title: Promyelocytic leukemia protein is a cell-intrinsic factor inhibiting parvovirus DNA replication.
abstract: Tripartite motif proteins are important viral restriction factors and affect processes ranging from uncoating to transcription to immune signaling. Specifically, the promyelocytic leukemia protein (TRIM19; also called PML) is a viral restriction factor inhibiting processes from uncoating to transcription to cell survival. Here we investigated PML's effect on adeno-associated virus (AAV), a parvovirus used for gene delivery. Although dependovirus (AAV) and autonomous parvovirus (minute virus of mice) replication centers can colocalize with PML, PML's functional effect on parvoviruses is unknown. Using PML knockout mice, we determined that PML knockout enhances recombinant AAV2 (rAAV2) transduction at a range of vector doses in both male and female mice. In fact, male and female PML knockout mice exhibited up to 56-fold and 28-fold increases in transduction, respectively. PML inhibited several rAAV serotypes, suggesting a conserved mechanism, and organ specificity correlated with PML expression. Mechanistically, PML inhibited rAAV second-strand DNA synthesis, precluding inhibition of self-complementary rAAV, and did not affect the prior steps in transduction. Furthermore, we confirmed the effect of human PML on rAAV transduction through small interfering RNA (siRNA)-mediated knockdown in HuH7 cells and determined that the highest level of inhibition was due to effects of PML isoform II (PMLII). Overexpression of PMLII resulted in inhibition of second-strand synthesis, vector production, and genome replication. Moreover, wild-type AAV2 production and infectivity were also inhibited by PMLII, demonstrating a PML interaction with wild-type AAV. These data have important implications for AAV-mediated gene therapy. Additionally, PMLII inhibition of AAV second-strand synthesis and replication, which are processes necessary for all parvoviruses, suggests implications for replication of other parvoviruses.
authors.affiliation: Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Mitchell
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Hirsch
authors.forename: Chengwen
authors.initials: C
authors.lastname: Li
authors.forename: R Jude
authors.initials: RJ
authors.lastname: Samulski
mesh_terms: D000818:Animals; D002460:Cell Line; D004261:DNA Replication; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D015536:Down-Regulation; D005260:Female; D006801:Humans; D008297:Male; D057507:Mice, 129 Strain; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010322:Parvoviridae Infections / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D000071156:Promyelocytic Leukemia Protein; D011995:Recombination, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human