Adeno-associated virus (AAV) is

Endonuclease G (endo G) is one of the most abundant nucleases in eukaryotic cells. It is encoded in the nucleus and imported to the mitochondrial intermembrane space. This nuclease is active on single-and double-stranded DNA. We genetically disrupted the endo G gene in mice without disturbing a conserved, overlapping gene of unknown function that is oriented tail to tail with the endo G gene. In these mice, the production of endo G protein is not detected, and the disruption abolishes the nuclease activity of endo G. The absence of endo G has no effect on mitochondrial DNA copy number, structure, or mutation rate over the first five generations. There is also no obvious effect on nuclear DNA degradation in standard apoptosis assays. The endo G null mice are viable and show no age-related or generational abnormalities anatomically or histologically. We infer that this highly conserved protein has no mitochondrial or apoptosis function that can discerned by the assays described here and that it may have a function yet to be determined. The early embryonic lethality of endo G null mice recently reported by others may be due to the disruption of the gene that overlaps the endo G gene.

Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1)proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions. Adeno-associated virus (AAV) is a nonpathogenic human parvovirus with a biphasic life cycle (reviewed in reference 45). The linear, single-stranded DNA genome of AAV possesses inverted terminal repeats (ITRs) at either end that fold into hairpin structures and serve as the origin of replication. There are two open reading frames that encode the replication (Rep) and structural (Cap) proteins. The two large Rep proteins (Rep68 and Rep78) are required for replication and possess multiple activities, including specific DNA binding and sitespecific endonuclease nicking at the viral ITR (45). Productive AAV infection in cell culture requires helper functions that can be supplied by coinfection with a second virus. In the absence of helper virus, the AAV genome stably integrates into the host genome. Helper viruses that enable efficient AAV replication include adenovirus (Ad) and viruses of the herpesvirus group, such as herpes simplex virus types 1 and 2 (HSV-1 and -2) (12), human herpesvirus 6 (51), and human cytomegalovirus (HCMV) (43). Replication of AAV can also be achieved in several cell lines by the addition of a variety of genotoxic agents (67-69), and autonomous replication has been demonstrated in cultured differentiating keratinocytes (44). A basic question in AAV biology is the nature of the helper effect supplied by the different helper viruses as well as by other permissive conditions. Genetic analysis using mutant helper viruses and expression of individual helper virus genes has defined gene products that are required for efficient AAV replication. The helper functions of Ad are well characterized and are supplied by E1a, E1b, E2a, E4, and the VA RNA (36, 50). Specific functions have been assigned for these proteins and their predominant role appears to be in regulation of gene expression for AAV proteins. T...

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