Enhanced replication and pathogenesis of Moloney murine leukemia virus in mice defective in the murine APOBEC3 gene

Abstract

Human APOBEC3G (hA3G), a member of the AID/APOBEC family of deaminases, is a restriction factor for human immunodeficiency virus (HIV). In the absence of the viral Vif protein hA3G is packaged into virions and during reverse transcription in a recipient cell it deaminates cytosines, leading to G-->A hypermutation and inactivation of the viral DNA. Unlike humans, who carry seven APOBEC3 genes, mice only carry one, mA3. Thus the role of mA3 in restriction of retroviral infection could be studied in mA3 -/- knockout mice, where the gene is inactivated. M-MuLV-infected mA3 -/- mice showed substantially higher levels of infection at very early times compared to wild-type mice (ca. 2 logs at 0-10 days), particularly in the bone marrow and spleen. Restriction of M-MuLV infection was studied ex vivo in primary bone marrow-derived dendritic cells (BMDCs) that express or lack mA3, using an M-MuLV-based retroviral vector expressing enhanced jellyfish green fluorescent protein (EGFP). The results indicated that mA3 within the virions as well as mA3 in the recipient cell contribute to resistance to infection in BMDCs. Finally, M-MuLV-infected mA3 +/+ mice developed leukemia more slowly compared to animals lacking one or both copies of mA3 although the resulting disease was similar (T-lymphoma). These studies indicate that mA3 restricts replication and pathogenesis of M-MuLV in vivo.

Figure 1. M-MuLV infection in C57/Bl6 and mA3−/− mice

Neonatal mice were inoculated i.p. with M-MuLV. At 6, 8, 10 and 12 days post inoculation, the animals were sacrificed and single cell suspensions were prepared from bone marrow, thymus and spleen. The levels of infection (infectious centers (ICs)/10⁶ cells plated) are shown. Each point represents results from one animal. Circles, C57/Bl6; diamonds, mA3−/−. Mean values are indicated by horizontal bars. A) Bone marrow, B) Spleen, C) Thymus.

Figure 2. M-MuLV infection in mA3+/− F2 mice

Neonatal mice from inter-crosses between two mA3 +/− mice were inoculated i.p. with M-MuLV. At 6, 8, 10, 12 and 14 days post-inoculation, the animals were sacrificed and single cell suspensions were prepared from bone marrow, thymus and spleen. Genotypes of the animals were determined. The levels of infection (ICs/10⁶ cells plated) are shown. Each point represents results from one animal. Squares, mA3+/+ mice; triangles, mA3+/− mice; circles, mA3−/− mice. Mean values are indicated by horizontal bars. A) Bone marrow, B) Spleen, C) Thymus.

Figure 3. M-MuLV infection of BMDCs

(A) Protein levels in LEGFP-N1 vector stocks Western produced in the absence (pcDNA 3.1) or presences of mA3HA or mA3Δexon5HA. Western blots for mA3 (anti-HA) and MuLV CA protein (anti-p30) are shown. All samples were run on the same gel (B) After standardization of viral stocks by RT-PCR for vector RNA, BMDCs from mA3 +/+ and −/− mice were infected by vector produced in the absence (light grey) or presence (dark grey) of mA3Δexon5HA. FACS analysis for EGFP expression after 24 hr determined infection levels as shown.

Figure 4. Pathogenesis by M-MuLV in mA3 −/− animals

Kaplan-Meier plots of survival of animals from mA3 +/− intercrosses after i.p. inoculation with M-MuLV. Dashed line, mA3−/− animals (n=13; median latency 208 days); dotted line, mA3+/− animals (n=17; median latency 178 days); and the solid line, mA3+/+ animals (n=10, median latency 269.5 days).

Figure 5. Kidney tumors in mA3 −/− and mA3+/− animals

(A) H&E staining of kidney tissue from an mA3−/− animal showing lymphocyte infiltrates (B) Immunohistochemistry for CD3 staining in the same kidney verified that infiltration was T-lymphoid. (C) H & E staining of a tumor from an mA3+/− animal also shows lymphocyte infiltrates in the kidney.