Bombyx mori nucleopolyhedrovirus ORF51 encodes a budded virus envelope associated protein.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF 51 (Bm51) is a gene present in many lepidopteran NPVs, but its function is unknown. In this study, Bm51 was characterized. Transcripts of Bm51 were detected from 4.5 through 72 hour post infection (h p.i.) by RT-PCR. The corresponding protein was detected from 6 to 72 h p.i. in BmNPV-infected BmN cells by western blot analysis using a polyclonal antibody against Bm51. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm51 encodes a 23-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. The protein was temporarily called BV-E23. In addition immunofluorescence microscopy demonstrated that the protein was present within the cytoplasm and nuclei in virus-infected cells. In conclusion, the available data suggest that Bm51 is a functional ORF of BmNPV and encodes a protein expressed in the early stage of the infection cycle that is associated with the BV envelope.

Gene therapy strategies for treating Epstein-Barr virus-associated lymphomas: comparison of two different Epstein-Barr virus-based vectors.

B cell lymphomas in immunocompromised patients frequently contain the Epstein-Barr virus (EBV) genome (MacMahon et al, 1991), suggesting that gene therapy strategies that target EBV-positive cells for destruction might be useful for the therapy of such tumors. We have previously shown that stable expression of the cytosine deaminase (CD) gene in EBV-positive lymphoblastoid cell lines induces cell killing in the presence of the prodrug 5-fluorocytosine, with a substantial bystander killing effect (Rogers et al., 1996). To promote specific killing of EBV-positive tumor cells, we have constructed two different EBV-based vectors containing the cytosine deaminase gene. The first vector (OriP-CD), which contains the intact EBV oriP enhancer/replication element, replicates as an episome specifically in EBV-positive cells and likewise enhances transcription in an EBV-specific manner. The OriP-CD vector cannot be packaged or spread from cell to cell. The second vector (OriLyt-CD) contains the EBV lytic origin of replication (oriLyt), the EBV packaging sequences (located in the viral termini), the oriP enhancer element (but not the complete replication origin), and the EBV BZLF1 gene (which induces expression of the EBV proteins required for replication of oriLyt). The OriLyt-CD vector is replicated through the oriLyt origin specifically in EBV-positive cells and packaged as an EBV pseudovirion. The packaged oriLyt-CD virion can subsequently infect cells containing the EBV receptor, CD21, and initiate another round of replication in EBV-positive cells. Here we demonstrate that each of these two different EBV-based gene therapy strategies induces specific killing of EBV-positive B cells in vitro (in the presence of 5-FC). The advantages and disadvantages of each strategy are discussed.

The Epstein-Barr virus (EBV) DNA polymerase accessory protein, BMRF1, activates the essential downstream component of the EBV oriLyt.

The EBV DNA polymerase accessory protein, BMRF1, is an essential component of the viral DNA polymerase and is required for lytic EBV replication. In addition to its polymerase accessory protein function, we have recently reported that BMRF1 is a transcriptional activator, inducing expression of the essential oriLyt promoter, BHLF1. Here we have precisely mapped the BMRF1-response element in the BHLF1 promoter. We demonstrate that a region of oriLyt (the "downstream component"), previously shown to be one of two domains absolutely essential for oriLyt replication, is required for BMRF1-induced activation of the BHLF1 promoter. Furthermore, the downstream component of oriLyt is sufficient to confer BMRF1-responsiveness to a heterologous promoter. The downstream component contains Sp1 binding sites, and confers Sp1-responsiveness to a heterologous promoter. A series of plasmids containing various protions of the oriLyt downstream component were constructed and analyzed for their ability to respond to the BMRF1 versus Sp1 transactivators. Although the BMRF1-responsive region of the downstream component overlaps the Sp1-responsive element, certain oriLyt sequences required for maximal BMRF1-responsiveness were not required for maximal Sp1-responsiveness. In particular, a site-directed mutation altering the downstream component sequence GATGG (located from -588 to -592 relative to the BHLF1 transcription initiation site) did not affect Sp1-responsiveness, but reduced BMRF-1-responsiveness by 75% and abolished oriLyt replication. Although BMRF1 possesses nonspecific DNA binding activity, were unable to demonstrate specific BMRF1 binding to the downstream component of oriLyt. Our results suggest that BMRF1-induced activation of the essential downstream component of oriLyt may play an important role in oriLyt replication.

Killing Epstein-Barr virus-positive B lymphocytes by gene therapy: comparing the efficacy of cytosine deaminase and herpes simplex virus thymidine kinase.

Epstein-Barr virus (EBV)-positive lymphomas are frequent among immunosuppressed patients. We have examined the feasibility of killing EBV-immortalized B lymphocytes by gene transfer involving the use of "suicide" genes whose expression in target cells renders them susceptible to killing by a prodrug. We examined two gene/prodrug pairs: the Escherichia coli cytosine deaminase (CD) gene with the prodrug 5-fluorocytosine (5-FC), and the herpes simplex virus thymidine kinase (HSV-TK) gene with the prodrug ganciclovir. Retroviral vectors and drug selection were used to obtain CD or HSV-TK expression in cells. Both the CD/5-FC and the HSV-TK/ganciclovir combinations yielded substantial killing of EBV-immortalized B lymphocytes in vitro, although the CD/5-FC regimen had a significantly greater therapeutic margin than the HSV-TK/ganciclovir combination. The CD/5-FC pair, but not the HSV-TK/ganciclovir pair, was shown to have a "bystander killing effect" in vitro. When only 30% of the cells expressed the suicide gene, scid mouse tumors regressed in both the CD/5-FC regimen and the HSV-TK/ganciclovir regimen, documenting an in vivo bystander effect with both regimens. However, a greater percentage of tumors completely regressed with the CD/5-FC regimen. Overall, the sum of our data indicates that the CD/5-FC combination is the more promising regimen for treatment of EBV-associated lymphomas in vivo.

A simplified new method for the identification of arboviruses: virus detection using enzyme immunoassay on cultured cells.

A simple new method is reported for the identification of arbovirus isolates and the titration of arboviruses. Horseradish peroxidase conjugated Staphylococcus aureus protein A was used for the indirect staining of virus antigens grown in an Aedes albopictus cell line, C6/36 cells. Thirty-five isolates from Culex tritaeniorhynchus mosquitoes were examined by this method and 20 of these were identified as Japanese encephalitis virus (JEV). These results were confirmed by immunofluorescent assay and plaque neutralization test. Comparative titration of JEV, Murray Valley encephalitis (MVE) and Kunjin viruses showed that this method was as sensitive as the chick embryo plaque assay. Specific enzymatic reactions on these virus-infected cells began to appear before day 3 and reached the end-point on day 5 post-infection of C6/36 cells, whereas the cytopathic effect (CPE) appeared about 2 days later than the positive enzymatic reactions. Fixation with 10% formalin for 0.5-8 h did not damage the positive reaction in infected cells and did not increase the background colour of uninfected cells.