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. 2019 Aug 13;10(4):e01678-19.
doi: 10.1128/mBio.01678-19.

A Protein Antagonist of Activation-Induced Cytidine Deaminase Encoded by a Complex Mouse Retrovirus

Gurvani B Singh  1 , Hyewon Byun  1 , Almas F Ali  1 , Frank Medina  1 , Dennis Wylie  2 , Haridha Shivram  1 , Andrea K Nash  1 , Mary M Lozano  1 , Jaquelin P Dudley  3
Affiliations

A Protein Antagonist of Activation-Induced Cytidine Deaminase Encoded by a Complex Mouse Retrovirus

Gurvani B Singh et al. mBio. .

Abstract

Complex human-pathogenic retroviruses cause high morbidity and mortality worldwide, but resist antiviral drugs and vaccine development due to evasion of the immune response. A complex retrovirus, mouse mammary tumor virus (MMTV), requires replication in B and T lymphocytes for mammary gland transmission and is antagonized by the innate immune restriction factor murine Apobec3 (mA3). To determine whether the regulatory/accessory protein Rem affects innate responses to MMTV, a splice-donor mutant (MMTV-SD) lacking Rem expression was injected into BALB/c mice. Mammary tumors induced by MMTV-SD had a lower proviral load, lower incidence, and longer latency than mammary tumors induced by wild-type MMTV (MMTV-WT). MMTV-SD proviruses had many G-to-A mutations on the proviral plus strand, but also C-to-T transitions within WRC motifs. Similarly, a lymphomagenic MMTV variant lacking Rem expression showed decreased proviral loads and increased WRC motif mutations relative to those in wild-type-virus-induced tumors, consistent with activation-induced cytidine deaminase (AID) mutagenesis in lymphoid cells. These mutations are typical of the Apobec family member AID, a B-cell-specific mutagenic protein involved in antibody variable region hypermutation. In contrast, mutations in WRC motifs and proviral loads were similar in MMTV-WT and MMTV-SD proviruses from tumors in AID-insufficient mice. AID was not packaged in MMTV virions. Rem coexpression in transfection experiments led to AID proteasomal degradation. Our data suggest that rem specifies a human-pathogenic immunodeficiency virus type 1 (HIV-1) Vif-like protein that inhibits AID and antagonizes innate immunity during MMTV replication in lymphocytes.IMPORTANCE Complex retroviruses, such as human-pathogenic immunodeficiency virus type 1 (HIV-1), cause many human deaths. These retroviruses produce lifelong infections through viral proteins that interfere with host immunity. The complex retrovirus mouse mammary tumor virus (MMTV) allows for studies of host-pathogen interactions not possible in humans. A mutation preventing expression of the MMTV Rem protein in two different MMTV strains decreased proviral loads in tumors and increased viral genome mutations typical of an evolutionarily ancient enzyme, AID. Although the presence of AID generally improves antibody-based immunity, it may contribute to human cancer progression. We observed that coexpression of MMTV Rem and AID led to AID destruction. Our results suggest that Rem is the first known protein inhibitor of AID and that further experiments could lead to new disease treatments.

Keywords: AID inhibitor; Apobec3; activation-induced cytidine deaminase; mouse mammary tumor virus; retroviruses.

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Figures

FIG 1
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
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
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
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
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
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
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.
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