Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product.

Abstract

We previously described use of the human parvovirus, adeno-associated virus (AAV), as a vector for transient expression in mammalian cells of the gene for chloramphenicol acetyltransferase (CAT). In the AAV vector, pTS1, the CAT gene is expressed under the control of the major AAV promoter p4o. This The human parvovirus, adeno-associated virus (AAV), replicates in mammalian cells in the presence of helper functions provided by adenovirus or herpesvirus (10). When molecular clones containing the entire AAV type 2 (AAV2) genome in bacterial plasmids are transfected into mammalian cells in the presence of adenovirus, the AAV genome is excised from the plasmid and replicated to produce infectious AAV particles (38,50,52). This facilitated both the genetic analysis of AAV (27,(51)(52)(53)59) and the development of AAV as a eucaryotic expression vector (26,60,61).The AAV genome contains three transcription promoters, P5, Pi9, and P40, which yield overlapping transcripts (11). Genetic analysis showed that a major AAV reading frame (orf-2) which is accessible from P40 transcripts codes for a major portion of the AAV capsid proteins (27,59). Mutations in this region (cap-) allow normal synthesis of duplex replicating form DNA but prevent synthesis of progeny single-stranded DNA and infectious particles (27,53,59). A second major open reading frame (orf-1) in the left half of the genome is apparently accessible from either p5 or P19 transcripts and codes for one or more proteins (rep) required for AAV DNA replication (27,53,59). Mutations in this region (rep-) are DNA negative but can be complemented. Deletion of both terminal palindromes results in a cis-dominant (ori-) defect which reflects the presence of the AAV replication origins in the terminal repeat sequences (3,4,25,51,53).

Parvovirus Gene Regulation

“…To test this possibility experiments have been done in which the transient expression of CAT activity has been measured when CAT constructs under control of the SV40 early promoter have been co-transfected with AAV plasmids. Transient expression of CAT activity is also blocked by the presence of a functional AAV rep gene (M. A. Labow, L. H. Graf & K. I. Berns, unpublished observations;Tratschin et al, 1986). Therefore, it seems likely that the major inhibitory effect on permanent transformation seen when AAV plasmids are co-transfected with selectable markers on separate plasmids is also at the level of inhibition of gene expression.…”

“…It was noted earlier that Tratschin et al (1986) had found that the regulatory effects of the AAV rep gene products were different when only the Ad E 1A and E 1B products were present in the cell compared to when there was a productive Ad infection. Among the additional Ad genes required for AAV replication is E4.…”

“…As discussed above, Tratschin et al (1986) have recently reported that upon transfection of HeLa cells using CAT constructs in which the bacterial gene for CAT is under control of the AAV p40 promoter, the products of the AAV rep gene enhance CAT expression. Without Ad co-infection the effect was marginal; with Ad co-infection a significant enhancement was observed.…”

“…Attenuation as a mechanism of regulation of transcription has been observed by Aloni and his colleagues in the case of MVM (Ben-Asher & Aloni, 1984). Another example of negative self-regulation is the observation by Tratschin et al (1986) that in Ad-transformed human cells (293 cells) the AAV left side gene products appear to inhibit p40-driven gene expression in trans.…”

Adeno-associated virus type 2-mediated inhibition of human immunodeficiency virus type 1 (HIV-1) replication: involvement of p78rep/p68rep and the HIV-1 long terminal repeat

“…The terminal repeats which form well defined secondary structures serve as origins of DNA replication; the cap gene encodes the three viral capsid proteins; the rep gene encodes four non-structural proteins with Mrs of 78000, 68000, 52000 and 40000 (Mendelson et al, 1986). These proteins are involved in AAV-2 DNA replication and in the regulation of AAV-2 gene expression (Tratschin et al, 1986;Beaton et al, 1989).…”