Promyelocytic Leukemia Protein Is a Cell-Intrinsic Factor Inhibiting Parvovirus DNA Replication

Abstract

e 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.A deno-associated virus (AAV) is a helper-dependent member of the Parvoviridae family, which, in addition to AAV, contains other viruses of clinical and veterinary importance, such as B19 parvovirus, human bocavirus, and canine parvovirus. AAV consists of an icosahedral capsid surrounding a single-stranded DNA genome carrying two genes, Rep and Cap, and has been developed as a gene delivery vector for gene therapy applications. For use as a vector or virus-like particle (recombinant AAV [rAAV]), the viral genes can be removed and replaced with a transgene cassette, with the terminal repeats being the only viral elements required in cis (1). Although clinical rAAV-mediated gene therapy has demonstrated increasing success in reaching efficacy goals, especially in restricted sites such as the eye (2), low transgene expression or loss of expression over time has repeatedly compromised efficacy in other clinical trials (3, 4). Therefore, efforts to increase the efficiency of rAAV transduction without increasing the vector dose are imperative.AAV's replication pathway involves receptor-mediated endocytosis, trafficking to th...

Emerging Role of PML Nuclear Bodies in Innate Immune Signaling

“…Evidence continues to accumulate that the restricting activity of NB proteins not only extends to further DNA viruses, including adeno-, papilloma-and parvoviruses, but also affects specific processes in the life cycle of cytoplasmically replicating RNA viruses (8)(9)(10)(11). While in vivo experiments with PML knockout mice demonstrated restriction of the arenavirus lymphocytic choriomeningitis virus (LCMV) and the rhabdovirus vesicular stomatitis virus (VSV) more than a decade ago, convincing data for an involvement of PML in HIV-1 restriction were only recently provided by two publications (4,5).…”

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies

“…However, since few spatiotemporal analyses for incoming genomes of other DNA viruses have been reported (9), it remains to be determined whether the encounter of PML-NB components is a general cellular defense against invading DNA viruses. In addition, the effects of depletion of PML-NB components have different effects in different viral systems: knockdown or knockout of certain PML-NB components resulted in enhancement, no effect, or suppression of viral propagation (8,(17)(18)(19)(20)(21)(22)(23), suggesting distinct responses against each virus. Furthermore, although colocalization has been observed, it is not clear how incoming viral genomes and/or viral DNA replication activities are connected to PML-NBs.…”

Recombinant adeno-associated virus vectors in the treatment of rare diseases

“…Deficiency in MyD88, an effector molecule of TLR9, results in decreased NAb responses to rAAV 36 . Though it is unclear if other cellular proteins promote immune responses like TLR9, promyelocytic leukemia protein (PML) or the DNA damage complex Mre11/Rad50/Nbs1 (MRN) were shown to inhibit rAAV second strand synthesis and/or inhibit gene expression independent of rAAV DNA synthesis through an unknown mechanism 37-39 . These studies highlight the need for continued investigation of cellular host anti-viral responses and a link to humoral and cell-mediated immunity against rAAVs.…”