Adeno-associated Virus Life Cycle Research Articles

Use of adeno-associated virus as a general transduction vector for mammalian cells.

Adeno-associated virus (AAV) is a human virus that can be propagated either as an integrated provirus or by lytic infection (Atchison et al. 1965; Hoggan et al. 1966, 1972). The ability to form a latent infection appears to be an integral part of the AAV life cycle. Except under special circumstances (Yacobson et al. 1987; Schlehofer et al. 1986; Yalkinoglu et al. 1988), AAV requires the presence of a helper virus to initiate a productive viral infection (Fig. 1). Members of either the herpes or adenovirus families can provide the necessary helper functions (Atchison et al. 1965; Melnick et al. 1965; Hoggan et al. 1966; Buller et al. 1981; McPherson et al. 1985) and vaccinia virus can provide at least partial helper function (Schlehofer et al. 1986). In the absence of a helper virus AAV produces no progeny virus, but instead integrates into a host chromosome (Hoggan et al. 1972; Berns et al. 1975; Handa et al. 1977; Cheung et al. 1980). With rare exceptions, AAV proviruses appear to be stable. However, if a cell line that is carrying an AAV provirus (Fig. 1) is subsequently superinfected with a helper virus, the AAV genome is excised and proceeds through a normal productive infection (Hoggan et al. 1972; Cheung et al. 1980). This ability to establish a latent infection which can later be rescued appears to be a mechanism for ensuring the survival of AAV in the absence of a helper virus. The unusual life cycle of AAV has led a number of parvovirus laboratories to explore the possibility of using AAV as a general mammalian transduction vector.

Requirement for either early region 1a or early region 1b adenovirus gene products in the helper effect for adeno-associated virus.

Several adenovirus early genes act together to promote growth of the helper-dependent adeno-associated virus (AAV). Data from several laboratories have implicated adenovirus early regions 1a, 1b, 2a, and 4 in the helper effect, as well as the small RNA polymerase III transcript, virus-associated RNA I. Although a subset of these must participate directly in the AAV life cycle, some may play an indirect role by influencing expression of the others. This paper is concerned particularly with the roles of early regions 1a and 1b in the helper effect. We introduced DNA fragments representing the various early regions into AAV-infected or uninfected Vero cells, by the manual microinjection procedure. After labeling the cells with [35S]methionine, we visualized immunoprecipitates of AAV or adenovirus proteins on sodium dodecyl sulfate-polyacrylamide gels. When over 200 copies of each DNA fragment per cell were injected, early regions 2a and 4 were themselves sufficient to provide the helper effect. At 100 copies per cell, however, a third gene became essential, and this could be either early region 1a or 1b. The role of early region 1a is easily explained by its known ability to stimulate transcription of the other early genes. The function of early region 1b is less clear, but it does not simply mimic the action of early region 1a. Instead, there appear to be at least two distinct regulatory pathways which can lead to expression of AAV. To investigate the sequence of regulatory interactions, we microinjected purified adenovirus mRNAs, or combinations of mRNA and DNA, into AAV-infected cells. Our results suggest that adenovirus early products enhance viral gene expression by several mechanisms which can operate independently, but whose effects may be cumulative.