The AKV murine leukemia virus is restricted and hypermutated by mouse APOBEC3

Abstract

APOBEC3 proteins are potent restriction factors against retroviral infection in primates. This restriction is accompanied by hypermutations in the retroviral genome that are attributable to the cytidine deaminase activity of the APOBEC3 proteins. Studies of nucleotide sequence diversity among endogenous gammaretroviruses suggest that the evolution of endogenous retroelements could have been shaped by the mutagenic cytidine deaminase activity of APOBEC3. In mice, however, APOBEC3 appears to restrict exogenous murine retroviruses in the absence of detectable levels of deamination. AKV is an endogenous retrovirus that is involved in causing a high incidence of thymic lymphoma in AKR mice. A comparative analysis of several mouse strains revealed a relatively low level of APOBEC3 expression in AKR mice. Here we show that endogenous mouse APOBEC3 restricts AKV infection and that this restriction likely reflects polymorphisms affecting APOBEC3 abundance rather than differences in the APOBEC3 isoforms expressed. We also observe that restriction of AKV by APOBEC3 is accompanied by G-->A hypermutations in the viral genome. Our findings demonstrate that APOBEC3 acts as a restriction factor in rodents affecting the strain tropism of AKV, and they provide good support for the proposal that APOBEC3-mediated hypermutation contributed to the evolution of endogenous rodent retroviral genomes.

FIG. 1.

Endogenous APOBEC3 mRNA expression in mice. (A) Total RNA (10 μg) was prepared from the spleens of various strains of mice (3 weeks old). (Top) Northern blots were probed with a fragment of the mouse APOBEC3 (mA3) cDNA encompassing exons 1 to 4. (Bottom) An actin cDNA probe served as a loading control. (B) Comparison of APOBEC3 expression in mice 3 days (3d) or 3 weeks (3w) old. (C) Pictogram showing the alternative splicing of exon 5 of mouse APOBEC3 mRNA. (D) Representation of the proportions of mA3 mRNA splicing variants in various mouse strains. mA3 was amplified from splenic cDNA using flanking primers to the coding sequence and was cloned into the FLAG-C3 plasmid. Exon 5-negative (−E5) and exon 5-positive (+E5) clones are represented as percentages of the total number of clones sequenced.

FIG. 2.

Restriction of MoMLV and AKV by APOBEC3 proteins. (A) The infectivities of MoMLV or AKV particles that were produced by cotransfecting subconfluent 293T cells with 1 μg of either pMOV-eGFP or pAKV-NB-eGFP proviral DNA together with 0.2 μg of one of the FLAG-APOBEC3-expressing vectors were determined by transferring the virus-containing supernatants to NIH 3T3 cells at 36 h posttransfection and monitoring the percentages of cells displaying eGFP fluorescence after a further 48 h. The results are displayed as the level of infection relative to that obtained with cotransfection of the empty vector. Error bars represent the standard errors of the means for six independent experiments (as in panel B). hA2, human APOBEC2; mA3, mouse APOBEC3; hA3G, human APOBEC3G. (B) AKV infectivity, monitored by eGFP fluorescence of infected NIH 3T3 cells as a function of the amount of an APOBEC-expressing plasmid cotransfected with 1 μg of pAKV-NB-eGFP proviral DNA into subconfluent 293T cells during viral production. (C) Packaging of mouse APOBEC3 into AKV and MoMLV virions. Subconfluent 293T cells were cotransfected with 1 μg of either pMOV-eGFP or pAKV-NB-eGFP proviral DNA and 0.2 μg of each of the FLAG-APOBEC3-expressing vectors. After 36 h, virus was collected from the supernatants by ultracentrifugation. Western blot analysis with a horseradish peroxidase-conjugated anti-FLAG antibody was performed on transfected-cell lysates (top) or on virions pelleted from the supernatants (center). (Bottom) Virion encapsidation immunoblots were stripped and reprobed with an anti-p30 (Gag) antibody. CTRL, control. (D) Effects of different mouse APOBEC3 isoforms on AKV infectivity. Assays were performed and results presented as in panel A. +E5, with exon 5; −E5, without exon 5. (E) The expression of the various FLAG-tagged APOBEC proteins in the transfected 293T cells used in panel D was assayed by Western blot analysis using a horseradish peroxidase-conjugated anti-FLAG antibody on transfected-cell lysates. The immunoblot was then stripped and reprobed with an anti-tubulin antibody.

FIG. 3.

APOBEC3-mediated hypermutation of MoMLV and AKV. (A) Pie charts depict the proportions of sequences with the indicated numbers of mutations. The total number of clones sequenced is given at the center of each pie. Below each pie, a line drawing depicts the distribution of G→A mutations along the eGFP gene in the first 10 mutated sequences analyzed in each data set. hA3G, human APOBEC3G; mA3, mouse APOBEC3. (B) Local sequence preference for deamination by hA3G or mA3, computed with respect to the deaminated cytidine (position zero) on the viral minus strand. “n” indicates the total number of mutations analyzed.

FIG. 4.

Restriction of AKV by endogenous mouse APOBEC3. (A, B, and C) Purified mouse splenocytes were infected with replicative MoMLV (A) or AKV (B and C) that had been produced by plasmid transfection into 293T cells. At 72 h postinfection, virus-containing culture supernatants were harvested from the cultured splenocytes and used to infect NIH 3T3 cells. Infection levels were determined by assessing the percentages of eGFP-positive cells 48 h later. Relative infection levels were established by setting the average percentage of eGFP-positive cells from the C57BL/6 mouse cohort to 1. Each point represents the mean of five independent infectivity measurements from a mouse splenocyte preparation. (D) Purified thymocytes from APOBEC3-deficient C57BL/6 mice were infected with AKV, and restriction of the virus was analyzed as for panel B. Each point represents the mean of four independent infectivity measurements from a mouse thymocyte preparation. (E) The pie chart depicts the proportions of sequences with the indicated numbers of mutations. The total number of clones sequenced is given at the center of the pie. A line drawing depicts the distribution of G→A mutations along the eGFP gene in the first 10 mutated sequences analyzed. mA3, mouse APOBEC3. (F) Local sequence preference for deamination by endogenous mouse APOBEC3, computed with respect to the deaminated cytidine (position zero) on the viral minus strand. “n” indicates the total number of mutations analyzed.

FIG. 5.

Restriction of MoMLV and AKV by rat APOBEC3. (A) Amino acid alignment of rat and mouse APOBEC3 proteins. Yellow boxes indicate major amino acid differences; blue boxes indicate conservative amino acid changes. Residues making up exon 5 and the zinc coordination motif of the first and second protein domains are underlined. (B) Encapsidation of rat APOBEC3 by MoMLV and AKV. FLAG-tagged APOBEC3 proteins were detected in transfected cell lysates or in virions pelleted from culture supernatants by Western blot analysis using a horseradish peroxidase-conjugated anti-FLAG antibody. The lysate immunoblots were stripped and reprobed with an anti-tubulin antibody, whereas the virion blots were reprobed with an anti-p30 (Gag) antibody. hA2, human APOBEC2; hA3G, human APOBEC3G; mA3, mouse APOBEC3; ratA3, rat APOBEC3. (C) The infectivities of MoMLV or AKV particles that were produced by cotransfecting subconfluent 293T cells with 1 μg of either pMOV-eGFP or pAKV-NB-eGFP proviral DNA together with 0.2 μg of either the rat, mouse, or human FLAG-APOBEC3-expressing vector (or the human APOBEC2 control) were determined by transferring the virus-containing supernatants to NIH 3T3 cells at 36 h posttransfection and monitoring the percentages of cells displaying eGFP fluorescence after a further 48 h.