Inhibition of adenovirus cytotoxicity, replication, and E2a gene expression by adeno-associated virus

Adeno-associated virus (AAV) and the other parvoviruses have long been known to inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we have begun an investigation into AAV's relationship with adenovirus (Ad), AAV's most efficient helper virus. AAV, but not UV-inactivated AAV, delayed Ad-induced cytotoxicity and inhibited Ad E2a gene expression. AAV, but not UV-inactivated AAV or a recombinant AAV vector, inhibited Ad DNA replication. To determine whether AAV or its replication (Rep) proteins alter Ad early gene expression, we measured steady state E2a mRNA levels in AAV and Ad coinfected cultures and in a cell line (Neo6) that inducibly expresses the Rep proteins. AAV, but not UV-AAV, and Rep expression resulted in diminution of E2a protein and mRNA levels. To determine whether the AAV Rep proteins directly affect the individual Ad early promoters, we constructed luciferase reporter plasmids containing each of the five early promoters. Cotransfection of Ad-luciferase and an AAV rep gene-expressing plasmid in HeLa cells demonstrated that Rep78 repressed the E1a, E2a, and E4 promoters but trans-activated the E1b and E3 promoters. In the presence of a cotransfected E1a-expressing plasmid, Rep78 repressed expression from all five promoters. These results indicate that Rep may have different effects on the Ad early promoters dependent upon the presence of the E1a trans-activating protein.     

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.     

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.     

Rescue of AAV by antibody-induced Fas-mediated apoptosis from viral DNA integrated in HeLa chromosome

Adenoassociated virus (AAV) provirus in latently infected cells is rescued by superinfection with adenovirus. We examined helper-independent rescue of AAV by Fas-mediated apoptosis from the HeLa lines with AAV DNA integrated in chromosome (HeLa/AAV). In HeLa/AAV, anti-Fas antibody was found to induce low-level production of AAV virions, as detected by the presence of AAV DNA protected from DNase digestion. The antibody induced higher virion production in Fas-enriched HeLa/AAV, which was generated by transfecting HeLa/AAV with an expression plasmid for Fas, than in HeLa/AAV, and the rescued virions were shown to be infectious as assayed along with adenovirus. The antibody also induced apoptotic DNA fragmentation, as detected by staining intracellular fragmented DNA and electrophoresis, in HeLa/AAV and, more markedly, in Fas-enriched HeLa/AAV. Furthermore, an inhibitor for caspase-8 suppressed both the AAV virion production and the DNA fragmentation. Thus, the integrated AAV DNA is likely to be induced to initiate a low level of replication when Fas-mediated apoptosis is activated in HeLa cells.     

Characterization of a nuclear localization signal in the C-terminus of the adeno-associated virus Rep68/78 proteins

Adeno-associated virus (AAV) replicates in the nucleus of infected cells, and therefore multiple nuclear import events are required for productive infection. We analyzed nuclear import of the viral Rep proteins and characterized a nuclear localization signal (NLS) in the C-terminus. We demonstrate that basic residues in this region constitute an NLS that is transferable and mediates interaction with the nuclear import receptor importin alpha in vitro. Mutant Rep proteins are predominantly cytoplasmic and are severely compromised for interactions with importin alpha, but retain their enzymatic functions in vitro. Interestingly, mutations of the NLS had significantly less effect on importin alpha interaction and replication in the context of Rep78 than when incorporated into the Rep68 protein. Together, our results demonstrate that a bipartite NLS exists in the shared part of Rep68 and Rep78, and suggest that an alternate entry mechanism may also contribute to nuclear localization of the Rep78 protein.     

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.     

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.     

Rep68 protein of adeno-associated virus type 2 interacts with 14-3-3 proteins depending on phosphorylation at serine 535

Rep78/68 proteins of adeno-associated virus type 2 (AAV-2) are involved in many aspects of the viral life cycle, including replication, gene expression, and site-specific integration. To understand the molecular mechanisms of the actions of Rep proteins, we searched for Rep68-interacting cellular proteins by utilizing a one-step affinity purification technique and identified two members of 14-3-3 proteins (14-3-3 epsilon and gamma). We found that phosphorylation of 535Ser at the carboxy terminus of Rep68 was critical for its association with 14-3-3. The association of 14-3-3 proteins to Rep68 resulted in reduction of the affinity of Rep68 for DNA. Furthermore, genome DNA replication of a recombinant mutant virus carrying a phosphorylation-deficient Rep68 (Ser535Ala) was more efficient than that of the wild-type virus. These results suggest that phosphorylation of Rep68 and subsequent association with 14-3-3 proteins regulates Rep-mediated functions during the AAV life cycle.     

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.     

Adeno-associated virus type 2 nonstructural protein Rep78 suppresses translation in vitro

Adeno-associated virus type 2 nonstructural protein Rep78 [621 amino acids (aa) long] affects the expression of various cellular and viral genes. In this study we examined the effects of Rep78 on expression of the luciferase gene from the human cytomegalovirus immediate-early promoter in HeLa cells and on translation of RNA encoding luciferase in rabbit reticulocyte lysate. When Rep78 and luciferase were coexpressed, the luciferase activity decreased despite increased levels of luciferase mRNA in the cells. Purified Rep78 or Rep68 fused with Escherichia coli maltose binding protein suppressed translation of luciferase RNA in vitro, but Rep52/40 fusion proteins did not. A mutated Rep78, which is 520 aa long and truncated at its C-terminus, did suppress the in vitro translation, whereas a similarly truncated Rep78 of 420 aa did not. The results indicate that Rep78/68 function to suppress gene expression through translation inhibition, which requires the N-terminal region contained within aa 1-520.     

Induction of apoptosis by cadmium and the adeno-associated virus Rep proteins.

The parvoviruses exert antiproliferative effects on transformed cells in culture. The development of cell lines that inducibly express the parvovirus nonstructural proteins have implicated these proteins in the limitation of cell growth. To study the host cell interactions of the nonstructural proteins we have developed a human 293 cell line that expresses the adeno-associated virus (AAV) rep gene upon induction with heavy metal salts. When induced with both Zn(2+) and Cd(2+), Rep protein expression correlates with a cell cycle block in S phase (Yang, Q., Chen, F., and Trempe, J. P. (1994). J. Virology 68,7169-7177). However when induced with Cd(2+) alone, the Rep proteins are expressed and the cells are killed. Production of a nucleosomal DNA repeat pattern and degradation of poly-ADP ribose polymerase (PARP) suggest that killing occurs by apoptosis. These results demonstrate that AAV Rep protein expression in chemically stressed cells is cytotoxic due to induction of apoptotic pathways.     

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.