The Adeno-Associated Virus Type 2 Rep Protein Regulates RNA Processing via Interaction with the Transcription Template

Abstract

The adeno-associated virus type 2 (AAV) large Rep proteins can act to increase the ratio of spliced to unspliced AAV RNA when they are targeted to the transcription template via a Rep binding element. The required Rep binding site is both location and orientation independent; however, Rep enhancement decreases as the distance between the promoter and the intron of the affected transcription unit increases. Only the AAV intron and an extended polyadenylation site must remain for the AAV transcription unit to manifest responsiveness to Rep. A number of promoters, when driving the AAV capsid gene transcription unit, were responsive to targeted Rep, though to various degrees. Transactivation of transcription initiation is not sufficient for the enhancement of RNA processing, because activation of the P40 transcription unit by other activators targeted to this transcription template did not result in enhancement of the ratio of spliced to unspliced AAV RNA. These results suggest that Rep may act as a trans regulator of RNA processing by modulating such functions coupled to RNA polymerase II (RNA pol II) transcription, perhaps by affecting the composition of the transcription complex either prior to or during elongation. These results reveal another way in which gene expression can be regulated by trans-acting proteins and help explain an important feature of the parvovirus life cycle.It has become increasingly clear that various features of pre-mRNA cap formation, splicing, and polyadenylation as well as mRNA export are coupled to the transcription of these RNAs (reviewed in references 3, 11, 12, 16, 31, 36, and 40). The C-terminal domain (CTD) of RNA polymerase II (RNA pol II) has been implicated in the coordination of these events (17,26,48), and most recently, domains of the CTD which can independently stimulate pre-mRNA processing events have been identified (14). It has also been shown that the same RNA, when transcribed from different promoters, can undergo different levels of processing (10,20) and, in addition, that the function of certain SR proteins in splicing is dependent upon promoter structure (9).One way in which trans-acting transcriptional activators can affect the transcriptional activity of RNA pol II is by changing the composition or the activity of the transcription machinery binding at the promoter (28, 47). That trans-acting factors may affect RNA processing by inducing changes in the transcription machinery binding at the promoter or during elongation has so far only been suggested (16,36,40).The human parvovirus adeno-associated virus type 2 (AAV) is a single-stranded DNA virus which utilizes a helper virus (typically adenovirus or herpesvirus) in the establishment of productive infection (reviewed in references 4 and 5). The genetic map (Fig. 1A) and the temporal profile of the appearance of AAV RNA (27) have been determined. The genome of approximately 4.7 kb is flanked by palindromic inverted terminal repeats (ITRs) that are required for viral replication and site-specific integration into...

Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

“…Since most of these factors associate with mRNAs during the process of transcription and mark immature mRNAs (37), this finding was not unexpected, as viral RCs are active sites of transcription, and thus newly synthesized viral mRNAs and associated complexes are probably major components of these intranuclear domains (81). Rep proteins themselves are involved in AAV genome transcription and have been shown to affect the ratio of spliced versus unspliced AAV mRNAs (47). An increasing number of studies have suggested that various hnRNPs are involved in the replication of numerous viruses, including herpesviruses (7,23,24,33).…”

Replication of Minute Virus of Mice DNA Is Critically Dependent on Accumulated Levels of NS2

“…The MVMp and MVMi parent plasmids used in this study were the original infectious clones derived by Merchlinsky et al (16) and Gardiner and Tattersall (11), and all subsequent derivatives were constructed from these plasmids. Mutations were made using standard PCR techniques, as described previously (21), and were sequenced to ensure that they were as expected. The mutation which increased NS2 interaction with Crm1 was K96E/L103P, as described by Lopez-Bueno et al (14).…”

Characterization of the Nonstructural Proteins of the Bocavirus Minute Virus of Canines

“…Those parvoviruses with separate capsid gene promoters, like the Parvovirus MVM (23) and the Dependovirus AAV (17,24,25), do so by transcriptional transactivation of those promoters. The erythroviruses (26) have a single promoter at the left-hand end of the genome and a polyadenylation site within an intron in the center of the genome.…”