Cellular proteins required for adeno-associated virus DNA replication in the absence of adenovirus coinfection.

We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3' primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase alpha-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase delta and epsilon, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase delta or polymerase epsilon for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.

In vitro replication of adeno-associated virus DNA.

The study of eukaryotic viral DNA replication in vitro has led to the identification of cellular enzymes involved in DNA replication. Adeno-associated virus (AAV) is distinct from previously reported systems in that it is believed to replicate entirely by leading-strand DNA synthesis and requires coinfection with adenovirus to establish completely permissive replication. In previous work, we demonstrated that two of the AAV nonstructural proteins, Rep78 and -68, are site-specific endonucleases and DNA helicases that are capable of resolving covalently closed AAV termini, a key step in AAV DNA replication. We have now cloned the AAV nonstructural proteins Rep78, Rep68, and Rep52 in the baculovirus expression system. Using the baculovirus-expressed proteins, we have developed an efficient in vitro AAV DNA replication system which mimics the in vivo behavior of AAV in every respect. With no-end AAV DNA as the starting substrate, the reaction required an adenovirus-infected cell extract and the presence of either Rep78 or Rep68. Rep52, as expected, did not support DNA replication. A mutant in the AAV terminal resolution site (trs) was defective for DNA replication in the in vitro assay. Little, if any, product was formed in the absence of the adenovirus-infected HeLa cell extract. In general, uninfected HeLa extracts were less efficient in supporting AAV DNA replication than adenovirus-infected extracts. Thus, the requirement for adenovirus infection in vivo was partially duplicated in vitro. The reduced ability of uninfected HeLa extracts to support complete DNA replication was not due to a defect in terminal resolution but rather to a defect in the reinitiation reaction or in elongation. Rep78 produced a characteristic monomer-dimer pattern of replicative intermediates, but surprisingly, Rep68 produced little, if any, dimer replicative form. The reaction had a significant lag (30 min) before incorporation of 32P-deoxynucleoside triphosphate could be detected in DpnI-resistant monomer replicative form and was linear for at least 4 h after the lag. The rate of incorporation in the reaction was comparable to that in the simian virus 40 in vitro system. Replication of the complete AAV DNA molecule was demonstrated by the following criteria. (i) Most of the monomer and dimer product DNAs were completely resistant to digestion with DpnI. (ii) Virtually all of the starting substrate was converted to heavy-light or heavy-heavy product DNA in the presence of bromo-dUTP when examined on CsCl density gradients.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of mutations in adeno-associated virus Rep protein in vivo and in vitro.

The adeno-associated virus (AAV) Rep protein is required for both viral DNA replication and transactivation of the AAV promoters. Here we report the effects of mutations in the rep gene on transcription and replication in vivo and terminal repeat binding and terminal resolution site (trs) endonuclease activities in vitro. In all, we examined 10 in-frame deletions and 14 amino acid substitution mutations at eight positions. The point mutations were targeted to regions that are highly conserved among the parvovirus nonstructural proteins and include the extended ATPase domain of the AAV Rep protein. The mutations identify at least two noncontiguous regions of Rep which are essential for terminal repeat binding (amino acids 134 to 242 and amino acids 415 to 490). Mutations in either region render the protein inactive for both DNA replication and transactivation. In addition, mutations within a putative ATPase region also cause defects in replication and transactivation in vivo as well as in the ATP-dependent trs endonuclease activity in vitro. These results suggest that Rep transactivates via a novel mechanism which may require both DNA binding and an enzymatic activity, namely, ATPase or DNA helicase activity.

[Expression of human lymphotoxin gene in CHO-DHFR- cells].

A recombinant plasmid p91-HuLT was constructed with a human lymphotoxin (HuLT) gene 2.4 kb EcoRI fragment and a mammalian cell expression vector plasmid p91023. The HuLT EcoRI DNA fragment covers entire coding sequence and some 3(1) non-translated region sequence of the gene, p91-HuLT DNA was used to transfect Chinese hamster cell line CHO-DHFR- cells by using calcium phosphate-DNA coprecipitation method, and DHFR+ transfectants were obtained. RNA dot blot hybridization analysis showed that the HuLT mRNA was produced by p91-HuLT in transfectants. The results of MTT dye reduction assay indicated that the DHFR+ cell lines we obtained constitutively synthesize and secrete the HuLT with the cytotoxic activity of at least 200 units per mL of medium.