Transcriptional Analysis of the Adeno-Associated Virus Integration Site

Abstract

The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate that AAVS1 is defined by a complex transcriptional environment and that the MBS85 promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectional MBS85 promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5 untranslated region of the gene and play a posttranscriptional role in the regulation of MBS85 expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.

Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells

“…While the MBS85 mRNA levels in iPS cells showed greater variation than CCR5 mRNA levels, they were, however, overall comparable to those in the HeLa-TZM-bl cell line. This implies that the MBS85 gene is transcribed at low levels in all cell lines, a finding that is in agreement with the ubiquitous expression of this gene 42 .…”

Evolution of a Species-Specific Determinant within Human CRM1 that Regulates the Post-transcriptional Phases of HIV-1 Replication

“…The nuclear pellet was washed twice in cold, low salt NB buffer, lysed in RLT buffer and spun through a Qiashredder column (Qiagen). The 32 P-labelled random primed probes for northern analyses were generated using HIV-1 NL4-3 nucleotides 8465-8892 or a β -actin PCR fragment [60] .…”

Adeno-Associated Virus Rep Represses the Human Integration Site Promoter by Two Pathways That Are Similar to Those Required for the Regulation of the Viral p5 Promoter

“…The plasmid contains the PPP1R12C promoter region from nucleotides (nt) −332 to +94 relative to the transcription start site (27). …”

“…AAV2 has the ability to site-specifically integrate its genome into a gene, PPP1R12C , that encodes a protein that is thought to be a component of the regulatory subunit of myosin light chain phosphatase ( 26 ). Analysis of the 5′ untranslated region (UTR) of this gene led to the observation that the minimal promoter region of PPP1R12C and the p5 promoter have two important cis -regulatory elements in common, namely, the RBS and YY1 sites ( 27 ). This observation, together with the fact that AAV2 establishes latency by integrating into a ubiquitously transcribed locus, provoked the question of whether AAV2 has evolved the ability to control transcription of the AAV2 integration target locus in order to aid the establishment of latency and secure viral rescue.…”