The HPV16 E1 Carboxyl Domain Provides a Helper Function for Adeno-Associated Virus Replication

Abstract

Background/Aims: Recombinant adeno-associated virus (rAAV) is now in the clinic, yet production of rAAV remains problematic. We previously determined that human papillomavirus type 16 (HPV16) E1 protein boosts rAAV yields and E1 enhances AAV Rep78’s replication-related biochemistries. Here, we deletion-mapped the helper domain within E1 to help glean its mechanism of action. Methods: Rep78-E1 interaction was analyzed by Gal4-based yeast two-hybrid (Y2H)-cDNA assay. rAAV DNA replication was studied by AAV/helper plasmid transfection into HEK293 cells and Southern blot. Gene expression analysis was made of AAV and E1 plasmid transfection, cDNA generation, and then quantitative polymerase chain reaction. NCBI protein BLAST was used for the homology analysis. Results: Gal4-Y2H- cDNA assay found in vivo Rep78-E1-binding activity across E1, but the carboxyl-third (amino acids [aa] 421–649) of E1 contained the predominant DNA replication helper domain. The amino-half of E1 (aa 1–337) inhibited transcription of rep (p5 promoter) and cap (p40, trending lower) from non-replicating helper plasmid by quantitative (q)RT-PCR. Conclusions: The aa 421–649 helper domain of HPV16 E1 includes the ATP-binding/helicase region of E1 which boosts rAAV production and has homology with the analogous region of parvovirus NS-1/Rep78 by NCBI protein BLAST, suggesting these biochemistries are responsible for the mechanism of action in E1 helper function.

The Interplay between Adeno-Associated Virus and Its Helper Viruses

“…The role of papillomaviruses, especially HPV-16, in AAV biology gained great interest in the past years after it was found that both HPV-16 and AAV can infect and reside in cervical cancer tissue and that AAV reduces the risk of developing HPV-16-induced cervical cancer [ 10 , 215 , 216 , 217 , 218 ]. A number of studies have addressed the molecular interaction between HPV-16 and AAV in coinfected cells, resulting in the identification of HPV-16 helper functions for AAV replication [ 219 , 220 , 221 , 222 , 223 ]. However, it turned out that HPV-16 may not be a complete helper virus for AAV, as AAV does not replicate very efficiently when solely HPV-16 helper factors are present.…”

“…Similarly, You et al found that the same set of HPV-16 proteins can enhance AAV replication in epithelial raft cultures [ 222 ]. The HPV-16 helper factor E1 has recently been investigated in more detail, and it has been found that its helper effect can be attributed specifically to the E1 carboxyl domain [ 221 ]. In vivo and in vitro experiments have revealed that the AAV Rep and the HPV-16 E1 proteins can directly interact and modulate the Rep ATPase activity [ 224 , 225 ].…”