HPV E1 up-regulates replication-related biochemistries of AAV Rep78

Human papillomavirus type 16 (HPV) E1 protein provides helper function for the adeno-associated virus type 2 (AAV) life cycle. E1 is the replication protein of HPV, analogous to AAV Rep78, but without the endonuclease/covalent attachment activity of Rep78. Previously we have shown that E1 and Rep78 interact in vitro. Here we investigated E1's effects on Rep78 interaction with AAV's inverted terminal repeat (ITR) DNA in vitro, using purified Rep78 and E1 proteins from bacteria. E1 enhanced Rep78-ITR binding, ATPase activity, Rep78-ITR-covalent linkage and Rep78-ITR-endonuclease activity (central to AAV replication). These enhancements occurred in a dose-dependent manner whenever assayed. However, overall Rep78-plus-E1 helicase activity was lower than Rep78's helicase activity. These data suggest that E1's broad-based helper function for the AAV life cycle (AAV DNA, mRNA, and protein levels are up-regulated by E1) is likely through its ability to enhance Rep78's critical replication-required biochemistries on ITR DNA.     

The adeno-associated virus Rep78 gene inhibits cellular transformation induced by bovine papillomavirus

Adeno-associated virus type 2 (AAV) is a helper dependent parvovirus which can inhibit the oncogenic and transforming potential of its helper viruses: adenoviruses (Ad) and herpesviruses. Here we have assayed AAV's ability to inhibit cellular transformation induced by bovine papillomavirus type 1 (BPV-1), a member of another family of DNA viruses. AAV was able to markedly inhibit BPV-1-induced transformation of contact-inhibited murine fibroblasts either by infection with virus particles or by DNA transfection. This inhibition was mapped to the full-length, unspliced, rep gene product, Rep78, of AAV. DNA replication by AAV or by BPV-1 was not required for inhibition of transformation to take place.     

Cloning, Expression, and Partial Purification of Rep78: An Adeno-Associated Virus Replication Protein

Using the vaccinia virus/T7-RNA polymerase transient protein expression system, the AAV Rep78 protein was expressed in mammalian cells. Rep78 protein was found localized primarily to the nucleus of cells. Maximal steady-state protein levels were reached as early as 12 hr postinfection, with no discernable increase at later time points. The Rep78 protein has been partially purified from nuclear extracts of the expression system. We have successfully used the cloned, purified Rep78 protein to complement an uninfected HeLa cell extract in an in vitro AAV DNA replication assay. Rep78-containing fractions are sufficient to make an uninfected HeLa cell extract competent for AAV DNA replication.     

Binding of the human papillomavirus type 16 E7 oncoprotein and the adeno-associated virus Rep78 major regulatory protein in vitro and in yeast and the potential for downstream effects

OBJECTIVE: Both human papillomavirus (HPV) and adeno-associated virus (AAV) are common anogenital viruses and likely co-infect the epithelium in vivo. However, whereas HPVs are positively associated with cervical cancer, AAV appears to be negatively associated. In tissue culture, AAV-encoded Rep78--which is essential for AAV--inhibits gene expression and oncogenic transformation by HPV-16/18 and bovine papillomavirus type 1. Here we observed whether the HPV-16 E7 oncoprotein might recognize and bind Rep78. Further, upon finding Rep78-E7 binding, we investigated some of the potential downstream effects such an interaction might have. E7 is capable of recognizing a variety of proteins, including RB105, TATA box-binding protein (TBP), TBP-associated factor (TAF)(II)110, E2F, cyclins A and D, and c-jun. Some of these interactions are likely responsible for E7's cancer-promoting activity. STUDY DESIGN/METHODS: Rep78-E7 interaction was investigated in vitro by West(far)-Western and affinity chromatography analysis and in vivo in living yeast by the GAL4 two-hybrid cDNA assay. Mapping of the E7 binding domain within Rep78 was carried out using a series of amino- and carboxy-truncated Rep78 proteins in a West(far)-Western assay. Downstream effects of the interaction were analyzed by competitive affinity chromatography (protein-protein) and competitive electrophoretic mobility shift assay (protein-DNA). RESULTS: E7 and Rep78 were found to interact both in vitro and in vivo (yeast) in all assays attempted. The E7 binding domain within Rep78 was found to reside within amino acids 121-370. Regarding downstream effects of this interaction, Rep78 was found to mildly inhibit E7-TAF(II)110 and E7-RB105 interaction in vitro but to have little affect on E7-TBP interaction. Finally, it was found that E7 was able to affect Rep78's interaction with AAV's terminal repeat (TR) DNA in vitro, reducing the formation of the largest sized Rep78-TR complexes in a dosage-dependent manner. CONCLUSIONS: These data suggest that the Rep78-E7 interaction may have repercussions for both viruses. The Rep78-E7 interaction may be a second mechanism, in addition to Rep78 regulation of the p97 promoter, by which AAV inhibits HPV-16 oncogenic transformation. These data also suggest that HPV-16 may affect the AAV life cycle by altering Rep78-TR interaction.     

Inhibition of bovine papillomavirus plasmid DNA replication by adeno-associated virus.

The helper-dependent human parvovirus adeno-associated virus type 2 (AAV) inhibits both the oncogenic transforming abilities and the DNA replication of its helper viruses, adenovirus (Ad), and herpes simplex virus (HSV). As AAV-2 also inhibits the transforming ability of bovine papillomavirus type 1 (BPV), AAV-2 was assayed for its ability to inhibit BPV plasmid DNA replication. Here we find that the AAV-2 Rep78 gene is able to trans-inhibit BPV plasmid DNA replication and that the AAV-2 terminal repeats (TR) are also cis-required for the full inhibitory effect of AAV-2. When both the AAV-2 Rep78 open reading frame and TRs are present the inhibition of BPV plasmid DNA replication is very strong.     

Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors

We have developed a simple protocol to transfect mammalian cells using linear polyethylenimine (PEI). Our linear PEI protocol is as effective as commercial reagents in the transfection of HeLa cells and XDC293 cells, a derivative of HEK293 cells, but at a fraction of the cost. Greater than 90% of XDC293 cells and 98% of HeLa cells transfected using our method were positive for EGFP expression as determined by flow cytometery. Our protocol should be useful for many different applications such as large-scale production of recombinant protein and viruses, which requires transient transfection of mammalian cells in large batches. We have used this protocol to produce recombinant adeno-associated virus (AAV) in XDC293 cells and in HeLa cells. This requires transient expression of three adenovirus gene-products (E2A, E4orf6, and VA RNAs) as well as the AAV replication (Rep78, Rep68, Rep52, and Rep40) and capsid (VP1, VP2, and VP3) proteins. Production of a recombinant AAV that expresses green fluorescent protein was assessed by quantitative PCR and by transduction of HeLa cells. Linear PEI is a better transfection reagent than calcium phosphate for the production of recombinant AAV in both HEK293 and HeLa cells. In addition, when both HeLa and XDC293 cells were by our method, HeLa cells in the absence of E1A generated three-fold more recombinant AAV than XDC293 cells, which constitutively express E1A.     

SUMO-1 modification regulates the protein stability of the large regulatory protein Rep78 of adeno associated virus type 2 (AAV-2)

The large Rep proteins Rep78 and Rep68 of the helper-dependent adeno associated virus type 2 (AAV-2) are essential for both site-specific integration of AAV DNA in the absence of helpervirus and productive AAV replication in the presence of helpervirus. We have identified UBC9, the E2 conjugating enzyme for the small ubiquitin-related polypeptide SUMO-1, as binding partner of the large Rep proteins in yeast two-hybrid analysis and in GST pulldown assays. Modification of the large Rep proteins with SUMO-1 could be demonstrated in immunoblot analysis and in immunoprecipitations, with the lysine residue at amino acid position 84 serving as the major attachment site. The largely sumolation-deficient Rep78 lysine to arginine point mutant showed a strongly reduced half-life as compared to the wild-type protein. This finding implicates a role for sumolation in the regulation of Rep78 protein stability that is assumed to be critical for the establishment and maintenance of AAV latency.     

Adeno-associated virus DNA replication is induced by genes that are essential for HSV-1 DNA synthesis

Adeno-associated virus (AAV) DNA replication is not detectable unless cells are coinfected with a helper adenovirus (Ad) or herpesvirus or unless AAV infection is carried out in certain established cell lines that have been treated with various metabolic inhibitors or uv irradiation. In helper-dependent infections, it has been shown that AAV DNA synthesis depends on one or more early Ad genes, whereas little is known concerning any herpesvirus gene that promotes AAV DNA synthesis. In this study we tested the ability of four cloned Xbal fragments of herpes simplex virus type 1 (HSV-1) DNA to induce AAV DNA synthesis in Vero cells. Cotransfections, which were carried out with pAV1 (an infectious AAV2 plasmid), revealed that AAV DNA synthesis could be optimally induced by three of these clones (C,D, and F) plus a clone of the HSV-1 ICP4 (IE 175) gene. ICP4, an immediate early gene, was presumably required to activate expression of other HSV genes. To help identify the additionally needed HSV genes, we tested Xbal C,D, and F subclones that contain genes previously found necessary for origin-dependent HSV DNA synthesis and found that at least five of these genes (UL 5, 8, 9, 29, and 30) contributed to the induction of AAV DNA synthesis. In contrast to their absolute requirement for HSV DNA synthesis, none of these genes were strictly necessary for AAV DNA replication. Because they are all known to specify proteins that are directly involved in HSV DNA synthesis, our results suggest that some or all of their products also may directly participate in the replication of AAV DNA.     

Adeno-associated virus induces apoptosis during coinfection with adenovirus

Adeno-associated virus (AAV) is a nonpathogenic parvovirus that efficiently replicates in the presence of adenovirus (Ad). Exogenous expression of the AAV replication proteins induces caspase-dependent apoptosis, but determining if AAV infection causes apoptosis during viral infection is complicated by Ad-mediated programmed cell death. To eliminate Ad-induced cytolysis, we used an E3 adenoviral death protein (ADP) mutant, pm534. AAV and pm534-coinfected cells exhibited increased cell killing compared to pm534 alone. Relative to cells infected with Ad alone, AAV and wild-type Ad-infected cells displayed decreased ADP expression, increased cytolysis until the third day of the infection, and decreased cytolysis thereafter. Biochemical and morphological characteristics of apoptosis were observed during coinfections with AAV and pm534 or Ad, including a moderate degree of caspase activation that was not present during infections with pm534 or Ad alone. AAV coinfection also increased extracellular pH. These studies suggest that AAV induces caspase-dependent and caspase-independent apoptosis.     

Adenovirus early region 1b gene function required for rescue of latent adeno-associated virus

Various early and late temperature-sensitive mutants of adenovirus (Ad) were able to support adeno-associated virus (AAV) DNA replication in either coinfections or in the rescue of AAV from latently infected human (Detroit 6) cells. Group I Ad host range mutants (hr 3, early region 1a) were unable to help AAV DNA replication in either case. Group II Ad host range mutants (hr 6, early region 1b), however, were able to help AAV DNA replication in coinfection but were unable to rescue AAV DNA replication in latently infected Detroit 6 cells. Thus, an Ad function(s), in addition to those required for AAV DNA replication, is needed for AAV rescue.     

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

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.