Effects of Adeno-Associated Virus on Adenovirus Replication and Gene Expression during Coinfection

Abstract

Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10-to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

Differential Contribution of Adeno-Associated Virus Type 2 Rep Protein Expression and Nucleic Acid Elements to Inhibition of Adenoviral Replication in cis and in trans

“…The AAV-mediated inhibition of adenoviral replication has been attributed to the expression of the Rep proteins (20,21,31), and based on these findings, the difficulties encountered in various attempts to incorporate the rep gene region into the context of replication-competent first-generation adenoviral vectors (11,12,32) were also assigned to Rep protein expression. A recent study by Sitaraman et al (22) strongly changed this prevailing picture of the rep gene-mediated inhibition of Ad/AAV hybrid vector propagation by showing the involvement of cis inhibitory effects.…”

“…Isolated Rep78 expressed under the control of heterologous promoters has been shown to inhibit early adenoviral gene expression and genome replication (20) as well as the maturation of adenoviral replication centers (21). Recently, however, synonymous codon pair recoding of Rep78 strongly challenged the solitary role of Rep protein expression in the rep gene-mediated inhibition of rAd replication by demonstrating the involvement of a cis inhibitory effect (22).…”

Computationally designed adeno-associated virus (AAV) Rep 78 is efficiently maintained within an adenovirus vector

“…Moderate success has been achieved with the use of complex homologous recombination strategies (17) and helper-dependent (18) or tightly regulated (19) expression systems, although the latter two approaches are additionally restricted by the helper-dependent nature of Rep 78-containing Ad. The mechanism of Rep 78/68-mediated Ad inhibition remains incompletely elucidated (20,21), although Rep 78 is known to inhibit Ad replication (22), and colocalizes to Ad replication centers to prevent their maturation (23). Furthermore, the complexity of these relationships is highlighted by the lack of Rep 78/68-associated Ad inhibition when delivered in trans, such as in Rep 78/68-expressing cell lines (24).…”

Ribozyme-mediated reduction of wild-type and mutant cartilage oligomeric matrix protein (COMP) mRNA and protein

“…Surprisingly, coinfection resulted in a net increase of both WT-and MT-COMP mRNA and protein. This increase in steady-state levels of COMP could be the result of interactions of gene products of the two independent adenoviruses that alter transcript processing, export, or stability or increase gene expression in unanticipated ways (Timpe et al 2006). Alternatively, Ribo56 is not able to recognize or cleave the target sequence when both are expressed from an adenovirus.…”