Replication but not transcription of simian virus 40 DNA is dependent on nuclear domain 10

DNA viruses from several families including herpes simplex virus type 1, adenovirus type 5, and simian virus 40 (SV40), start their transcription and replication adjacent to a specific nuclear domain, ND10. We asked whether a specific viral DNA sequence determines the location of these synthetic activities at such restricted nuclear sites. Partial and overlapping SV40 sequences were introduced into a beta-galactosidase expression vector, and the beta-galactosidase transcripts were localized by in situ hybridization. Transcripts derived from control plasmids were found throughout the nucleus and at highly concentrated sites but not at ND10. SV40 genomic segments supported ND10-associated transcription only when the origin and the coding sequence for the large T antigen were present. When the large T-antigen coding sequence was eliminated but the T antigen was constitutively expressed in COS-7 cells, the viral origin was sufficient to localize transcription and replication to ND10. Deletion analysis showed that only the large T-antigen binding site II (the core origin) was required but the T antigen was needed for detectable transcription at ND10. Large T antigen expressed from plasmids without the viral core origin did not bind or localize to ND10. Blocking of DNA replication prevented the accumulation of transcripts at ND10, indicating that only sites with replicating templates accumulated transcripts. Transcription at ND10 did not enhance total protein synthesis of plasmid transcripts. These findings suggest that viral transcription at ND10 may only be a consequence of viral genomes directed to ND10 for replication. Although plasmid transcription can take place anywhere in the nucleus, T-antigen-directed replication is apparently restricted to ND10.