Persistence of Systemic Murine Norovirus Is Maintained by Inflammatory Recruitment of Susceptible Myeloid Cells

Abstract

Viral persistence can contribute to chronic disease and promote virus dissemination. Prior work demonstrated that timely clearance of systemic murine norovirus (MNV) infection depends on cell-intrinsic type I interferon responses and adaptive immunity. We now find that the capsid of the systemically replicating MNV strain CW3 promotes lytic cell death, release of interleukin-1α, and increased inflammatory cytokine release. Correspondingly, inflammatory monocytes and neutrophils are recruited to sites of infection in a CW3-capsid-dependent manner. Recruited monocytes and neutrophils are subsequently infected, representing a majority of infected cells in vivo. Systemic depletion of inflammatory monocytes or neutrophils from persistently infected Rag1^-/- mice reduces viral titers in a tissue-specific manner. These data indicate that the CW3 capsid facilitates lytic cell death, inflammation, and recruitment of susceptible cells to promote persistence. Infection of continuously recruited inflammatory cells may be a mechanism of persistence broadly utilized by lytic viruses incapable of establishing latency.

Keywords: inflammation; monocytes; neutrophils; norovirus; persistence.

Figure 1.. The MNV capsid determines lytic cell death and inflammatory cytokine release.

A–B. Diagram of genomes and structures of capsid chimeric viruses used in this study. Strains with the VP1 gene (highlighted region) of CW3 (blue) or CR6 (red) have otherwise identical CW3^D94E (A) or CR6 (B) genomes as depicted. C–I. WT (C–F) or Stat1^−/− (G–I) BMDCs were infected at a MOI of one with the indicated MNV strains. 24hr post-infection, cytokines were detected in cell-free supernatants by protein array (C) or ELISA (D, G); Il1a transcripts (F, I) and MNV genomes (E, H) were quantitated from cells by qPCR. J–K. FACS analysis of cell death was performed on BV2 cells and BMDCs infected at a MOI of one with the indicated MNV strains for 17 hrs or treated with 0.75 µM staurosporine for 3.5 hrs. L. BV2 cells were infected at an MOI of 10, 1, or 0.1 with the indicated MNV strains for 12–14 hrs and LDH was detected in the supernatant. M–N. Stat1^−/− mice were infected with the indicated MNV strain for 48 hrs and serum was collected for protein array (M) and IL-1α ELISA (N). Data is from three to five experiments, with the exception of protein arrays, which were performed once. Statistical significance was determined by one-way (D–I, K) or two-way (L) ANOVA with Tukey’s multiple comparison test. *= p≤0.05, ^**= p≤0.01, ^***= p≤0.001, ^****= p≤0.0001. See also figure S1.

Figure 2.. The MNV capsid determines inflammatory myeloid cell recruitment to infected tissues.

WT (A–C) or Stat1^−/− (D–G) mice were infected with CW3^D94E that used the indicated capsid, and MLNs were isolated 48 hours later. Single-cell suspensions were analyzed by FACS (A–F), and tissue sections were analyzed by immunofluorescence microscopy (G). Scale bar in is 10 microns. Data is from two (G) or four (A–F) experiments with points representing individual mice. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test. *= p≤0.05, ^**= p≤0.01, ^****= p≤0.0001. See also figure S2

Figure 3.. Requirements for inflammatory monocyte and neutrophil recruitment following MNV infection.

A. WT mice were infected with indicated CW3^D94E strains, and Ccl2, Ifnb1, and Ifnl2/3 were quantitated from the MLNs 48 hours later. B–D. WT mice or the indicated knockout mice were infected with CW3 (B–C) or CW3^D94E (D–E) and single cell suspensions of MLNs were analyzed by FACS 48 hours later. F. WT (closed symbols) or Il1r1^−/− (open symbols) mice were injected intraperitoneally with PBS or 200ng of recombinant IL-1α. Peritoneal exudate cells (PECs) were analyzed by flow cytometry 24 hours after injection. G–H. Capsid-domain chimeric viruses (depicted in G) were used to infect WT (closed circles) and Ifnlr1^−/− (open circles) mice, and single cell suspensions of MLNs were analyzed by FACS 48 hours post-infection. Data is from two experiments with points representing individual mice. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test (A, F, H) or by t-test (B–D). *= p≤0.05, ^**= p≤0.01, ^**= p≤0.001, ^****= p≤0.0001. See also figure S3

Figure 4.. MNV preferentially infects recruited monocytes and neutrophils.

Stat1^−/− mice were infected orally using CW3^D94E with the indicated capsid (A–B) or with VP1^CW3 (C–F); MLNs and PP were isolated 48 hours later. A–E. Single-cell suspensions were analyzed using intracellular NS1–2 FACS staining to detect MNV-infected cells. Representative plots are shown in A and C. B. Percent infected of total MLN and PP cells. D. Proportion of the indicated cell types among total and infected cells. E. Percentage of the indicated cell types staining with intracellular NS1–2. F. MLN tissue sections were analyzed by immunofluorescence microscopy for the presence of Gr-1/NS1–2 double-positive cells (arrowheads). Scale bar is 10 microns G–H. Stat1^−/− mice were injected intraperitoneally with 10⁶ PFU CW3^D94E-VP1^CR6 concurrent with 200ng of recombinant IL-1α or PBS as a control. Peritoneal exudate cells (PECs) were analyzed using intracellular NS1–2 FACS staining to detect MNV-infected cells. Data is combined from two (F), three (G–H), or four (A–E) experiments for a total of five to 11 mice per group. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test (B, E) or two-way ANOVA with Sidak’s multiple comparison test (G–H) *= p≤0.05, ^**= p≤0.01, ^**= p≤0.001. See also figure S4.

Figure 5.. Monocyte and neutrophil recruitment is sustained during systemic MNV persistence.

Rag1^−/− mice were infected with CW3^D94E for 21 days. A–D. Single-cell suspensions of MLNs (A, C) and spleens (B, D) were isolated. IMs and neutrophils were analyzed by FACS (A–B); MNV genomes and Ccl2 transcripts were quantitated by qPCR (C–D). Data is from three experiments with points representing individual mice. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test *= p≤0.05, ^**= p≤0.01, ^**= p≤0.001, ^****= p≤0.0001. See also figure S5.

Figure 6.. Sustained monocyte and neutrophil recruitment supports systemic MNV persistence.

Rag1^−/− mice were infected with CW3^D94E for 21 days followed by daily intraperitoneal injection of 20 µg depleting antibodies or isotype control for five days as depicted in panel A. The extent of IM and neutrophil depletion was determined by FACS analysis of blood cells (F) and viral titers were quantitated in the indicated tissues (G) by plaque assay. Data is from two to three experiments with points representing individual mice. Statistical significance was determined by Kruskal-Wallis with Dunn’s multiple comparison test. *= p≤0.05, ^****= p≤0.0001.