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The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under control of the Rous sarcoma virus long terminal repeat in the episomal replicating vector pRP-RSV, which contains the origin of replication and early region of the human papovavirus BK as well as a cDNA for a mutant mouse dihydrofolate reductase that is resistant to methotrexate. gB-1 (0.15 to 0.25 pg per cell per 24 h) was constitutively secreted into the culture medium of pRP-RSV-gBs-transformed human 293 cells. Treatment of transformed cells with methotrexate at high concentrations (0.6 to 6 microM) increased gB-1 production 10- to 100-fold, because of an amplification of the episomal recombinant. Mice immunized with secreted gB-1 produced HSV-1- and HSV-2-neutralizing antibodies and were protected against HSV-1 lethal, latent, and recurrent infections. Constitutive expression of secreted gB-1 in human cells may establish a system to develop diagnostic material and a subunit vaccine for HSV infections.

Hamster kidney cells were transformed, with comparable efficiency, by circular or linear molecules of complete BK virus (BKV) genome and by agarose gel-purified fragments of BKV DNA obtained by single or double digestions with various restriction endonucleases. Only fragments containing the complete early region of BKV DNA displayed transforming activity. Analysis by blot hybridization of the arrangement of viral DNA sequences in a cloned cell line transformed by a 3.8-kilobase fragment, obtained after sequential digestion of BKV DNA with HhaI and BamHI, showed the presence of seven viral integrations into the cellular DNA. Apparently all of the integrated viral molecules contained the entire early region of BKV DNA. Large T antigen, small t antigen, and the 56,000-dalton nonviral Tau antigen were detected in transformed cells by immunoprecipitation. The pattern of integration of viral sequences in transformed cells was constant over many generations. Likewise, large T antigen was always detected in transformed cells at various passage levels. These results may suggest that all of the sequences of the early region coding for large T antigen are required for transformation by BKV. Alternatively, subgenomic segments of the BKV DNA early region may be unable to transform because the appropriate polyadenylation site, necessary to obtain a complete functional transcriptional unit, is removed by the restriction enzyme cleavage.

The early events in the interaction of simian virus 40 (SV40) with permissive cells were investigated. Evidence is presented that 30 min after infection intact virions penetrate the nuclei of infected cells. The uncoating of the virus is carried out in the nuclei with a complete dissociation of the viral genome from the protein coat. Opening of the circular parental deoxyribonucleic acid (DNA), i.e., conversion of component I to component II of SV40 DNA, takes place after uncoating, followed by the appearance of a new component sedimenting faster than component I at alkaline p H.