Parvovirus minute virus of mice interacts with sites of cellular DNA damage to establish and amplify its lytic infection

Abstract

We have developed a generally adaptable, novel high-throughput Viral Chromosome Conformation Capture assay (V3C-seq) for use in trans that allows genome-wide identification of the direct interactions of a lytic virus genome with distinct regions of the cellular chromosome. Upon infection, we found that the parvovirus Minute Virus of Mice (MVM) genome initially associated with sites of cellular DNA damage that in mock-infected cells also exhibited DNA damage as cells progressed through S-phase. As infection proceeded, new DNA damage sites were induced, and virus subsequently also associated with these. Sites of association identified biochemically were confirmed microscopically and MVM could be targeted specifically to artificially induced sites of DNA damage. Thus, MVM established replication at cellular DNA damage sites, which provide replication and expression machinery, and as cellular DNA damage accrued, virus spread additionally to newly damaged sites to amplify infection. MVM-associated sites overlap significantly with previously identified topologically-associated domains (TADs).

The NS1 protein of the parvovirus MVM Aids in the localization of the viral genome to cellular sites of DNA damage

“…Induction of DNA damage in these cells via an HU pulse led to the spread of γH2AX over wide areas (purple histogram and, cf. Fig 1D , panel 3), as previously characterized by others for γH2AX induction [ 26 , 31 ], and consistent with previously published findings during MVM infection [ 21 ]. NS1 broadly relocalized to the γH2AX domains in both analyses (blue histogram).…”

“…As shown in Fig 1A , during wild-type MVM (MVM WT ) infection in U2OS osteosarcoma cells, which are fully permissive for MVM in an NS2-independent manner, NS1 co-localized with cellular sites of DNA damage monitored by γH2AX staining, as a part of APAR bodies ( Fig 1A , panel 1), consistent with previously published observations [ 15 , 20 , 21 ]. Upon induction of DNA damage by laser micro-irradiation in U2OS cells 16 hours post infection (16 hpi), a significant portion of NS1 relocalized to the laser micro-irradiated stripe as monitored by γH2AX staining ( Fig 1A , panel 2).…”

“…As shown for representative samples on mouse chromosomes 17 and 19, where the MVM genome was previously found to localize within megabase-sized domains during early stage of infection ([ 21 ] and described below), NS1 binds to distinct sites on Chr17 and Chr19 (red histogram), and colocalization of NS1 with γH2AX (green histogram) is evident when viewed both from the whole-chromosome view ( Fig 2A left panels), as well as in magnified finer detail ( Fig 2A right panels). Induction of DNA damage in these cells via an HU pulse led to the spread of γH2AX over wide areas (purple histogram and, cf.…”

“…At early stages of infection, MVM induces cellular DNA damage which results in a DDR in host cells, driven by signaling from the ATM kinase and virally mediated inactivation of the CHK1 kinase [ 15 , 19 ]. APAR bodies are also sites where cellular DNA Damage Response (DDR) sensor and response proteins accumulate and associate with replicating viral DNA [ 15 , 20 , 21 ]. As virus replication proceeds, MVM programs the degradation of p21 through the CRL4 Cdt2 ubiquitin ligase pathway, and downregulates Cyclin B1 expression in part by dysregulating the transcription factor FOXM1, ultimately resulting in a potent pre-mitotic cell cycle block [ 22 – 25 ].…”

“…Importantly, rather than merely recruiting essential proteins to replication centers, the MVM genome localizes to cellular genomic sites that contain high concentrations of replication and repair proteins. High-throughput chromosome conformation capture assays (which we have termed V3C-seq) have mapped the primary localization sites of the replicating MVM genome with cellular sites of DNA damage replete with such factors, many of which coincide with Early Replicating Fragile sites (ERFs, [ 21 , 26 ]). As replication proceeds, MVM induces and then spreads to additional sites of DNA damage.…”