In vitro replication of adeno-associated virus DNA

Abstract

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)

High-level expression of adeno-associated virus (AAV) Rep78 or Rep68 protein is sufficient for infectious-particle formation by a rep-negative AAV mutant

“…1A). This is plausible, because the small Rep proteins are not able to support AAV DNA replication (10,21,28). As expected, transfection of pTAV2-3 alone into HeLa cells did not lead to infectious virus production ( Fig.…”

“…Rep78 and Rep68 indeed possess the same biochemical activities required for AAV DNA replication (12)(13)(14). To date, only one qualitative difference between these two proteins could be demonstrated by in vitro replication studies, revealing that Rep68 preferentially generates monomer RF1 DNA, whereas Rep78 yields about equal amounts of RF1 and RF2 DNA (21).…”

“…two proteins alone is sufficient (21,31). Rep52 and Rep40 are not required for DNA replication, but they seem to be necessary for the efficient accumulation of progeny single-stranded DNA (5).…”

Adeno‐Associated Virus Rep78/Rep68 Promotes Localized Melting of the Rep Binding Element in the Absence of Adenosine Triphosphate

“…Rep68 was purified to homogeneity from baculovirus‐infected SF9 cells as previously described 7, 37. Rep68 was purified by sequential chromatography on phenyl‐Sepharose, ssDNA–cellulose, and DNA affinity matrices (fraction A) or by sequential chromatography on phenyl‐Sepharose, ssDNA‐cellulose, and diethylaminoethyl‐cellulose (fractions V and S).…”

Next generation agents (synthetic agents): Emerging threats and challenges in detection, protection, and decontamination

“…Adeno-associated virus (AAV) are nonenveloped icosahedral parvoviruses containing ssDNA with intact inverted terminal repeats (ITR), resulting in the formation of important secondary structures in viral genome. Further, co-infection with helper virus (like adenovirus or herpes simplex virus, HSV) assists its replication with host cell polymerase (Ni et al, 1994). An in vitro course of action for the packaging of AAV was studied by Zhou and Muzyczka (1998).…”