Article Figures & Data
Figures
- FIG 1
MMTVs lacking Rem expression have a reduced incidence and increased latency of mammary tumors. (A) Diagram of the Rem precursor and the cleavage products, SP and Rem-CT. Rem is synthesized as a precursor that is cleaved by signal peptidase at the ER membrane into SP (white box) and Rem-CT (yellow box). The ΔSU and ΔTM designations have been used to indicate that Rem represents an in-frame deletion of the Env protein. Env is translated from a singly spliced MMTV mRNA, whereas Rem is translated from a doubly spliced viral mRNA. (B) Western blotting of transfected 293T cell extracts with Rem-CT-specific antibody. Positions of the tagged Rem precursor and cleaved Rem-CT (tagged and untagged) are shown in the upper panel. The tagged precursor has GFP on the N terminus and a T7 tag on the C terminus. The bottom panel shows the same extracts with actin-specific antibody. (C) Strategy for generation of Rem-null MMTV and TBLV proviruses. The thick gray boxes represent long terminal repeats (LTRs); the thinner boxes show open reading frames. The Rem coding region is shown in red. The SD mutation (designated by an X) eliminates the downstream SD site needed to generate the doubly spliced rem mRNA and the singly spliced sag mRNA from the internal env promoter. (D) MMTV-WT and MMTV-SD produce equivalent amounts of Gag in tissue culture cells. Stably transfected XC rat cells expressing MMTV-WT or MMTV-SD were used to harvest cell extracts. Western blots were incubated with CA-specific (upper panel) or GAPDH-specific (lower panel) antibody. Western blotting with CA-specific antibody showed similar amounts of Gag precursor (Pr77) expression in cell extracts. (E) BALB/c mice develop mammary tumors with lower incidence and increased latency after infection with the Rem-null (MMTV-SD) virus. Kaplan-Meier plots reveal a difference in mammary tumor development between mice infected with MMTV-WT (blue line) and those infected with MMTV-SD (green line) (see P value). (F) MMTV-SD-induced mammary tumors have reduced proviral DNA levels relative to MMTV-WT-induced tumors. PCRs were performed using DNA from three mammary tumors derived by inoculation of three independent BALB/c mice with either MMTV-WT or MMTV-SD, and the results were compared with those using uninfected BALB/c DNA containing endogenous Mtv proviruses. Statistical significance of data from comparisons between columns is indicated by an asterisk (P < 0.05).
- FIG 2
Mutational analysis of proviral envelope genes from BALB/c mammary tumors induced by MMTV-WT or MMTV-SD (Rem-null). The number of C mutations within different motifs on either proviral strand is presented graphically for each clone. The number of clones (n) is indicated. Sequences were obtained from independent clones from five tumors in different animals. MMTV-SD proviruses recovered from mammary tumors were classified as non-recombinant (retaining the inoculated MMTV-SD sequence) or recombinant (carrying the wild-type SD2 sequence after recombination with endogenous Mtvs). (A) The number of mutations/clone in the WRC motif typical of AID mutation hot spots. (B) The number of mutations/clone in the SYC motif. (C) The number of mutations/clone in the TYC motif typical of mA3 mutation hot spots. (D) The number of mutations/clone in the ATC motif. Statistical significance of results of nonparametric Mann-Whitney tests is indicated on the scatter plots. The mutant C residue in each motif is underlined.
- FIG 3
Mutational analysis of proviral envelope gene from BALB/c thymic tumors induced by TBLV-WT or TBLV-SD (Rem-null). (A) Kaplan-Meier plots for thymic tumor development in BALB/c mice injected with TBLV-WT (blue line) or TBLV-SD (red line) are shown with P value. (B) TBLV-SD-induced thymic tumors have reduced viral DNA levels relative to TBLV-WT-induced tumors. PCRs were performed using DNA from three thymic tumors derived by inoculation of three independent BALB/c mice with either TBLV-WT or TBLV-SD, and the results were compared with those from uninfected BALB/c DNA containing endogenous Mtv proviruses. Statistical significance of data from comparisons between columns is indicated by an asterisk (P < 0.05). NS = not significant. (C) Analysis of the average number of G-to-A mutations above a 3% threshold within proviruses obtained from three independent TBLV-WT-induced or TBLV-SD-induced tumors after PCR and Illumina sequencing. (D) Analysis of the average number of T-to-C mutations above a 3% threshold within proviruses obtained from three independent TBLV-WT-induced or TBLV-SD-induced tumors after PCR and Illumina sequencing. (E to H) Analysis of independent clones obtained after PCR and Sanger sequencing. TBLV-SD proviral clones recovered from T-cell tumors were classified as non-recombinant (retaining the inoculated TBLV-SD sequence) or recombinant (carrying the wild-type SD2 sequence after recombination with endogenous Mtvs). The number of C mutations within different motifs on either proviral strand is presented graphically for each clone. The number of clones (n) is indicated. Sequences were obtained from independent clones from three tumors in different animals. (E) The number of mutations/clone in the WRC motif typical of AID mutation hot spots. (F) The number of mutations/clone in the SYC motif. (G) The number of mutations/clone in the TYC motif typical of mA3 mutation hot spots. (H) The number of mutations/clone in the ATC motif. Statistical significance of results of nonparametric Mann-Whitney tests is indicated on the scatter plots. The mutant C residue in each motif is underlined. (I and J) Spearman’s correlation coefficient (r) indicating high correlation between number of recombinants and number of mutations in WRC (I) and TYC (J) motifs. Correlation coefficient P values are shown on the graph.
- FIG 4
Mutational analysis of the proviral envelope gene from Aicda−/− BALB/c mammary tumors induced by MMTV-WT or MMTV-SD (Rem-null). (A) Kaplan-Meier plots for mammary tumor development in Aicda−/− BALB/c mice injected with MMTV-WT (blue line) or MMTV-SD (green line) are shown with P value. (B) Proviral loads in MMTV-WT-induced and MMTV-SD-induced mammary tumors relative to endogenous Mtv proviruses in uninfected Aicda−/− mice. Values for three independent tumors from different animals were determined as described for Fig. 1F. Although the values were not significantly different (NS), the trend indicated that MMTV-SD proviral loads were higher than the endogenous Mtv levels. (C to F) MMTV-SD proviruses recovered from mammary tumors were classified as non-recombinant (retaining the inoculated MMTV-SD sequence) or recombinant (carrying the wild-type SD2 sequence after recombination with endogenous Mtvs). The number of C mutations within different motifs on either proviral strand is presented graphically for each clone. Clone numbers (n) are indicated. Sequences were obtained from independent clones from five tumors in different animals after PCR and Sanger sequencing. (C) The number of mutations/clone in the WRC motif typical of AID mutation hot spots. (D) The number of mutations/clone in the SYC motif. (E) The number of mutations/clone in the TYC motif typical of mA3 mutation hot spots. (F) The number of mutations/clone in the ATC motif. Statistical significance of results of nonparametric Mann-Whitney tests is indicated on the scatter plots. The mutant C residue in each motif is underlined.
- FIG 5
Comparison of distributions of mutations/clone within proviral envelope genes between MMTV-WT-induced and MMTV-SD-induced mammary tumors in either wild-type or Aicda−/− BALB/c mice. The number of clones (n) is indicated. Sequences were obtained from independent clones from five tumors in different animals. (A) The number of G-to-A mutations on the proviral plus strand by Sanger sequencing. (B) The number of C-to-T mutations on the proviral plus strand by Sanger sequencing. (C to F) The number of C mutations within different motifs on either proviral strand is presently graphically for each clone. (C) The number of mutations/clone in the WRC motif typical of AID mutation hot spots. (D) The number of mutations/clone in the SYC motif. (E) The number of mutations/clone in the TYC motif typical of mA3 mutation hot spots. (F) The number of mutations/clone in the ATC motif. Statistical significance of results of nonparametric Mann-Whitney tests is indicated on the scatter plots. The mutant C residue in each motif is underlined.
- FIG 6
Rem antagonizes AID. (A) mA3-HA is detectable in MMTV virions. Cells (293T) were cotransfected with MMTV-expressing and mA3-HA-expressing plasmids. Western blots for both cell extracts (C.E.) and the 100-fold concentrated cell supernatant (with or without subtilisin) are shown. Blots were incubated with either MMTV CA-specific antibody (upper panel) or HA-specific antibody (lower panel). (B) AID is not detectable in MMTV virions. Cells (293T) were cotransfected with MMTV-expressing and AID-expressing plasmids. Western blots for both cell extracts (C.E.) and the 100-fold concentrated cell supernatant (with or without subtilisin) are shown. Blots were incubated with either MMTV CA-specific antibody (upper panel) or AID-specific antibody (lower panel). (C) SP is not packaged into wild-type virions. Concentrated TBLV-WT virions were treated for the indicated times with subtilisin to remove cellular proteins external to the virion envelope. Western blots of supernatants were incubated with MMTV CA-specific antibodies (upper panel) or SP-specific antibodies (lower panel). (D) Cotransfection of Rem-expressing provirus leads to reduced mAID-GFP levels, whereas cotransfection of a Rem-null provirus does not. Either TBLV-WT or TBLV-SD expression plasmid was transfected into Jurkat cells in the presence or absence of murine AID tagged with GFP. Western blots were performed with GFP-specific antibody (upper panel) or actin-specific antibody (lower panel). (E) Rem expression leads to decreased mAID-GFP levels and is dependent on the proteasome. Cells (293 line) were cotransfected with expression plasmids for mAID-GFP and either GFP-tagged or untagged Rem. Samples in even-numbered lanes were prepared from cells treated with the proteasomal inhibitor MG-132. Western blots of cell extracts were incubated with GFP-specific or actin-specific antibody (upper and lower panels, respectively). (F) Rem expression does not affect mA3-HA levels. Cells (293 line) were transfected with the indicated amount of mAID-GFP expression vector or mA3-HA in the presence or absence of the indicated amount of untagged Rem expression plasmid. The blot shown at upper left was incubated with GFP-specific antibody, and that shown at upper right was incubated with HA-specific antibody. Actin-specific antibody (lower panels) was used to verify protein loading.
- FIG 7
Model for Apobec-mediated mutations during MMTV replication in vivo. MMTV in maternal milk infects dendritic cells (DCs) in the gut of newborn animals. Virus is transmitted from DCs to T cells, which then likely transmit MMTV to B cells. MMTV-encoded superantigen (Sag) expression on mature B cells (gray lines) is required for efficient MMTV transmission from infected B and T lymphocytes to mammary cells during puberty. Gray lines also depict proteins mediating B-cell and T-cell interactions. Repeated proviral insertions into mammary cells are required to get MMTV insertional activation of proto-oncogenes and mammary tumors. Apobec-mediated proviral mutations, including those induced by AID, occur in the absence of the antagonist Rem during both MMTV-SD replication and TBLV-SD replication in lymphocytes. Recombination within lymphocytes of endogenous Mtvs with SD mutants allows recovery of Rem expression. TLR, Toll-like receptor.
Tables
- TABLE 1
Proviral mutation frequency in BALB/c mammary tumors induced by MMTV-WT and MMTV-SD
Mutation
typeMutation frequencya Fold increase
(SD/WT)MMTV-WT MMTV-SD G to A 0.36 1.28 3.6 A to G 0.56 1.22 2.2 C to T 0.08 0.66 8.3 T to C 1.08 1.38 1.3 Transitions 2.08 4.54 2.2 Transversions 0.56 1.00 1.8 WRC 0.12 0.59 4.9 SYC 0.08 0.28 3.5 TYC 0.00 0.53 – ATC 0.12 0.19 1.6 ↵a Number of mutations/number of clones was determined on the basis of Sanger sequencing of 1,100 bp of the plus strand of at least five proviral env gene clones obtained from each of five independent BALB/c tumors induced by MMTV-WT (n = 25) or MMTV-SD (n = 32). Frequencies of WRC, SYC, TYC, and ATC-motif mutations were calculated from both strands. W = A or T; R = A or G; S = G or C; Y = C or T. For statistical analysis of the data, see scatter plots in Fig. 2. Boldface data highlight the greatest differences between MMTV-WT and MMTV-SD sequences typical of AID-mediated mutagenesis.
- TABLE 2
Proviral mutation frequency in BALB/c T-cell lymphomas induced by TBLV-WT and TBLV-SD
Mutation
typeMutation frequencya Fold increase
(SD/WT)TBLV-WT TBLV-SD G to A 2.02 2.98 1.5 A to G 0.60 1.27 2.1 C to T 0.08 0.73 9.1 T to C 0.86 1.53 1.8 Transitions 3.56 6.51 1.8 Transversions 0.10 0.71 7.1 WRC 0.26 0.67 2.6 SYC 0.14 0.69 4.9 TYC 0.84 1.65 2.0 ATC 0.18 0.47 2.6 ↵a Number of mutations/number of clones was determined on the basis of Sanger sequencing of 1,100 bp of the plus strand of at least fifteen proviral env gene clones obtained from each of three independent BALB/c tumors induced by TBLV-WT (n = 50) or TBLV-SD (n = 55). Frequencies of WRC, SYC, TYC, and ATC-motif mutations were calculated from both strands. W = A or T; R = A or G; S = G or C; Y = C or T. For statistical analysis of the data, see scatter plots in Fig. 3. Boldface data highlight the greatest differences between MMTV-WT and MMTV-SD sequences typical of AID-mediated mutagenesis.
- TABLE 3
Proviral mutation frequency in BALB/c Aicda−/− mammary tumors induced by MMTV-WT and MMTV-SD
Mutation
typeMutation frequencya Fold increase
(SD/WT)MMTV-WT MMTV-SD G to A 0.80 1.65 2.1 A to G 0.56 0.75 1.3 C to T 0.12 0.10 0.8 T to C 0.64 1.28 2.0 Transitions 2.12 3.78 1.8 Transversions 0.20 0.25 1.3 WRC 0.08 0.08 1.0 SYC 0.08 0.73 9.1 TYC 0.64 0.28 0.4 ATC 0.12 0.33 2.8 ↵a Number of mutations/number of clones was determined on the basis of Sanger sequencing of 1,100 bp of the plus strand from at least five proviral env gene clones obtained from five independent BALB/c Aicda−/− tumors induced by MMTV-WT (n = 25) or MMTV-SD (n = 40). Frequencies of WRC, SYC, TYC, and ATC-motif mutations were calculated from both strands. W = A or T; R = A or G; S = G or C; Y = C or T. For statistical analysis of the data, see scatter plots in Fig. 4. Boldface data highlight the greatest differences between MMTV-WT and MMTV-SD sequences typical of AID-mediated mutagenesis.
FIG S1
Rem-null MMTV proviruses lack rem mRNA synthesis and retain SP activity. (A) RNA from XC fibroblast cells stably expressing either MMTV-WT or MMTV-SD and from untransfected cells was used for RT-PCR analyses. Primers were designed for the MMTV LTR (upper panel) or the Gapdh gene (lower panel). (B) Transient transfections of MMTV-WT and MMTV-SD in XC rat fibroblasts. Cells were transfected and incubated in the presence or absence of dexamethasone (Dex) as indicated to stimulate the hormone-inducible MMTV LTR. SP activity as measured by a Renilla luciferase reporter assay is preserved by the Rem-null MMTV provirus. NS, not significant. Transfection of a CMV promoter-driven Rem expression plasmid was used as a positive control. The majority of SP activity was derived from Env cleavage, with results indicating no difference in Env production levels between MMTV-WT and MMTV-SD. Download FIG S1, TIF file, 0.6 MB.
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FIG S2
MMTV-SD recombinant proviruses lack stop codons. Amino acid differences between the MMTV-WT and MMTV-SD recombinant proviruses in the env gene were determined for individual clones by Sanger sequencing. Proviral gene mutations in the absence of Rem yielded recombinants that lack stop codons within the env open reading frame of the MMTV-SD proviruses. Download FIG S2, JPG file, 2.0 MB.
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FIG S3
TBLV-SD proviruses retain SP activity and produce equivalent amounts of virus in tissue culture cells. (A) RNA from human Jurkat cells stably expressing either TBLV-WT or TBLV-SD and from untransfected cells was used for RT-PCRs. Primers were designed for the TBLV LTR (upper panel) or the GAPDH gene (lower panel). (B) Transient transfections of TBLV-WT and TBLV-SD in human Jurkat cells. The SP activity of TBLV-SD proviruses (as measured by a Renilla luciferase reporter assay) was preserved. SP was synthesized primarily from env mRNA, which was more abundant than rem mRNA. Transfection of a CMV-promoter-driven Rem expression plasmid was used as a positive control. The asterisk indicates significance (P < 0.05). (C) Stably transfected Jurkat cells expressing TBLV-WT or TBLV-SD were used to harvest supernatants or for preparation of cytosolic extracts. Western blotting showed similar amounts of Gag expression in whole-cell lysates and released virus in supernatants from cells transfected with expression plasmids for TBLV-WT or TBLV-SD. Western blots were incubated with CA-specific or actin-specific antibody. Arrows indicate the positions of cleaved capsid or capsid precursor (Pr77Gag). Download FIG S3, TIF file, 1.4 MB.
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TABLE S1
Reversion of the SD site in TBLV-SD proviruses detected by Illumina sequencing. Download Table S1, DOCX file, 0.02 MB.
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FIG S4
Analysis of high-throughput data obtained from Illumina sequencing of products obtained by PCR of TBLV-induced tumor DNA. Reads were analyzed from tumor-derived TBLV-WT proviruses (gray columns) or TBLV-SD proviruses (black columns) (each obtained from three independent tumors as described in Materials and Methods). The TBLV data were obtained from a mixture of PCR products derived from the pol, env, and 3′ LTR regions on the proviral plus strand. For each reference base of TBLV (upper panels) or Gapdh (lower panels), the alternative base is given on the x axis. None of the Gapdh changes were significantly different between the TBLV-WT and TBLV-SD proviruses. G-to-A changes were highly statistically significantly different (P = 6.3 × 10−8) between the TBLV-WT and TBLV-SD proviruses, but T-to-C transitions also were statistically significantly different (P = 0.026). Although TBLV-SD proviruses tended to have greater numbers of other transition and transversion mutations, these changes were not significantly different by the mixed-effect logistic regression model. (A) Data averaged for proviruses from all three TBLV-WT and TBLV-SD tumors. (B) Data for proviruses from individual tumors. Download FIG S4, TIF file, 1.0 MB.
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FIG S5
TBLV-SD revertant proviruses have stop codons. Amino acid differences between TBLV-WT and TBLV-SD recombinant proviruses in the env gene were determined for individual clones by Sanger sequencing. Unlike MMTV-SD recombinants, increased viral gene mutations in the absence of Rem resulted in increased numbers of stop codons within the env open reading frame of the TBLV-SD recombinant proviruses. Stop codons are shown with an asterisk and are boxed in red. Download FIG S5, JPG file, 1.6 MB.
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TABLE S2
Mutation frequency in the c-Myc gene from TBLV-WT and TBLV-SD proviruses from thymic tumors determined by Sanger sequencing. Download Table S2, DOCX file, 0.01 MB.
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FIG S7
Correlation analysis of the number of recombinants and sum of mutations in different motifs from MMTV-induced tumors and TBLV-induced tumors from wild-type BALB/c mice. Recombinants that repaired the SD mutations within C residues of the indicated motifs were analyzed. (A to D) Spearman’s correlation coefficient (r) data indicating high correlation between the number of recombinants and the sum of mutations in WRC (A), TYC (B), SYC (C), and ATC (D) motifs, respectively, as determined by Sanger sequencing of proviral envelope genes. Correlation coefficient P values are shown on the graph. Download FIG S7, TIF file, 0.3 MB.
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FIG S6
Kaplan-Meier survival plots for MMTV-infected mice on the BALB/c background. Aicda-sufficient BALB/c mice were infected with MMTV-WT or MMTV-SD (blue or green lines, respectively). Aicda-deficient BALB/c mice were infected with MMTV-WT or MMTV-SD (red or purple lines, respectively). No significant differences were observed (P values are shown) for MMTV-WT-infected mice (A) or MMTV-SD-infected mice (B) in the presence or absence of AID. Download FIG S6, TIF file, 0.2 MB.
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FIG S8
Correlation analysis of the number of recombinants and sum of mutations in different motifs from MMTV-induced tumors from Aicda−/− mice. Recombinants that repaired the SD mutations within C residues of the indicated motifs were analyzed. (A to D) Spearman’s correlation coefficient (r) data indicating high correlation between the number of recombinants and the sum of mutations in the WRC (A), TYC (B), SYC (C), and ATC (D) motifs, respectively, as determined by Sanger sequencing of proviral envelope genes. A correlation coefficient P value could be calculated only for the SYC motif. Download FIG S8, TIF file, 0.3 MB.
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