Autophagy Genes Enhance Murine Gammaherpesvirus 68 Reactivation from Latency by Preventing Virus-Induced Systemic Inflammation

Abstract

Host genes that regulate systemic inflammation upon chronic viral infection are incompletely understood. Murine gammaherpesvirus 68 (MHV68) infection is characterized by latency in macrophages, and reactivation is inhibited by interferon-γ (IFN-γ). Using a lysozyme-M-cre (LysMcre) expression system, we show that deletion of autophagy-related (Atg) genes Fip200, beclin 1, Atg14, Atg16l1, Atg7, Atg3, and Atg5, in the myeloid compartment, inhibited MHV68 reactivation in macrophages. Atg5 deficiency did not alter reactivation from B cells, and effects on reactivation from macrophages were not explained by alterations in productive viral replication or the establishment of latency. Rather, chronic MHV68 infection triggered increased systemic inflammation, increased T cell production of IFN-γ, and an IFN-γ-induced transcriptional signature in macrophages from Atg gene-deficient mice. The Atg5-related reactivation defect was partially reversed by neutralization of IFN-γ. Thus Atg genes in myeloid cells dampen virus-induced systemic inflammation, creating an environment that fosters efficient MHV68 reactivation from latency.

Graphical abstract

Figure 1

Multiple Autophagy Genes Promote MHV68 Reactivation from Macrophages (A–D) Fip200^f/f-LysMcre, beclin 1^f/f-LysMcre, and Atg14^f/f-LysMcre mice (A); Atg7^f/f-LysMcre, Atg16l1^f/f-LysMcre, Atg5^f/f-LysMcre, and Atg3^f/f-LysMcre mice (B); Atg4b^−/− (C); and p62^−/− (D) and wild-type littermates were infected with 1 × 10⁶ PFU MHV68 intraperitoneally (i.p.) for 42 days to determine frequency of virus reactivation in peritoneal exudate cells (PECs) during latent infection using LDA analysis. Data points indicate the percentage of wells that were positive for cytopathic effect (CPE) on a mouse embryonic fibroblast (MEF) monolayer at a given cell dilution. All LDA experiments are n = 3 or 4 with 3–5 mice pooled per experiment. Data are the mean ± SEM and p values were obtained by paired t test over all dilutions. Only significant comparisons are indicated. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.001.

Figure 2

Autophagy Genes Control MHV68 Reactivation from Macrophages, Not from B Cells Atg5^f/f, Atg5^f/f-LysMcre, and Atg5^f/f-CD19cre mice were infected with 1 × 10⁶ PFU MHV68 i.p. for 16 days, and LDA was performed on PECs and splenocytes to measure virus reactivation. Data are combined from three biological replicate experiments with 3–5 mice pooled per experiment. Data are the mean ± SEM and p values were obtained by paired t test over all dilutions. Only significant comparisons are indicated. ^∗∗∗∗p < 0.001.

Figure 3

Atg5 Is Not Required for Viral Replication or Establishment of Latent Infection (A and B) Atg5^f/f and Atg5^f/f-LysMcre BMDMs (A) or PECs (B) were infected with MHV68 at an MOI of 5 PFU/cell or 0.05 PFU/cell for 1 hr. Viral titers at the indicated time points were measured by plaque assay on an NIH 3T12 fibroblast monolayer. Data are the mean ± SEM, and a paired t test over all time points yielded no significant differences. (C) Atg5^f/f and Atg5^f/f-LysMcre mice were infected with 1 × 10⁶ PFU MHV68 per mouse i.p., and splenic viral titers were measured by plaque assay at times indicated. Three to five mice per genotype were used for each time point. The limit of detection by plaque assay is 50 PFU and is represented by the dotted horizontal line (A–C). Data were analyzed by a paired t test at each time point, and no significant differences were observed. (D) Atg5^f/f and Atg5^f/f-LysMcre mice were infected with MHV68-M3FL, injected with D-luciferin, and imaged at the indicated times. Data were analyzed by a paired t test at each time point, and no significant differences were observed. (E) PECs were 3-fold serially diluted for a limiting dilution nested PCR assay specific for MHV68 ORF72 to measure the frequencies of MHV68 genome-containing PECs. For the limiting dilution PCR, n = 4 and data are the mean ± SEM. Data are analyzed by a paired t test over all dilutions, and no significant differences observed. (F) Atg5^f/f and Atg5^f/f-LysMcre mice were infected with 1 × 10⁶ PFU MHV68 i.p. for 28 days, and LDA analysis was performed on PECs to measure virus reactivation. Data are combined from three biological replicate experiments with 3–5 mice pooled per experiment. PECs from Atg5^f/f and Atg5^f/f-LysMcre mice were mixed at a ratio of 1:1 and plated at the indicated ratios. Data are the mean ± SEM.

Figure 4

Atg5 Deficiency Increases Virus-Induced IFN-γ-Dependent Macrophage Activation (A) Gene set enrichment analysis was performed on RNA-seq data from naive and latent adherent peritoneal macrophages from Atg5^f/f and Atg5^f/f-LysMcre mice. (B) Transcript levels of the indicated genes were measured in latently infected peritoneal macrophages from Atg5^f/f and Atg5^f/f-LysMcre mice. Data are analyzed by the non-parametric Mann-Whitney U test. ^∗∗p < 0.01, ^∗∗∗p < 0.005. (C) Latently infected peritoneal macrophages were stained for intracellular iNOS and analyzed by flow cytometry. Each point represents a single mouse, and data are analyzed by the non-parametric Mann-Whitney U test. ^∗∗p < 0.01.

Figure 5

Elevated IFN-γ in Atg5^f/f-LysMcre Mice Drives Macrophage Activation (A) Serum levels of IFN-γ were measured by Luminex xMAP technology 42 days after infection. Each data point represents one mouse. Data were analyzed by a one-way ANOVA with Kruskal-Wallis test with multiple comparisons. ^∗p < 0.05, ^∗∗∗p < 0.005. (B) 40,000 PECs pooled from three Atg5^f/f mice or Atg5^f/f-LysMcre mice were plated onto a monolayer of MEFs for 24 hr in a 96-well plate. IFN-γ levels in the supernatants were measured by a sandwich ELISA. The data are four biological replicate experiments with 24 technical replicate wells per sample. Each data point represents a technical replicate and was analyzed by the non-parametric Mann-Whitney U test. ^∗∗∗p < 0.005.

Figure 6

Increased T Cell-Derived IFN-γ Inhibits MHV68 Reactivation in Atg5^f/f-LysMcre Mice (A) Total numbers of peritoneal CD4, CD8, and NK cells were measured by flow cytometry in latently infected Atg5^f/f and Atg5^f/f-LysMcre mice. Each point represents a single mouse, and data are analyzed by the non-parametric Mann-Whitney U test. No significant differences were observed. (B) Absolute cell numbers and % of cells of IFN-γ-positive CD4 were measured in latently infected Atg5^f/f and Atg5^f/f-LysMcre PECs using intracellular staining after 5 hr of Brefeldin A and monensin A treatment ex vivo. Data are three independent biological replicate experiments and analyzed by non-parametric Mann-Whitney U test. ^∗p = 0.02. (C) CD4 and CD8 T cells were depleted 2 days prior to harvesting PECs. Latently infected PECs were plated onto MEFs for 24 hr, and IFN-γ supernatant levels were measured by ELISA. Efficiency of depletion was measured by flow cytometry. Data were analyzed by a one-way ANOVA with Kruskal-Wallis test with multiple comparisons. ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.001.

Figure 7

IFN-γ Neutralization Rescues MHV68 Reactivation in PECs from Atg5^f/f-LysMcre Mice IFN-γ was neutralized with 250 μg/mouse i.p. every 7 days during the course of MHV68 infection for 28 days in Atg5^f/f and Atg5^f/f-LysMcre, and virus reactivation in PECs was measured ex vivo in the presence of anti-IFN-γ antibody. Data were combined from three biological experiments with 3–5 mice pooled per experiment and represented with the mean ± SEM.