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The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds α v β 3 and α v β 5 integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing α v β 5 integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.

DNA polymerase beta (pol beta) is a key player in DNA base excision repair (BER). Here, we describe the complex formation of pol beta with the protein arginine methyltransferase 1 (PRMT1). PRMT1 specifically methylated pol beta in vitro and in vivo. Arginine 137 was identified in pol beta as an important target for methylation by PRMT1. Neither the polymerase nor the dRP-lyase activities of pol beta were affected by PRMT1 methylation. However, this modification abolished the interaction of pol beta with proliferating cell nuclear antigen (PCNA). Together, our results provide evidence that PRMT1 methylation of pol beta might play a regulatory role in BER by preventing the involvement of pol beta in PCNA-dependent DNA metabolic events.

Alterations in DNA repair lead to genomic instability and higher risk of cancer. DNA base excision repair (BER) corrects damaged bases, apurinic sites, and single-strand DNA breaks. Here, a regulatory mechanism for DNA polymerase beta (Pol beta) is described. Pol beta was found to form a complex with the protein arginine methyltransferase 6 (PRMT6) and was specifically methylated in vitro and in vivo. Methylation of Pol beta by PRMT6 strongly stimulated DNA polymerase activity by enhancing DNA binding and processivity, while single nucleotide insertion and dRP-lyase activity were not affected. Two residues, R83 and R152, were identified in Pol beta as the sites of methylation by PRMT6. Genetic complementation of Pol beta knockout cells with R83/152K mutant revealed the importance of these residues for the cellular resistance to DNA alkylating agent. Based on our findings, we propose that PRMT6 plays a role as a regulator of BER.

Preclinical studies using various cell culture and animal systems highlight the potential of recombinant rodent parvoviruses (recPVs) for cancer therapy. Production of these viruses is, however, not efficient and this hampers the clinical applications of these agents. In this study, we show that the adenovirus genes E2a, E4(orf6) and VA RNA increase the production of recPVs by more than 10-fold and reduce the time of production from 3 to 2 days in HEK293T cells. The helper effects of these genes can be observed with different recPVs, regardless of the nature and size of the inserted transgene. Furthermore, we generated a recombinant Adenovirus 5 carrying the parvovirus VP transcription unit. This helper, named Ad-VP, allows recPVs to be efficiently produced through a protocol based only on cell infection, making possible to use cell lines, such as NB324K, which are good producers of parvoviruses but are hardly transfectable. Hence, we could further improve viral titers and reduce time and costs of production. This Ad-VP helper-based protocol could be scaled up to a bioreactor format for the generation of the large amounts of recPVs needed for future clinical applications.

We describe a novel regulatory mechanism for DNA polymerase beta (Polbeta), a protein involved in DNA base excision repair (BER). Polbeta colocalized in vivo and formed a complex with the transcriptional coactivator p300. p300 interacted with Polbeta through distinct domains and acetylated Polbeta in vitro. Polbeta acetylation was furthermore observed in vivo. Lysine 72 of Polbeta was identified as the main target for acetylation by p300. Interestingly, acetylated Polbeta showed a severely reduced ability to participate in a reconstituted BER assay. This was due to an impairment of the dRP-lyase activity of Polbeta. Acetylation of Polbeta thus acts as an intranuclear regulatory mechanism and implies that p300 plays a critical regulatory role in BER.