Friend virus utilizes the BMP4-dependent stress erythropoiesis pathway to induce erythroleukemia

Abstract

More than 50 years of genetic analysis has identified a number of host genes that are required for the expansion of infected cells during the progression of Friend-virus-induced erythroleukemia. In this report, we show that Friend virus induces the bone morphogenetic protein 4 (BMP4)-dependent stress erythropoiesis pathway in the spleen, which rapidly amplifies target cells, propagating their infection and resulting in acute splenomegaly. This mechanism mimics the response to acute anemia, in which BMP4 expressed in the spleen drives the expansion of a specialized population of stress erythroid progenitors. Previously we demonstrated that these progenitors, termed stress BFU-E, are targets for Friend virus in the spleen (A. Subramanian, H. E. Teal, P. H. Correll, and R. F. Paulson, J. Virol. 79:14586-14594, 2005). Here, we extend those findings by showing that Friend virus infects two distinct populations of bone marrow cells. One population, when infected, differentiates into mature erythrocytes in an Epo-independent manner, while a second population migrates to the spleen after infection, where it induces BMP4 expression and acts as a reservoir of virus. The activation of the stress erythropoiesis pathway in the spleen by Friend virus results in the rapid expansion of stress BFU-E, providing abundant target cells for viral infection. These observations suggest a novel mechanism by which a virus induces a stress response pathway that amplifies target cells for the virus, leading to acute expansion of infected cells.

FIG. 1.

f/f mice have decreased numbers of target cells in the spleen and bone marrow. (A) BALB/c-f/f and BALB/c-f/+ bone marrow (left) and spleen (right) cells were mock infected or infected with FVP and were plated in the indicated cytokines. BFU-E were scored. (B) RT-PCR analysis of Sf-Stk expression in MEPs that were sorted from BALB/c-f/f and BALB/c spleen. Bars represent the averages ± standard deviations from one representative experiment of four independent experiments.

FIG. 2.

PHZ-induced acute anemia expands FV target cells in the spleen but not the bone marrow. (A) BALB/c and BALB/c-f/f mice were treated with PHZ to induce acute anemia. Thirty-six hours and 4 days after treatment, spleen cells were harvested and either mock infected or infected with FVP. The cells then were plated in methylcellulose medium containing Epo, SCF, and IL-3 (mock infected) or SCF and IL-3 (FVP infected), and BFU-E were scored. (B) BALB/c mice were treated with PHZ to induce anemia, and bone marrow cells were harvested 36 h after treatment. The cells were mock infected or infected with FVP and were plated in methylcellulose medium containing Epo, SCF, and IL-3 (mock infected) or SCF and IL-3 (FVP infected), and BFU-E were scored. Significant differences as measured by t tests are indicated. Bars represent the averages ± standard deviations from one representative experiment of four independent experiments.

FIG. 3.

BMP4 treatment increases the number of target cells in the spleen, and its expression is induced in the spleen by FVP infection. (A) BALB/c spleen cells were mock infected or infected with FVP and were plated in methylcellulose medium containing Epo, SCF, and IL-3 (mock infected) or SCF and IL-3 (FVP infected), with or without added BMP4 (15 ng/ml). BFU-E were scored. Significant differences as measured by t tests are indicated. Bars indicate averages ± standard deviations from one representative experiment of three independent experiments. (B) Spleen sections of BALB/c mice isolated on the indicated days postinfection with FVP. The sections are stained with anti-BMP4 antibodies. The lower images are bright-field pictures.

FIG. 4.

FVP-infected bone marrow cells transplanted into f/f recipients cannot propagate the infection in the spleen. BALB/c control bone marrow cells were infected in vitro with FVP and were transplanted into BALB/c-f/f or BALB/c control recipients. (A) On the indicated days, spleens were removed and weighed to determine splenomegaly. Significant differences, as measured by t tests, are indicated. (B) Spleen sections were stained with anti-BMP4 antibodies to determine BMP4 expression. (C) Sections from spleens isolated from BALB/c mice infected with FVP on day 12 (top) or day 15 (bottom) after infection were stained with anti-BMP4 and mAB34, which recognizes FV-infected cells. BMP4 staining is shown in red, mAB34 staining in green, and the overlap is shown in yellow. A bright-field image is included in the lower left panel for each day.

FIG. 5.

CD31⁺ Kit⁺ Sca1⁻ Lin⁻ cells are FV target cells in the bone marrow. (A) The scatter plot on the left depicts flow cytometry analysis of BALB/c Lin⁻ Sca1⁻ cells analyzed for the expression of CD31 and Kit. The box indicates the CD31⁺ Kit⁺ Sca1⁻ Lin⁻ population. The graph on the right depicts CD31⁺ Kit⁺ Sca1⁻ Lin⁻ cells that were sorted from BALB/c bone marrow either mock infected or infected with FVP and plated in methylcellulose medium containing Epo, SCF, and IL-3 (mock infected) or SCF and IL-3 (FVP infected), and BFU-E were scored. (B) Spleen CD31⁺ Kit⁺ Sca1⁻ Lin⁻ cells were mock infected or infected with FVP and were plated in methylcellulose medium containing Epo, SCF, and IL-3 (mock infected) or SCF and IL-3 (FVP infected), and BFU-E were scored. (C) The scatter plot on the left shows the flow cytometry analysis of BALB/c-f/f Lin⁻ Sca1⁻ cells analyzed for expression of CD31 and Kit. The box indicates the CD31⁺ Kit⁺ Sca1⁻ Lin⁻ population. The graph on the right depicts CD31⁺ Kit⁺ Sca1⁻ Lin⁻ cells that were sorted from BALB/c-f/f bone marrow and either mock infected or infected with FVP and plated in methylcellulose medium containing Epo, SCF, and IL-3 (mock infected) or SCF and IL-3 (FVP infected), and were BFU-E scored. (D) RT-PCR analysis of Sf-Stk expression in CD31⁺ Kit⁺ Sca1⁻ Lin⁻ cells isolated from BALB/c-f/f and BALB/c control mice. Significant differences, as measured by t tests, are indicated. The bars represent averages ± standard deviations from one representative experiment of three independent experiments.

FIG. 6.

CD31⁺ Kit⁺ Sca1⁻ Lin⁻ cells act as IC cells. (A) Bone marrow cells from BALB/c control mice were sorted into CD31⁺ Kit⁺ Sca1⁻ Lin⁻ (CD31⁺) or MEP (Kit⁺ CD34⁻ FcγR^lo IL-7Rα⁻ Sca1⁻ Lin⁻) populations, and spleen MEPs were isolated. The sorted cells were infected in vitro with FVP and transplanted into BALB/c control mice. Fourteen days later, the spleens were removed and weighed to test for splenomegaly. Significant differences are indicated by t tests. The data represent the averages ± standard deviations from three independent experiments. (B) Spleen sections from mice transplanted with FVP-infected bone marrow CD31⁺ and spleen MEP cells were stained with anti-BMP4 antibodies. Bright-field images are presented in the lower panels.

FIG. 7.

CD41 expression marks the IC cells present in the bone marrow CD31⁺ Kit⁺ Sca1⁻ Lin⁻ population. (A) Flow cytometry analysis of CD41 expression in the bone marrow CD31⁺ Kit⁺ Sca1⁻ Lin⁻ population of cells. (B) Bone marrow CD31⁺ Kit⁺ Sca1⁻ Lin⁻ cells were sorted into CD41⁺ and CD41⁻ populations. Each population was tested for the ability to form Epo^ind BFU-E following infection in vitro with FVP. Cells were mock infected or infected with FVP and were plated in methylcellulose medium containing the indicated cytokines. BFU-E were scored. (C) Bone marrow CD31⁺ Kit⁺ Sca1⁻ Lin⁻ cells were sorted into CD41⁺ and CD41⁻ populations. Each population was tested for the ability to propagate infection in the spleen after infection with FVP in vitro and were transplanted into BALB/c control mice. Spleens were removed and weighed 14 days posttransplantation (left). Pictures of representative spleens are shown (right). (D) RT-PCR analysis of Sf-Stk expression in CD31⁺ Kit⁺ Sca1⁻ Lin⁻ cells sorted into CD41⁺ or CD41⁻ populations. The expression was analyzed in each population from both BALB/c and BALB/c-f/f mice. Significant differences, as measured by t tests, are indicated. The bars represent the averages ± standard deviations from one representative experiment of two independent experiments.

FIG. 8.

BALB/c-f/f mice exhibit a defect in IC cells in the bone marrow. (A) Flow cytometry analysis of CD41⁺ cells in the CD31⁺ Kit⁺ Sca1⁻ Lin⁻ cells in the bone marrow of BALB/c-f/f mice. (B) BALB/c-f/f and BALB/c control bone marrow cells were infected in vitro with FVP and transplanted into BALB/c control mice. On the indicated days, spleens were isolated and weighed. Significant differences, as measured by t tests, are indicated. The bars represent averages ± standard deviations from one representative experiment of two independent experiments.

FIG. 9.

CD31⁺ Kit⁺ CD41⁺ Sca1⁻ Lin⁻ cells are mobilized into the peripheral blood during infection with FVP in vivo. Peripheral blood mononuclear cells from uninfected (top) and infected (bottom) BALB/c control mice were analyzed by flow cytometry for CD31⁺ Kit⁺ CD41⁺ Sca1⁻ Lin⁻ cells.