lentiviruses.

Human T-cell lymphotropic virus type III shares sequence homology with a family of pathogenic lentiviruses.

The etiologic agent of the acquired immune deficiency syndrome, human T-cell lymphotropic virus type III (HTLV-III), has recently been shown to morphologically resemble and share sequence homology with visna virus, a pathogenic lentivirus. Molecular hybridization, heteroduplex mapping, and DNA sequence analyses were used to compare HTLV-III to other lentiviruses of domestic animals, including visna, caprine arthritis encephalitis, and equine infectious anemia viruses. Hybridization results showed that a substantial amount of sequence homology exists between each of these viruses and HTLV-III. In addition, a closer relationship was found between visna and caprine arthritis encephalitis viruses than for any of the other lentiviruses studied. These results, along with nucleotide and amino acid sequence comparisons, have been used in a comprehensive effort to derive a systematic relationship for lentiviruses and to provide further evidence for classifying HTLV-III with the Lentivirinae subfamily of retroviruses. This relationship predicts that similarities in biology and disease process can be expected between HTLV-III and other Lentivirinae members.

Sequence homology between cloned caprine arthritis encephalitis virus and visna virus, two neurotropic lentiviruses.

Caprine arthritis encephalitis virus (CAEV) is an exogenous, nononcogenic retrovirus which causes neurological disease and crippling arthritis in goats. A complete CAEV genome was cloned from unintegrated viral DNA in two fragments of 9.4 and 0.4 kilobases in length, respectively. The biological activity of these clones was tested by ligation of the fragments followed by transfection onto goat synovial membrane cells; infectious virus was recovered. Cloned CAEV and visna virus, a related neurotropic virus of sheep, were compared by heteroduplex and molecular hybridization analyses. These data demonstrated that the greatest overall conservation of nucleotide sequences occurred in the gag and pol gene regions and two smaller regions, sor and the putative tat gene. The region of greatest divergence occurred in the env gene and, in particular, was localized primarily in the region coding for the glycosylated outer membrane protein. These findings and the recently demonstrated genetic relationship of visna virus, CAEV, and human T-cell lymphotropic virus type III, the etiologic agent of the acquired immune deficiency syndrome, may have important implications concerning the biological properties of these related viruses for human and veterinary medicine.

Genetic variation among lentiviruses: homology between visna virus and caprine arthritis-encephalitis virus is confined to the 5' gag-pol region and a small portion of the env gene.

Visna virus of sheep and arthritis-encephalitis virus of goats are serologically related but genetically distinct retroviruses which cause slowly progressive diseases in their natural hosts. To localize homologous regions of the DNAs of these two viruses, we constructed a physical map of caprine arthritis-encephalitis virus DNA and aligned it with the viral RNA. Cloned probes of visna virus DNA were then used to localize regions of homology with the caprine arthritis-encephalitis virus DNA. These studies showed homology in the 5' region of the genome encompassing U5 and the gag and pol genes and also in a small region in the env gene. These findings correlate with biological data suggesting that the regions of the DNA which are homologous may be responsible for virus group characteristics such as the closely related virus core antigens. Regions which did not show homology such as large sections in the env gene may represent unique sequences which control highly strain-specific characteristics such as the neutralization antigen and specific cell tropisms.