Interactions of parvoviruses with the cellular DNA damage response and CTCF

Abstract

Nuclear DNA viruses must be able to simultaneously access cellular factors that aid their life cycle while evading inhibitory factors in order to sustain their expression and replication. Establishment of nuclear sites where particular DNA viruses are able to initiate their replication has likely evolved over time to optimize these needs. Parvoviruses are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. They contain a linear single-stranded DNA genome of ~5 kb with Inverted Terminal Repeats (ITRs) at each end that serve as origins of replication. Minute Virus of Mice (MVM), the prototype virus of the genus Protoparvovirus, is an autonomously replicating mouse parvovirus that is lytic in murine cells and human transformed cells. Unlike most parvoviruses, Adeno-associated virus (AAV) in the genus Dependoparvovirus cannot support their own replication and require the coinfection of a helper virus, such as Adenovirus or Herpesvirus, in order to propagate. Parvoviruses establish replication factories in the nucleus, where ongoing replication and expression takes place. Due to their limited genetic capacity, parvoviruses depend heavily on cellular functions and must interact with host factors (and helper virus factors in case of AAV) either positively or negatively to productively replicate. Parvovirus infection induces a cellular DNA damage response (DDR) in cells, either in response to viral DNA or viral proteins expressed in the host nucleus during infection. Using a novel adaptation of high-throughput circular chromosome conformation capture assay (4C) for use in trans (which we have termed V3C-seq), we have mapped the primary localization sites of the replicating MVM genome with cellular sites of DNA damage, replete with replication and repair factors. As replication proceeds, MVM induces and then spreads to additional sites of DNA damage forming large interaction domains termed Virus Association Domains (VADs). Furthermore, we have shown that the essential viral non-structural protein NS1 possess the intrinsic ability to localize to cellular DNA damage sites and could localize to those sites both the MVM genome and a heterologous DNA molecule engineered to contain NS1 binding sites. These results suggested that NS1 may function as a bridging molecule, helping the MVM genome localize to sites of DNA damage to facilitate ongoing virus replication. Similar V3C-seq assays to map AAV genome localization show that both replicating and non-replicating AAV2 genomes in the absence of helper virus colocalize with cellular sites of DNA damage. The AAV non-structural protein Rep68/78, when ectopically expressed in the absence of viral infection or during AAV2 infection in the absence of helper proteins also localizes to cellular sites of DNA damage. Strikingly however, recombinant AAV gene therapy vector genomes derived from AAV do not colocalize with AAV and Rep at cellular DDR sites, suggesting the Inverted Terminal Repeat origins of replication shared between them are insufficient to localize the genome to cellular sites of DNA damage. Our findings suggest that AAV Rep78 protein plays another critical role in the AAV life cycle by aiding in the establishment of viral replication centers in proximity to cellular sites of DNA damage. Our V3C-seq analysis on MVM also revealed that the VADs overlap significantly with previously identified topologically-associated domains (TADs). The boundaries of TADs are enriched for binding sites of CCCTC-binding factor (CTCF), a highly conserver 11-zinc-finger DNA-binding protein which play a key role in genome organization. In silico inspection of the MVM genome revealed the presence of two potential CTCF binding sites and Chromatin Immunoprecipitation (ChIP) assays showed that CTCF associates specifically with the viral genome. In our attempt to determine the role of the host factor CTCF during MVM infection, we found that the multifunctional host factor CTCF is required for proper engagement of the spliceosome at the MVM small intron and for the first steps of processing of P4 promoter-generated pre-mRNAs.