Dynamic association of gammaherpesvirus DNA with core histone during de novo lytic infection of primary cells

Abstract

Association of herpesvirus DNA with histones has important implications for lytic and latent infections; thus herpesviruses arbitrate interactions with histones to productively infect host cells. While regulation of alpha and betaherpesvirus chromatin during lytic infection has been actively investigated, very little is known about interaction of gammaherpesvirus DNA with histones upon de novo lytic infection. Murine gammaherpesvirus-68 (MHV68) is a rodent pathogen that offers a tractable system to study gammaherpesvirus lytic infection in primary cells. In this study we report that MHV68 promoter and orilyt sequences underwent dynamic association with histone H3 during de novo lytic infection of primary macrophages and fibroblasts. Similar to HSV-1, the degree of MHV68 DNA association with histone H3 was dependent on the multiplicity of infection and was further regulated by viral DNA synthesis. Our work sets a precedent for future studies of gammaherpesvirus chromatin during de novo lytic infection.

Figure 1. MHV68 DNA sequences undergo dynamic association with histone H3 during de novo lytic infection of primary bone marrow macrophages

Primary bone marrow macrophages were infected at an indicated MOI with wild type MHV68, and chromatin was harvested throughout a single cycle of replication and subjected to ChIP using anti-histone H3 or a negative control antibody (IgG), as indicated (A–D, F). Efficiency of immunoprecipitation of indicated MHV68 DNA sequences was normalized to the efficiency of immunoprecipitation of GAPDH (A) using the ΔΔCt method and is presented as relative enrichment over GAPDH (B–D, F). E. Total RNA was harvested at indicated times post infection and converted to cDNA in the presence or absence of reverse transcriptase (RT). Abundance of RTA sequences was measured by real time PCR and is presented as fold difference between the corresponding plus RT and minus RT samples. F. Primary macrophages were infected at an MOI of 1, chromatin harvested at indicated times, and subjected to ChIP using anti-histone H3 antibody. G, H. Viral DNA was measured in input samples by real time PCR at indicated time points post infection and normalized to corresponding GAPDH levels. For each panel, data were pooled from 2–5 independent experiments, with error bars representing one standard error of the mean.

Figure 2. Histone H3 associates early and dynamically with viral DNA sequences in de novo lytic MHV68 infection of primary murine embryonic fibroblasts

Primary murine embryonic fibroblasts (MEFs) were infected at an MOI of 1 with wild type MHV68, chromatin was harvested at the specified time points during a single round of viral replication, and subjected to ChIP using anti-histone H3 or a negative control antibody (IgG) as indicated (A–C). Efficiency of immunoprecipitation of indicated MHV68 DNA sequences was normalized to the efficiency of immunoprecipitation of GAPDH (A) using the ΔΔCt method and is presented as relative enrichment over GAPDH (B, C). D. Viral DNA was measured in input samples by real time PCR at indicated time points post infection and normalized to corresponding GAPDH levels. For each panel, data were pooled from 2–5 independent experiments, with error bars representing one standard error of the mean.

Figure 3. Viral DNA synthesis is not required for partial removal of histone H3 from template MHV68 DNA and does not eliminate association of histone H3 with newly synthesized MHV68 DNA

Primary bone marrow macrophages were infected at MOI 10 with wild type MHV68 and treated with PBS or 1 g/ml of cidofovir for the duration of the experiment. Chromatin was harvested at 30 and 48h post infection and subjected to ChIP using anti-H3 (A, B). Efficiency of immunoprecipitation of indicated MHV68 DNA sequences was normalized to the efficiency of immunoprecipitation of GAPDH using ΔΔCt method. C. Viral DNA was measured in input samples by real time PCR at indicated time points post infection and normalized to corresponding GAPDH, with relative levels observed at 30h post infection in cidofovir treated cells set to 1. D. Relative number of MHV68 sequences associated with histone H3 under indicated conditions. This number was determined based on efficiency of immunoprecipitation with histone H3 antibody and relative viral DNA levels (calculated in C). For each panel, data were pooled from 2–3 independent experiments and error bars presented as one standard error of the mean.