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. 2018 May 14;92(11):e00168-18.
doi: 10.1128/JVI.00168-18. Print 2018 Jun 1.

Deaminase-Dead Mouse APOBEC3 Is an In Vivo Retroviral Restriction Factor

Spyridon Stavrou  1   2 , Wenming Zhao  2 , Kristin Blouch  3 , Susan R Ross  3   2
Affiliations

Deaminase-Dead Mouse APOBEC3 Is an In Vivo Retroviral Restriction Factor

Spyridon Stavrou et al. J Virol. .

Abstract

The apolipoprotein B editing complex 3 (APOBEC3) proteins are potent retroviral restriction factors that are under strong positive selection, both in terms of gene copy number and sequence diversity. A common feature of all the members of the APOBEC3 family is the presence of one or two cytidine deamination domains, essential for cytidine deamination of retroviral reverse transcripts as well as packaging into virions. Several studies have indicated that human and mouse APOBEC3 proteins restrict retrovirus infection via cytidine deaminase (CD)-dependent and -independent means. To understand the relative contribution of CD-independent restriction in vivo, we created strains of transgenic mice on an APOBEC3 knockout background that express a deaminase-dead mouse APOBEC3 due to point mutations in both CD domains (E73Q/E253Q). Here, we show that the CD-dead APOBEC3 can restrict murine retroviruses in vivo Moreover, unlike the wild-type protein, the mutant APOBEC3 is not packaged into virions but acts only as a cell-intrinsic restriction factor that blocks reverse transcription by incoming viruses. Finally, we show that wild-type and CD-dead mouse APOBEC3 can bind to murine leukemia virus (MLV) reverse transcriptase. Our findings suggest that the mouse APOBEC3 cytidine deaminase activity is not required for retrovirus restriction.IMPORTANCE APOBEC3 proteins are important host cellular restriction factors essential for restricting retrovirus infection by causing mutations in the virus genome and by blocking reverse transcription. While both methods of restriction function in vitro, little is known about their role during in vivo infection. By developing transgenic mice with mutations in the cytidine deamination domains needed for enzymatic activity and interaction with viral RNA, we show that APOBEC3 proteins can still restrict in vivo infection by interacting with reverse transcriptase and blocking its activity. These studies demonstrate that APOBEC3 proteins have evolved multiple means for blocking retrovirus infection and that all of these means function in vivo.

Keywords: APOBEC3; MLV; cytidine deamination domains; retrovirus; transgenic mice; virus pathogenesis.

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Figures

FIG 1
FIG 1
Schematic diagram of the mouse APOBEC3 protein and the amino acids mutated in the CD domains (A) and the transgenic construct used to generate the mice (B). The mA3CDmut transgene was crossed onto an APOBEC3 KO background to generate mice that express only the mA3CDmut transgene and not the endogenous mouse APOBEC3. CMV, cytomegalovirus.
FIG 2
FIG 2
Expression of the mA3CDmut transgene. (A) RT-qPCR analysis of RNA isolated from different tissues of the mA3CDhigh and mA3CDlow strains. Shown for comparison are the endogenous APOBEC3 levels in nontransgenic C57BL/6 mice (WT). The mice used for this analysis were uninfected. RNA levels are the average of four individual mice. Error bars denote standard deviations. L. Intest, large intestine; S. Intest, small intestine; M. Gland, mammary gland; mA3, mouse APOBEC3. (B) Transgene expression in MMLV and MMTV in vivo targets of infection. T cells, B cells, peripheral dendritic cells (pDCs), and macrophages (Mac) were purified from mice of each genotype by cell sorting. RNA isolated from the purified cells was analyzed by RT-qPCR for transgene expression. Shown are the averages for cells isolated from four individual mice. Error bars denote standard deviations.
FIG 3
FIG 3
Mouse APOBEC3 (E73Q/E253Q) restricts murine retrovirus infection in vivo. (A) Newborn mice were infected with MMLV, and at 6 dpi, virus titers in spleens were measured. Each point represents the titer obtained from an individual mouse. The horizontal bar in each group represents the average. The transgenic mice were derived from four litters each; the knockout mice are the littermates of the transgenic mice. (B) Four APOBEC3 KO, WT, mA3CDlow, and mA3CDhigh mice were infected with MMTV subcutaneously, and at 4 dpi DNA was isolated from the draining lymph nodes and subjected to RT-qPCR with MMTV-specific primers as previously described (8). Error bars represent standard deviations. ***, P ≤ 0.0001; NS, not significant (one-way analysis of variance).
FIG 4
FIG 4
MMLV WT and MLVgGag virus loads in infected mice. (A) Mice were infected with equal amounts of virus and killed at 18 dpi. Levels of ICs in cells isolated from the spleens of APOBEC3 KO, WT, mA3CDlow, and mA3CDhigh mice infected with MMLV WT and MLVgGag virus were determined. The transgenic mice were derived from two to three litters each; the knockout mice are the littermates of the transgenic mice. (***, P ≤ 0.0001; **, P ≤ 0.001; NS, not significant; Mann Whitney t test). (B) Glyco-Gag mutant virus reverts in mA3CDhigh mice. Mice of the indicated genotype were infected with MLVgGag, and at 6 weeks postinfection, MMLV DNA was isolated from spleens and thymuses of the infected mice, PCR-amplified, and sequenced. WT MMLV has a TAT(Y) codon. Reversion frequency shows the average percentage of sequenced clones in each mouse that showed reversion. The data for the C57BL/6 group and 6/8 of the APOBEC3 KO mice (asterisks) were taken from Stavrou et al. (30).
FIG 5
FIG 5
mA3CDmut is not packaged inside MLV virions. Newborn WT, mA3CDhigh, mA3CDlow, and KO mice were infected with MMLV. Splenic extracts and isolated virions were analyzed by Western blotting. mA3CDmut was detected with precleared anti-mouse APOBEC3 antiserum. Shown is a representative Western blot from two mice per genotype, as indicated, infected with MMLV. This experiment was repeated three times using virions and lysates from different infected litters and gave similar results. The knockout mice are the littermates of the transgenic mice. CA, capsid; A3, APOBEC3.
FIG 6
FIG 6
Expression of mA3CDmut in BMDCs restricts incoming MLV. (A) Infection of BMDCs isolated from KO, WT, mA3CDlow, and mA3CDhigh mice with MMLV isolated from KO mice. RT-qPCR analysis of genomic DNA was performed with MMLV-specific primers and normalized to GAPDH levels. BMDCs were isolated according to standard procedures. Shown are the results of four independent experiments with three technical replicates in each experiment. Error bars indicate standard deviations. *, P ≤ 0.05; **, P ≤.0.0001 (Mann Whitney t test). (B) Mouse APOBEC3 expression levels in BMDCs of the different mice. Shown is RT-PCR analysis of mouse APOBEC3 normalized to GAPDH from BMDCs isolated from two mice of each genotype. Error bars show variations between the two samples.
FIG 7
FIG 7
Coimmunoprecipitation of APOBEC3 and MLV RT. 293T cells were cotransfected with MMLV-RT-myc and mA3/mA3CDmut-HA. (A) Lysates were immunoprecipitated with an anti-myc antibody, and Western blots were probed with an anti-HA antibody. Red fluorescent protein (RFP) was used as a control. Shown is a representative of three different experiments. (B) Quantitative analysis of the amount of immunoprecipitated protein relative to that of the input protein, using ImageJ analysis software (NIH), from three independent experiments. (C) Prior to immunoprecipitation as described for panel A, the lysates were treated with RNase A. Shown is a representative Western blot of three different experiments. IP, immunoprecipitation.
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