Multiple human papillomavirus genes affect the adeno-associated virus life cycle

Abstract

The risk of cervical cancer, one of the most prevalent cancers in the world, is determined by two viruses. Human papillomavirus (HPV) is the main risk factor for developing cervical cancer. However, although little known, it is well substantiated that the human Parvovirus adeno-associated virus type 2 (AAV), and its encoded Rep78 protein, interacts with HPV and lowers the risk of cervical cancer. HPV also contributes to AAV inhibition by serving as a helper virus for AAV and stimulating higher AAV replication levels. Here we surveyed four HPV-16 early genes, E1, E2, E6 and E7, for their ability to increase/decrease the basal level of AAV replication in stratifying squamous epithelium (the epithelial raft culture system). It was found that the HPV-16 E1, E2 and E6 genes were able to help/enhance AAV-2 replication in epithelial raft cultures. Under these conditions, with all the HPV genes being expressed from the AAV p5 promoter, E1 appeared to have the strongest enhancing effect on AAV DNA replication (Southern blot), RNA expression (RT-PCR), protein expression (Western blot) and AAV virion production (2 plate-Southern blot). Further study of E1 mutants showed that the carboxy-half of E1, the putative helicase/ATPase domain, was the main contributor of helper activity. These data are important for understanding the HPV-AAV interaction and its effect on modifying cervical cancer risk. These data also suggest the possibility that the identified HPV helper genes may be useful in the generation of recombinant (r)AAV virions for gene therapy, as rAAV is increasing in popularity for such purposes.

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

“…But this was a mild phenotype, not a strong inhibition. This finding of inhibition of AAV transcription is in contrast to our previous studies where E1 was found to be a significant upregulator of rep and cap expression [24]. However, these earlier studies of E1 help were in a fully productive system with wt AAV (simultaneous DNA replication and virion production).…”

“…This basic work was first carried out in the laboratory in 1984 [6-9] in primary cells/animal models [13-19], and AAV is now one of the top clinical gene therapy vectors in use today [20-22]. While wt AAV will replicate in differentiating keratinocytes [23, 24] and certain cell lines (often with genotoxic stress) [25-28] in the laboratory, in tissue culture, most cell types AAV require co-infection by a helper virus, such as adenovirus (Ad), herpes simplex virus (HSV), or human papillomavirus (HPV), to allow productive AAV infection to occur [24, 29-31]. However, a recent study has shown that AAV is most often not found with associated Ad helper virus in clinical isolates, suggesting that AAV is likely a type of highly tropic (“picky”) regular autonomous parvovirus [32].…”

“…In HPV, it is known that E1, E2, and E6 provide helper functions [24, 38], and it has been found that HPV16 E1 protein binds Rep78 in vitro and affects its replication-related biochemistries [37, 39]. The HPV16 E1 gene is the origin of the binding protein for HPV’s own DNA replication, analogous to the AAV Rep78 protein, but lacks the analogous endonuclease and covalent attachment activities of the large AAV Rep proteins [40, 41].…”

“…The HPV16 E1 gene is the origin of the binding protein for HPV’s own DNA replication, analogous to the AAV Rep78 protein, but lacks the analogous endonuclease and covalent attachment activities of the large AAV Rep proteins [40, 41]. The E1 helper protein enhances both autonomous wt AAV replication in differentiating keratinocytes and wt and rAAV production in HEK293 cells, the latter in conjunction with the Ad helper gene set [24, 38]. However, it is unclear how this E1-stimulated enhancement of Rep78-DNA replication biochemistry actually takes place.…”

A Recombinant AeDNA Containing the Insect-Specific Toxin, BmK IT1, Displayed an Increasing Pathogenicity on Aedes albopictus

“…However, expression levels declined sharply when the NS1 gene was deleted from the recombinant genome, presumably because of the multiple activities that this densovirus replication initiator protein possesses. These activities include sequence-and strand-specific nicking activity, 19 ATP-dependent helicase activity, 20 and especially promoter transregulation 21,22 that influences the expression of target genes. If NS1 is preserved in its entirety, the size of the inserted sequence is limited to approximately 1,000 bases; this limit excludes most genes for insecticidal proteins.…”