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

Adeno-associated virus (AAV) codes for four closely related nonstructural proteins (Rep) required for AAV DNA replication and gene regulation. In vitro studies have revealed that either Rep78 or Rep68 alone is sufficient for AAV DNA replication. Rep52 and Rep40 are not required for DNA replication but have been reported to enhance the efficiency of accumulation of single-stranded progeny DNA. Previous studies on rep-expressing cell lines had indicated that only a subset of the four Rep proteins are required for the production of infectious AAV. We therefore set out to determine the minimal set of Rep proteins sufficient for the generation of infectious AAV. Transient cotransfections in HeLa cells of constructs for high-level expression of individual Rep proteins with a rep-negative AAV genome revealed that either Rep78 or Rep68 alone could complement for a full replication cycle yielding infectious virus. This result was confirmed by transfection studies in the cell line HeM2, which selectively expresses Rep78 at rather low levels under the control of the glucocorticoid-responsive mouse mammary tumor virus long terminal repeat (C. Hölscher, M. Hörer, J. A. Kleinschmidt, H. Zentgraf, A. Bürkle, and R. Heilbronn, J. Virol. 68:7169-7177, 1994). Increasing the level of Rep78 expression by transfection of a glucocorticoid receptor expression construct resulted in a higher level of DNA replication of a cotransfected rep-negative AAV genome and in the production of infectious rep-negative AAV particles. We further report on the generation of a new rep-expressing cell line, HeCM1, which was obtained by stable supertransfection of a construct for constitutive Rep40 expression into HeM1 cells (Hölscher et al., J. Virol. 68:7169-7177). Transfection of rather large amounts of rep-negative AAV DNA led to detectable virus production in HeCM1 cells even in the absence of the cotransfected glucocorticoid receptor expression construct, but higher yields were obtained after increasing the Rep78 level by coexpression of the glucocorticoid receptor. These data demonstrate that all Rep functions required for the productive replication of AAV in HeLa cells are contained within both Rep78 and Rep68.