Location, nucleotide sequence, and regulation of the p51 late gene of the hz-1 insect virus: identification of a putative late regulatory element.

An Hz-1 insect virus (Hz-1V) late gene encoding, a predicted polypeptide of 51 kDa was isolated from a cDNA library and mapped to the HindIII-J region (40-44.6 map units) of the viral genome. The p51 gene was characterized by DNA sequence, Northern blot, and primer extension analyses. The 1,152 bp open reading frame (ORF) is transcribed as a 1.8 kb RNA between 8 and 18 h post-infection (hpi) with maximum expression at 12 hpi. Homology was not detected between the nucleotide sequence upstream of the p51 ORF and the baculovirus conserved late promoter element NTAAG. Primer extension analysis detected one major late transcription initiation site at -205 nucleotides relative to the start of the p51 ORF and seven minor late initiation sites at positions upstream of this primary site. Comparison of the upstream regulatory regions of the p51 gene and the Hz- 1V p34 late gene revealed a region of significant homology comprised of the 9 bp sequence TTATAGTAT. The primary p51 transcription initiation site and all p34 transcription initiation sites were mapped to different nucleotides within this nonanucleotide sequence. This 9 bp motif was not observed in the ORFs of these genes, and no significant homology was detected between this motif and the 5' regulatory regions of any other characterized genes. The results of our study suggest that this conserved sequence may serve an important role in the regulation of Hz-1V late genes.

Expression of a human, neutralizing monoclonal antibody specific to puumala virus G2-protein in stably-transformed insect cells.

We cloned the heavy- and light-chain antibody genes of a human X (humanxmouse) trioma secreting a neutralizing, IgG monoclonal antibody to the G2-protein of Puumala virus. The antibody genes were inserted separately into plasmid transfer vector pIEI-4 such that the genes were under control of the baculovirus immediate early gene promoter, IEI. Trichoplusia ni (TN) cells were co-transfected with these constructs and a selection plasmid containing a neomycin-resistance gene. Cloned transformants expressing the IgG monoclonal antibody were identified by ELISA of transfected TN cell culture supernatants. TN cell lines were established from four selected clones, of which one was chosen for detailed analysis. Specificity of the insect cell-expressed human antibody was determined by ELISA with Puumala virus-infected cell lysates and by immune-precipitation of radiolabeled Puumala virus proteins. The expressed IgG retained the ability to neutralize Puumala virus in plaque-reduction neutralization assays. Using competitive polymerase chain reaction methods, multiple copies of integrated heavy- and light-chain antibody genes were detected in the insect cell genome. The transformed insect cells were stable and continuously expressed biologically active IgG. We conclude that this methodology provides an alternative eukaryotic source for the generation of human antibodies.

Nucleotide sequence, temporal expression, and transcriptional mapping of the p34 late gene of the Hz-1 insect virus.

A late gene of the Hz-1 insect virus (Hz-1V) encoding a predicted polypeptide of 34 kilodaltons (kDa) was isolated from a cDNA library and mapped to the HindIII-T region (50.3 to 52.4 map units) of the viral genome. The p34 gene was characterized by DNA sequence, Northern blot, and primer extension analyses. The 765-bp open reading frame (ORF) is transcribed in the clockwise direction as a 1.2-kb RNA. Primer extension analysis detected two late transcription initiation sites at -16 and -17 nt relative to the start of the p34 ORF. Transcription initiation was observed between 4 and 18 hr postinfection (hr p.i.) with maximum expression at 12 hr p.i. No nucleotide sequence homology was detected between the regulatory region of the p34 gene and the baculovirus conserved late promoter motif NTAAG. This observation was substantiated by results obtained from an investigation of Hz-1V late gene expression using a transient expression assay system which suggested that Hz-1V late gene promoters do not resemble the baculovirus late promoter motif. This is the first molecular analysis of Hz-1V late gene expression and offers a basis by which to compare Hz-1V to other insect viruses.