Purified tomato spotted wilt virus particles support both genome replication and transcription in vitro

Abstract

Purified Tomato spotted wilt virus particles were shown to support either genome replication or transcription in vitro, depending on the conditions chosen. Transcriptional activity was observed only upon addition of rabbit reticulocyte lysate, indicating a dependence on translation. Under these conditions RNA molecules of subgenomic length were synthesized that hybridized to strand-specific probes for the N and NSs genes. Cloning of these transcripts demonstrated the presence of nonviral leader sequences at their 5' ends, confirming the occurrence of genuine viral transcription initiation known as "cap snatching." Sequence analyses revealed that both alpha- and beta-globin mRNA, present in the reticulocyte lysate, as well as added Alfalfa mosaic virus (AMV) RNA sequences, were utilized as cap donors. Moreover, an artificially produced N mRNA containing an AMV-derived leader was shown to be used as cap donor, indicating that resnatching of viral mRNAs takes place in vitro.