Replication of the pathogenic human parvovirus B19 is restricted to erythroid progenitor cells. Although blood group P antigen has been reported to be the cell surface receptor for parvovirus B19, a number of nonerythroid cells, which express P antigen, are not permissive for parvovirus B19 infection. We have documented that P antigen is necessary for parvovirus B19 binding but not sufficient for virus entry into cells. To test whether parvovirus B19 utilizes a cell surface coreceptor for entry, we used human erythroleukemia cells (K562), which allow parvovirus B19 binding but not entry. We report here that upon treatment with phorbol esters, K562 cells become adherent and permissive for parvovirus B19 entry, which is mediated by ␣5␤1 integrins, but only in their high-affinity conformation. Mature human red blood cells (RBCs), which express high levels of P antigen, but not ␣5␤1 integrins, bind parvovirus B19 but do not allow viral entry. In contrast, primary human erythroid progenitor cells express high levels of both P antigen and ␣5␤1 integrins and allow ␤1 integrin-mediated entry of parvovirus B19. Thus IntroductionTwo parvoviruses of human origin, the nonpathogenic adenoassociated virus 2 (AAV) and the pathogenic parvovirus B19, have been studied extensively. 1,2 Recombinant AAV vectors have gained attention as a potentially useful alternative to the more commonly used recombinant retroviral and adenoviral vectors in human gene therapy. 3 AAV possesses a broad host range that transcends the species barrier because it utilizes the ubiquitously expressed cell surface heparan sulfate proteoglycan as a primary receptor for viral binding. 4 We and others have reported the requirement of fibroblast growth factor receptor 1 (FGFR1) and ␣V␤5 integrin as coreceptors for viral entry. 5,6 Parvovirus B19, on the other hand, has been shown to have an extremely limited tissue-tropism, and the virus replication is restricted to human erythroid progenitor cells, 7 presumably because (1) the blood group P antigen (synonym "globoside"; "globotetraosylceramide") is used as primary cellular receptor for parvovirus B19, 8 (2) putative intracellular factors, largely restricted to human erythroid cells, are required for optimal transcriptional activation of the B19 promoter at map unit 6 (B19p6) and viral replication, 9,10 and (3) B19 capsid protein expression in nonpermissive cells is impaired due to a block in full-length transcription of the viral genome, atypical mRNA splicing, and impaired ribosome loading of structural gene transcripts. [11][12][13] However, P antigen expression is not restricted to erythroid cells, and a number of P antigen-positive nonerythroid cells are nonpermissive for a successful infection by parvovirus B19. 2 In addition, mature red blood cells (RBCs), which are known to express high levels of P antigen, are unlikely to be ideal targets for B19 infection and replication because they lack nuclei. Similarly, B19p6 promoter-driven expression analyses were carried out with plasmid DNA transfections, 11...

Here, we report that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein 52 (FKBP52). FKBP52 was purified by using a prokaryotic expression plasmid containing the human cDNA. The purified protein could be phosphorylated at both tyrosine and serine or threonine residues, and only the phosphorylated forms of FKBP52 were shown to interact with the AAV single-stranded D-sequence probe. Furthermore, in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibited AAV second-strand DNA synthesis by greater than 90%. Serine-or threoninephosphorylated FKBP52 caused Ϸ40% inhibition, whereas dephosphorylated FKBP52 had no effect on AAV second-strand DNA synthesis. Deliberate overexpression of FKBP52 effectively reduced the extent of tyrosine phosphorylation of the protein, resulting in a significant increase in AAV-mediated transgene expression in human and murine cell lines. These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may have important implications for the optimal use of AAV vectors in human gene therapy.Adeno-associated virus type 2 (AAV) is a small, nonpathogenic, single-stranded DNA-containing virus which requires coinfection with a helper virus, usually adenovirus, for its optimal replication (1, 31). In the absence of coinfection with the helper virus, the wild-type AAV establishes a latent infection in which the viral genome integrates into human chromosomal DNA in a site-specific manner (22,23,45). The nonpathogenic nature of AAV coupled with the remarkable site specificity of integration prompted the development of recombinant AAV vectors for gene transfer and gene therapy. Although recombinant AAV genomes do not appear to integrate site specifically, AAV vectors have been successfully used for gene delivery to a wide variety of cells and tissues in vitro and in vivo (2, 3, 11, 12, 16-19, 21, 32-36, 47, 48, 51, 55, 57), as well as in phase I clinical trials for gene therapy of cystic fibrosis and hemophilia B (11, 18). However, the transduction efficiencies of AAV vectors vary greatly in different cell types. Studies from two independent laboratories have suggested that following infection, the viral second-strand DNA synthesis is a ratelimiting step in efficient transduction by AAV vectors (8,9). We have documented that a host cell protein, designated the single-stranded D-sequence binding protein (ssD-BP), interacts specifically and preferentially with the D sequence within the inverted terminal repeat (ITR) at the 3Ј end of the AAV genome and, in its tyrosine-phosphorylated form, prevents viral second-strand DNA synthesis, resulting in inhibition of AAV-mediated transgene expression. ssD-BP is phosphorylated at tyrosine residues by the epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK), and the phosphorylation state of ssD-BP corre...