Summary auto-generated
This study characterized a previously unknown 2.4 kilobase DNA segment from pseudorabies virus (PrV), a herpesvirus that causes disease in pigs. The researchers sequenced this genomic region located between genes encoding viral proteins ICP4 and ICP22 homologues. Northern blot analysis identified a 1.8 kilobase messenger RNA transcript that undergoes RNA splicing (removal of two introns) before translation. The transcript represents the mRNA for the ICP22 homologue protein. Most importantly, the researchers identified functional elements for viral DNA replication within this sequence: three copies of the origin-binding protein recognition motif GTTCGCAC, each separated by AT-rich sequences. In vitro replication assays confirmed this region functions as an active origin of viral DNA replication (OriS). The findings demonstrate that PrV has a structurally distinct organization compared to other alphaherpesvirus family members, specifically possessing three OriS elements in its inverted repeat sequences plus two additional replication origins elsewhere in the genome. This unique arrangement may contribute to PrV's rapid replication and broad host range compared to related herpesviruses.
Key findings
- Sequencing closed a genomic gap in pseudorabies virus by characterizing a 2.4 kb DNA fragment containing regulatory and transcriptional elements
- Identified a spliced 1.8 kb immediate-early mRNA transcript encoding the viral ICP22 homologue protein, similar to other alphaherpesviruses
- Discovered a functional origin of viral DNA replication (OriS) containing three directly repeated copies of the origin-binding protein recognition sequence GTTCGCAC, an unusually high copy number compared to related herpesviruses
- In vitro replication assays confirmed the cloned fragment contains functional replication origin activity when expressed in virus-infected cells
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Abstract
The DNA sequence of a 2·4 kbp fragment located in the internal and terminal inverted repeat sequences of the pseudorabies virus genome determined in this study closes a gap between the previously described genes for the ICP4 and ICP22 homologues. The novel sequence contains no conserved herpesvirus open reading frames. Northern blot and cDNA analyses revealed a viral immediate-early transcript of 1·8 kb, which is spliced by the removal of two small introns close to its 5' end and which presumably represents the mRNA of the downstream open reading frame encoding the ICP22 homologue. Upstream of the transcribed region, an imperfect set of three directly repeated sequences was identified. Each of them contains a complementary pair of the alphaherpesvirus origin-binding protein recognition motif GTTCGCAC, spaced by AT-rich sequences. In vitro studies confirmed that the DNA fragment analysed includes a functional origin of viral DNA replication.