Encapsidation of adeno-associated virus type 2 Rep proteins in wild-type and recombinant progeny virions: Rep-mediated growth inhibition of primary human cells

Abstract

The adeno-associated virus type 2 (AAV) arrests the growth of primary human fibroblasts in vitro at high particle-to-cell ratios. To test the role of AAV gene expression in the observed growth inhibition, primary human cells were infected, under identical conditions, with wild-type (wt) AAV or with recombinant AAV that lacked all viral promoters and coding sequences. Significant, dose-dependent growth inhibition of primary human cells was observed with both wt and recombinant AAV at particle-to-cell ratios equal to or exceeding 10(4). In contrast, neither virus affected the growth of immortalized human cells even at a 10-fold-higher particle-to-cell ratio. AAV-induced growth arrest could be overcome by reculturing cells after treatment with trypsin. Even after reculturing, cells still harbored the proviral AAV genome. Thus, neither integration nor expression of the AAV genome appears to be required for the virus-induced growth-inhibitory effect on primary human cells. The growth-inhibitory effect of AAV was hypothesized to be mediated by virion-associated AAV Rep proteins, since these proteins have been reported to inhibit cellular DNA synthesis. Rep proteins tightly associated with wt as well as recombinant AAV could be detected on Western blots. Coinfection by adenovirus was necessary and sufficient for ample replication of recombinant AAV genomes lacking the rep gene. Although wt AAV-like particles arose during production of the recombinant AAV stocks, their low-titer levels were insufficient to cause the observed growth inhibition. AAV rep gene expression from these contaminating particles was not required for replication of the recombinant AAV genomes, which could be detected even in the absence of de novo Rep protein synthesis. Exposure of recombinant AAV to anti-AAV Rep protein antibodies did not abrogate viral infectivity. These results suggest that biologically active Rep proteins are encapsidated in mature progeny AAV particles. AAV Rep protein-mediated growth inhibition of primary human cells has implications in the use of AAV-based vectors in human gene therapy.