ENCAPSIDATION OF ADENOASSOCIATED VIRUS TYPE-2 REP PROTEINS IN WILD-TYPE AND RECOMBINANT PROGENY VIRIONS - REP-MEDIATED GROWTH-INHIBITION OFPRIMARY HUMAN-CELLS

The adeno-associated virus type 2 (AAV) arrests the growth of primary human fibroblasts in vitro at high particle-to-cell ratios, To test th e role of AAV gene expression in the observed growth inhibition, prima ry human cells were infected, under identical conditions, with wild-ty pe (wt) AAV or with recombinant AAV that lacked all viral promoters an d coding sequences, Significant, dose-dependent growth inhibition of p rimaly human cells was observed with both wt and recombinant AAV at pa rticle-to-cell ratios equal to or exceeding 10(4), In contrast, neithe r virus affected the growth of immortalized human cells even at a 10-f old-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, neit her integration nor expression of the AAV genome appears to be require d for the virus-induced growth-inhibitory effect on primary human cell s, 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 asso ciated with wt as well as recombinant AAV could be detected on Western blots, Coinfection by adenovirus was necessary and sufficient for amp le replication of recombinant AAV genomes lacking the rep gene, Althou gh wt AAV-like particles arose during production of the recombinant AA V stocks, their low-titer levels were insufficient to cause the observ ed growth inhibition, AAV rep gene expression from these contaminating particles was not required for replication of the recombinant AAV gen omes, which could be detected even in the absence of de novo Rep prote in synthesis, Exposure of recombinant AAV to anti-AAV Rep protein anti bodies did not abrogate viral infectivity, These results suggest that biologically active Rep proteins are encapsidated in mature progeny AA V particles, AAV Rep protein-mediated growth inhibition of primary hum an cells has implications in the use of AAV-based vectors in human gen e therapy.