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J Virol. Apr 2009; 83(7): 3029–3038.
Published online Jan 19, 2009. doi:  10.1128/JVI.02536-08
PMCID: PMC2655539

Expression of Murine APOBEC3 Alleles in Different Mouse Strains and Their Effect on Mouse Mammary Tumor Virus Infection[down-pointing small open triangle]


Recent work has shown that mouse APOBEC3 restricts infection by mouse mammary tumor virus (MMTV) and murine leukemia virus (MLV) and that there are polymorphic APOBEC3 alleles found in different inbred mouse strains. For example, C57BL/6 mice, which are resistant to Friend MLV (F-MLV), encode a APOBEC3 gene different from that encoded by F-MLV-susceptible BALB/c mice; the predominant RNA produced in C57BL/6 mice lacks exon 5 (mA3−5) and encodes a protein with 15 polymorphic amino acids. It has also been reported that BALB/c mice produce only a variant RNA that lacks exon 2 (mA3−2). In this study, we examined the effect of these polymorphic APOBEC3 proteins on MMTV infection. We found that the major RNA made in C57BL/6 and B10.BR mice lacks exon 5 but that BALB/c and C3H/HeN mice predominantly express an RNA that contains all nine exons. In addition to producing the splice variant, C57BL/6 and B10.BR cells and tissues had levels of mA3 RNA fivefold higher than those from BALB/c and C3H/HeN mice. A cloned C57BL/6-derived mA3 protein lacking exon 5 inhibited MMTV infection better than a cloned full-length protein derived from 129/Ola RNA when packaged into MMTV virions. We also tested dendritic cells derived from different inbred mouse strains for their abilities to be infected by MMTV and showed that susceptibility to infection correlated with the presence of the exon 5-encoding allele. In vivo susceptibility to infection cosegregated with the inherited mA3 allele in a C57BL/6 × BALB/c backcross analysis. Moreover, virus produced in vivo in the mammary tissue of mA3 knockout and BALB/c mice was more infectious than that produced in the tissue of C57BL/6 mice. These data indicate that mA3 plays a role in the genetics of susceptibility and resistance to MMTV infection.

APOBEC3 (apolipoprotein B mRNA-editing complex; A3) proteins are host cellular restriction factors that have antiviral activities against exogenous and endogenous viruses, as well as retrotransposons (13, 17). Following incorporation of A3 protein into budding human immunodeficiency virus type 1 (HIV-1) virions and infection of cells, A3 deaminates cytosine residues in the minus strand of the nascent viral DNA during reverse transcription, converting deoxycytidine to deoxyuracil; this results in G-to-A transitions in the retroviral genome and is referred to as cytidine deamination-dependent restriction (11, 17). Cytidine deamination-independent inhibition of infection has also been described previously (27).

While much is known about the role that human A3 proteins play in restricting virus infection and retrotransposition, this has largely been defined in tissue culture cells, and less is known about their in vivo function. The human genome contains seven A3 genes, the mouse genome contains a single copy of the A3 gene (11, 16, 21). This has allowed several groups to study retrovirus infection in mice with targeted deletion of the A3 gene, as well as genetic variations of this gene in different inbred mouse strains. For example, we showed several years ago that murine A3 (mA3) inhibits mouse mammary tumor virus (MMTV) replication in mice and that this inhibition was not due to cytidine deamination (28). More recently, several groups have shown that mA3 inhibits Friend murine leukemia virus (F-MLV) and Moloney MLV (M-MLV) (22, 37, 40); mA3-mediated restriction of F-MLV also appeared to be independent of cytidine deamination (40). It has long been known that different inbred strains of mice are resistant or susceptible to infection by F-MLV (38). One of the resistance genes, recovery from Friend virus 3 (Rfv3), maps to the genomic location where mA3 is encoded (10, 18, 20, 25), and thus, two groups tested whether there were polymorphic differences between F-MLV-resistant and -susceptible mice and if mA3 restricted F-MLV. Indeed, restriction of F-MLV infection depended on the mA3 allele expressed. F-MLV-resistant strains, such as C57BL/6, predominantly express an mRNA lacking exon 5 (C57-mA3−5) encoding a ~49-kDa protein, while susceptible mice, such as BALB/c mice, express an RNA (BALB-mA3+5) encoding an ~51-kDa protein (40); in one study, it was also reported that BALB/c mice made an mA3−2 RNA that would encode an ~38-kDa protein (37). Additionally, the two alleles potentially encode proteins with 15 different polymorphic amino acids.

Inbred mouse strains differ in their responses to MMTV infection as well (reviewed in references 30 and 35). Because we showed previously that mA3 played an important role in restricting MMTV infection, we examined here whether there was also a difference in the abilities of the different allelic variants to restrict MMTV infection. We first sequenced several introns and exons of the mA3 genes present in a number of different MMTV-susceptible and -resistant mouse strains and showed that some but not all MMTV-resistant strains contain a C57-mA3−5-like allele. We also show that, like the case with F-MLV infection, C57-mA3−5 more potently inhibited MMTV infection than that from strain 129 mice (129-mA3+5) when packaged into virions produced in transfected cells and that virions made in the mammary tissues of C57BL/6 mice were less infectious than those made in BALB/c mice. Finally, we show that increased susceptibility to MMTV infection, at least at early times after infection, genetically cosegregates with the BALB-mA3+5 allele.


Mice and mouse DNA.

B10.BR-H2k H2-T18a/SgSnJ (B10.BR) mice were purchased from the Jackson Laboratory. BALB/c (BALB/cAnNCrl), C3H/HeN (C3H/HeNCrl), and C57BL/6 (C57BL/6NCrl) mice were purchased from the National Cancer Institute (NCI). The mA3-deficient mice, which were backcrossed 10 generations onto a C57BL/6J background, have been previously described (28). (BALB/c × C57BL/6)F1 and [(BALB/c × C57BL/6)F1 × BALB/c]G2 progeny were generated at the University of Pennsylvania. Mice were housed according to the policies of the Institutional Animal Care and Use Committee of the University of Pennsylvania. In addition to DNA obtained from the mouse strains described above, DNAs from the other strains of mice were obtained as follows: NAMRU DNA was isolated from NMuMG normal murine mammary gland epithelial cells; A.BY, MOLF/EiJ, Pera/EiJ, YBR/EiJ, I/LnJ, MA/MyJ, C57BL/10J, C57BR/cdJ, C57L/J, C58/J, WSB/EiJ, SB/LeJ, SPRET/EiJ, and SWR/J DNAs were purchased from the Jackson Laboratory; 129/Ola DNA was a kind gift from Nika Lovsin.

Cell culture and transfection.

Bone marrow-derived dendritic cells (BMDCs) were generated according to published procedures (23). The DCs were cultured for eight days at 37°C with 5% CO2 in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.05 mM 2-mercaptoethanol, and 20 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (Peprotech, Inc., Rocky Hill, NJ). mA3−/− murine embryonic fibroblasts (MEFs; MEFs from mA3−/− mice immortalized with simian virus 40 [SV40] T antigen), 293T, 293T-mTfR1 (293T cells that stably express MMTV entry receptor transferrin receptor 1 [mTfr1]) (44), and CGRES6 (CrFK cells stably transfected with pGR102ES, a green fluorescent protein [GFP]-tagged molecular MMTV clone) (19) were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin; the 293T-mTfR1 and CGRES media were supplemented with Geneticin (100 μg/ml), and MEF medium was supplemented with 0.05 mM 2-mercaptoethanol.

Virus production.

All transient transfections were performed using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Inc.). The C57-mA3−5 and 129-mA3+5 expression plasmids were previously described (36). For the production of GR102ES virus, 293T cells were cotransfected with pGR102ES, RSVGR, and hemagglutinin (HA)-tagged C57-mA3−5, 129-mA3+5, or empty vector (pcDNA3.1), or CGRES cells stably producing GR102ES were transfected with HA-tagged C57-mA3−5, 129-mA3+5, or pcDNA3.1. For all cells, 24 h posttransfection, virion production was induced by the addition of 0.5 μM dexamethasone. Twenty-four hours postinduction, the virus-containing culture supernatants were harvested, treated with 20 U/ml of DNase I (Roche, Inc., Nutley, NJ) for 30 min at 37°C to remove any residual plasmid DNA, and pelleted through a 30% sucrose cushion in phosphate-buffered saline by centrifugation at 105,000 × g for 1 h. All cell lysates and virus preparations were analyzed by Western blot analysis with anti-HA and anti-MMTV antisera to ensure that similar levels of virions were produced, as previously described (28). Milk- or tumor-isolated virions were isolated from mA3−/−, C57BL/6, and BALB/c mice infected with the MMTV(RIII) strain as previously described (15) and were analyzed by Western blotting using anti-MMTV antisera and by reverse transcription—real-time PCR to examine viral RNA levels (see below).

Infection of DCs, MEFs, and 293T-mTfR1 cells.

DCs, MEFs, and 293T-mTfR1 cells were infected with GR102ES or GR102ES with C57-mA3−5 or 129-mA3+5 as indicated in the figure legends. Control cells were treated with 3 mg/ml of the reverse transcriptase inhibitor azidothymidine (AZT; Sigma, Inc.) at 37°C for 2 h prior to infection. DCs and MEFs were infected by the spinoculation method as previously described (12). The cells were cultured for 24 h after infection prior to harvesting. In some experiments, DCs were matured with 100 ng/ml of lipopolysaccharide (LPS; Sigma, Inc., St. Louis, MO) for 24 h prior to infection. Differentiation into mature DCs was assessed by flow cytometry detection of cell surface expression of CD40 and CD86 (not shown).

Infection of mice.

In vivo infection of mA3−/−, C57BL/6, BALB/c, F1, and G2 mice was performed with MMTV(RIII). Briefly, three mA3−/−, C57BL/6, BALB/c, and F1 mice each and 11 G2 mice received a single subcutaneous footpad injection of virus. Ninety-six hours after injection, the mice were sacrificed and the draining lymph nodes from each mouse were harvested and used for DNA isolation. In addition, RNA isolated from the lymph nodes or spleen was used to determine the genotype of the mice, using primers for exons 4 and 7 (see below).

Infectivity assays.

DNA was isolated from the draining lymph nodes of infected mice by using DNeasy (Qiagen, Inc.) according to the manufacturer's instructions. DNA was used for real-time quantitative PCR (RT-qPCR) to detect integrated proviruses, as previously described, using primers specific to the MMTV(RIII) long terminal repeat and were normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (12, 28). In some experiments, fluorescence-activated cell sorter (FACS) analysis was used to detect infection of DCs, MEFs, and mA3-transfected 293T-mTfR1 cells by pGR102ES virus; data are presented as percentages of GFP-positive cells. All infectivity assays were done in triplicate.

Western blots.

Western blots of virus preparations or cell lysates from transient transfections to test for mA3 packaging and expression were probed with anti-HA (Invitrogen, Inc.), and antitubulin antibodies (Neomarkers, Fremont, CA). Western blots of tumor virus preparations were probed with anti-total MMTV (National Cancer Institute Biochemical Carcinogenesis Branch Repository, Bethesda, MD). The species-appropriate horseradish peroxidase-conjugated secondary antibody was used, followed by detection with ECL reagents (Amersham Biosciences, Inc.) or the Odyssey infrared imaging system (LI-COR Biosciences).

Reverse transcription-RT-qPCR to measure mA3 levels and examine alternative splicing.

Total RNA was isolated from the DCs of mA3−/−, C57BL/6, BALB/c, and F1 mice or spleens of mA3−/−, C57BL/6, BALB/c, F1, G2, B10.BR, and C3H/HeN mice using the RNeasy minikit (Qiagen, Inc.) according to manufacturer's instructions. Isolated RNA was treated with DNase I (Qiagen, Inc.) and reverse transcribed with the First Strand cDNA synthesis kit (GE Healthcare/Amersham Biosciences). To look at alternatively spliced mRNAs, the cDNAs were subjected to PCR using the primers shown in Fig. Fig.22 (primer sequences are found in Table S1 in the supplemental material). For reverse transcription-RT-qPCR, the cDNA was amplified with mA3 exon 6/exon 7-specific primers (see Table S2 in the supplemental material); all amplifications were normalized to GAPDH levels. RT-qPCR was carried out using the ABI 7900 instrument as previously described (28).

FIG. 2.
Alternatively spliced mA3 RNAs in different mouse strains. (A) Schematic of the mA3 gene containing nine exons. Shown are the locations and directions of the PCR primers used for amplification. (B) Reverse transcription PCR analysis of spleen RNA from ...

Sequencing of mA3 genomic DNA and cDNA from different mouse strains.

Genomic DNA from different mouse strains was PCR amplified, analyzed by agarose gel electrophoresis, and sequenced using the primers shown in Table S2 in the supplemental material. Sequences were aligned and compared using Multialign software to look for nucleotide polymorphisms. Sequences from Celera (accession no. NW_001030577) and NCBI (accession no. NT_039621) were also used for this comparison.

Statistical analyses.

All error bars presented represent standard deviations. The statistical significance of differences between groups was tested using the paired two-tailed Student t test.


Genetic deletions and polymorphisms observed in mA3 genes from different inbred mouse strains.

Sequence analyses of genomic DNA, as well as genetic and functional studies of RNAs, have identified several allelic forms of the mA3 gene in different mouse strains. These include an allele encoding the full-length mRNA with intact exon 5 (mA3+5) and one encoding a shorter form lacking exon 5 (mA3−5) (1, 24, 25). In addition to demonstrating the deletion of exon 5 in some strains of mice, Takeda et al. (in 2008) showed that there were polymorphic differences between the mA3 coding sequences of the BALB/c and C57BL/6 strains (40). To investigate if these or additional polymorphisms exist in different inbred mouse strains, we sequenced DNA around and including exon 5 from a total of 20 different inbred mouse strains (Fig. (Fig.1A;1A; see Fig. S1 in the supplemental material). Sequence analysis revealed that there are only two allelic versions of the mA3 exon 5 found in the different Mus strains and species (M. musculus domesticus, M. musculus musculus, M. spretus, and M. musculus molossinus) examined here (Fig. (Fig.1A).1A). Interestingly, there is a point mutation in a putative splice branch selection sequence as well as a deletion of four nucleotides in the intron between exons 4 and 5 of all mice of the black subline, as well as two other strains (MOLF/EiJ and WSB/EiJ) (Fig. (Fig.1B;1B; see Fig. S1 in the supplemental material). This group includes C57BL/6 and B10.BR mice, which both predominantly express the mA3−5 mRNA (see Fig. Fig.2;2; see below). Similarly, BALB/c and C3H/HeN mice retain common polymorphisms in exon 3, and these are different from those found in C57BL/6 and B10.BR mice (not shown).

FIG. 1.
Allelic differences in the exon 5 region of mA3 genes of different inbred mouse strains. (A) The strain genealogy is based on that documented by Beck et al. (3). Sequence data are presented in Fig. S1 in the supplemental material. Circled strains have ...

In addition to studying the mA3−5 RNA produced in C57BL/6 mice, Santiago and colleagues showed that BALB/c and A.BY mice expressed a mA3−2 splice variant, and their data suggested that this variation was responsible for the susceptibility of these strains to F-MLV (37). To determine if there were genetic differences between the different strains around exon 2, we used primers for exons 1, 2, and 3 to PCR amplify genomic DNA from two C57BL-derived strains (C57BL/6 and B10.BR) as well as C3H/HeN, BALB/c, 129/Ola, and A.BY mice. The exon 1/3 PCR products from the C57BL-derived strains were approximately 5.2 kb in length, while those from the four other strains were approximately 3.9 kb (Fig. (Fig.1C).1C). This size difference was due, at least in part, to a deletion of approximately 535 bp, as well as several smaller deletions of intronic sequence between exons 2 and 3 of the mA3 genes in C3H/HeN, BALB/c, 129/Ola, and A.BY mice (Fig. (Fig.1C).1C). Sequence analysis of exons 1, 2, and 3 and the surrounding introns again distinguished the C57BL-derived mice from the other strains but did not reveal any significant polymorphic differences between the BALB/c, 129/Ola, A.BY, and C3H/HeN genes (see Fig. S2 in the supplemental material). As reported by Takeda et al., there were several polymorphisms in exon 2 of BALB/c mice compared to that of C57BL/6 mice (40); exon 2 of C3H/HeN mice was identical to those of BALB/c and 129/Ola, while the B10.BR exon was the same as that found in C57BL/6 mice. Importantly, the splice donors and acceptors for exons 1, 2, and 3 were identical in all the strains (see Fig. S2 in the supplemental material). Thus, at least for the strains examined here, there appear to be only two mA3 alleles present in common inbred mouse strains.

We next used a PCR-based assay to determine which mRNAs were made by the different strains that are resistant (C57BL/6 and B10.BR) or susceptible (BALB/c and C3H/HeN) to MMTV infection. Using eight different primer pairs within the mA3 exons to detect alternative splicing of exon 2 (exon 1/3, 2/5, and 2/7 primers; Fig. Fig.2A)2A) and exon 5 (exon 2/5, 2/7, 4/5, and 4/7 primers; Fig. Fig.2A)2A) in splenic RNA from these four mouse strains, we found that C57BL-derived strains made high levels of the mA3−5 mRNA and low levels of the mA3+5 mRNA; the converse was true for C3H/HeN and BALB/c mice (exon 2/5, 2/7, 4/5, and 4/7 primers; Fig. Fig.2B).2B). Additionally, we found that all four mouse strains predominantly produced a mA3+2 mRNA (Fig. (Fig.2B,2B, exon 1/3 and exon 2/7 panels); the mA3−2 mRNA was produced at very low levels in BALB/c and C3H/HeN mice (Fig. (Fig.2B,2B, exon 1/3 panel). Taken together, these data show that there are two mA3 RNAs expressed in inbred mouse strains, which encode either an mA3+5 protein or an mA3−5 protein.

Packaged C57-mA3−5 is a more efficient inhibitor of MMTV in tissue culture.

Previously, we showed that 129-mA3+5 protein is efficiently incorporated into MMTV virions and inhibited infection (28, 29). Several groups have shown that 129-mA3+5 and C57-mA3−5 are incorporated into MLV virions and that C57-mA3−5 inhibits infection better than 129-mA3+5 (7, 40). To determine if C57-mA3−5 was also incorporated into MMTV virions, we cotransfected both mA3 isoforms along with the GFP-tagged MMTV molecular clone GR102ES into 293T cells. Virions isolated from these cells were first examined by Western blotting. We found that the C57-mA3−5 isoform was packaged into MMTV virion, although at slightly lower levels than the 129-mA3+5 isoform (Fig. (Fig.3A3A).

FIG. 3.
Packaging of 129-mA3+5 and C57-mA3−5 into MMTV virions. (A) Western blot of virions produced by cells expressing the 129-mA3+5 or C57-mA3−5 variants. The table shows the relative amounts of mA3 and p27 proteins in the virus ...

We next tested if C57-mA3−5 restricted MMTV infection. GR102ES virions produced without mA3 or containing 129-mA3+5 or C57-mA3−5 were used to infect 293T cells stably expressing mTfR1. At 24 h after infection, fivefold and twofold fewer cells were infected with the C57-mA3−5 virions than with virions without mA3 or those containing the 129-mA3+5 isoform, respectively; this was also the case at 48 h postinfection (Fig. (Fig.3B).3B). Virions containing the 129-mA3+5 isoform were also twofold less infectious than those without mA3, indicating that this isoform, while less effective, still retains anti-MMTV activity.

To test if packaged C57-mA3−5 was also more restrictive in mouse cells, we infected primary DCs and SV40-immortalized MEFs from mA3−/− mice with MMTV containing the two mA3 alleles. Results similar to those obtained with the 293T cells were observed (Fig. 4A and B). Additionally, the C57-mA3−5-containing virus seemed to inhibit infection of both 293T and MEFs better than it did infection of DCs; this cell-type-specific difference was not examined further. To study the efficiency of virus spread in mouse cells, we also monitored infection levels of the mA3−/− MEFs at 96 and 192 h postinfection. We observed that both forms of mA3 maintained their relative inhibitory effects compared to virus lacking mA3, with C57-mA3−5 still inhibiting better than 129-mA3+5 96 h after infection (Fig. (Fig.4B).4B). However, when infection was extended up to 192 h (8 days) in the mA3−/− cells, the initial effects of either form of mA3 were abolished and levels of infection reached those of virions lacking mA3 (Fig. (Fig.4B).4B). At this time point, all of the newly synthesized virions would lack either form of mA3, and thus, infection resembles that caused by mA3-lacking virions.

FIG. 4.
Packaged C57-mA3−5 restricts infection of mA3−/− BMDCs (A) and MEFs (B) better than 129-mA3+5. Cells were analyzed by FACS at 24 h (A) or at the indicated times (B) after infection. AZT-treated cells were harvested at 24 ...

mA3 RNA is expressed at higher levels in the BMDCs of C57BL-derived mice.

Next, we investigated the effect of mA3−5 and mA3+5 on MMTV infection when expressed at physiological levels. To this end, we produced BMDCs from the C57BL-derived mice, B10.BR and C57BL/6 (which express mA3−5), two strains that express the mA3+5 allele, C3H/HeN and BALB/c, and (C57BL/6 × BALB/c)F1 mice. First, we used reverse transcription-RT-qPCR to measure the relative levels of mA3 RNA, using primers to a nonpolymorphic region of the gene (exon 6/7) and found that C3H/HeN and BALB/c mice expressed ~5-fold lower levels of mA3 RNA than either B10.BR or C57BL/6 mice, while the (C57BL/6 × BALB/c)F1 mice expressed intermediate levels (Fig. (Fig.5A).5A). Similar results were seen when the same assay was used to determine mA3 RNA levels in spleen (not shown).

FIG. 5.
C57BL-derived mice express higher levels of mA3 in BMDCs and are more resistant to MMTV infection. (A) RNA was isolated from BMDCs from mA3−/− (−/−), B10.BR, C3H/HeN (C3H), C57BL/6 (BL/6), BALB/c (BALB), and (C57BL/6 × ...

The DCs were then infected with GR102ES virus lacking mA3, and infection was assayed at 24 h after infection by determining the percentages of GFP-positive cells. The DCs from strains expressing mA3+5 were more susceptible to MMTV than were their mA3−5-expressing counterparts (Fig. (Fig.5B).5B). Interestingly, the BMDCs from the F1 progeny of BALB/c and C57BL/6 mice that expressed intermediate levels of mA3−5 were relatively resistant to infection by MMTV compared to the mA3+5-expressing BALB/c parental mice, indicating that resistance to MMTV infection was dominant (Fig. 5A and B). These data suggest that the effectiveness with which mA3 restricts MMTV infection correlates with either the particular allele expressed or the level of mA3 expressed in different inbred strains.

LPS induces mA3 expression and virus restriction in different inbred mouse strains.

We showed previously that mA3-mediated restriction of MMTV infection could be induced by LPS or alpha interferon treatment of DCs ex vivo or in vivo; this effect was mostly due to mA3, since mA3−/− DCs and mice did not show increased restriction upon LPS treatment (22, 29). To determine whether such treatment would induce expression of the mA3−5 and mA3+5 RNAs to similar levels, we treated C57BL/6, BALB/c, (C57BL/6 × BALB/c)F1, and mA3−/− BMDCs with different concentrations of LPS. LPS treatment induced mA3 expression in all of the mA3-positive mice (Fig. (Fig.6A),6A), and concomitantly, there was decreased infection by MMTV (Fig. (Fig.6B).6B). However, mA3 expression remained lower and MMTV infection higher in BALB/c DCs at all LPS concentrations. At the highest LPS concentration (10 μg/ml), the level of MMTV infection in BMDCs from BALB/c mice was similar to that seen in untreated C57BL/6 BMDCs, showing that increasing the level of expression of the mA3+5 allele results in better restriction. BMDCs from (C57BL/6 × BALB/c)F1 and C57BL/6 mice were infected to similar extents at all LPS concentrations, again suggesting that the C57BL/6 allele is dominant.

FIG. 6.
LPS-induced restriction of MMTV infection in BMDCs. BALB, BALB/c; B6, C57BL/6. (A) BMDCs from the different mice were cultured for 24 h with the indicated concentrations of LPS. RNA was analyzed by RT-qPCR. RNA from mA3−/− mice showed ...

Susceptibility to in vivo infection segregates with the inherited mA3 allele.

Next, to determine whether the inherited mA3 allele and in vivo infection levels genetically cosegregated, we generated [(C57BL/6 × BALB/c)F1 × BALB/c]G2 backcross mice and infected them with MMTV(RIII), along with mA3−/−, BALB/c, C57BL/6, and F1 controls. Ninety-six hours after subcutaneous virus inoculation, the mice were sacrificed and their draining lymph nodes were used for infection analysis, while their spleens were used for mA3 genotyping and phenotyping. Out of 11 G2 mice generated, three had the same phenotype as their BALB/c parent with high viral load and mA3+5 RNA expression (mouse no. 2, 6, and 9; Fig. 7A and B), while the remaining 8 mice showed the same lower infection levels and mA3−5 RNA expression as F1 mice (no. 1, 3, 4, 5, 7, 8, 10, and 11; Fig. 7A and B). Genotypic analysis of DNA confirmed the RNA analysis (not shown). This experiment was repeated with an additional nine G2 mice (six genotyped as BALB/c and three genotyped as F1) with similar results (not shown). Thus, the C57-mA3−5 allele appears to play an important role in restricting MMTV infection, at least at early times after infection.

FIG. 7.
C57BL/6 resistance to MMTV infection is semidominant in vivo. (A) (BALB/c × C57BL/6 [B6])F1 mice were backcrossed to BALB/c, and the 11 offspring were subcutaneously injected with MMTV(RIII). Four days after infection, the mice were sacrificed ...

MMTV virions isolated from mouse strains expressing different alleles of the mA3 gene differ in their infectivity.

The mammary gland is the ultimate target in vivo for MMTV infection, and the natural route of virus transmission is through milk from infected mothers to nursing pups. The virus also causes mammary tumors, which can be used as a source of infectious virus (15). We showed previously that mA3 was expressed in the mouse mammary gland, at least at the RNA level (28). Thus, we next tested whether the mA3 isoform expressed in different inbred mice altered the infectivity of mammary gland-produced virus.

Virus was isolated from MMTV(RIII)-induced tumors that arose in BALB/c (mA3+5) and C57BL/6 (mA3−5) mice, as well as mA3−/− mice. Serial dilutions of these virions were then used to infect 293T cells stably expressing mTfR1. An aliquot of each virus preparation was also used for reverse transcription-RT-qPCR to determine the level of viral RNA. After normalization of the infection results to the level of viral RNA in each virus preparation, we found that the virus from mA3−/− mice infected cells at the highest level, while virus isolated from C57BL/6 mice was the least infectious; virus isolated from BALB/c mice was closer to mA3−/− virus in infectivity (Fig. (Fig.8A8A).

FIG. 8.
Virus isolated from C57BL/6 (B6) mice is less infectious than that from BALB/c (BALB) or mA3−/− mice. (A) Virions were purified from the tumors of MMTV(RIII)-infected BALB/c, C57BL/6, and mA3−/− mice and RNA was isolated ...

Next, to determine whether different inbred strains of mice expressed different levels of mA3 in mammary tumors, we isolated RNA from MMTV(RIII)-induced tumors that developed in C57BL/6, BALB/c, and mA3−/− mice and subjected it to reverse transcription-RT-qPCR. As we saw for lymphoid tissue and DCs, C57BL/6 mammary tumors had more mA3 RNA than did those from BALB/c mice (Fig. (Fig.8B).8B). Reverse transcription PCR analysis of these RNAs showed that mammary tissue expressed the same mA3 isoform as lymphoid tissue (Fig. (Fig.8B,8B, inset). These data show that virus produced in vivo in mA3−5-expressing mice is less infectious than that produced in mA3+5-expressing mice, similar to what was seen with virions produced in tissue culture cells (Fig. (Fig.3).3). However, because there are currently no good immunological reagents for detecting endogenous mA3, we cannot distinguish whether the virus made in C57BL/6 mice is less infectious because of the mA3 isoform or the higher levels of expression.


Viruses that persist in their natural hosts generally are not highly pathogenic, in large part the result of a wide array of host antiviral responses, including the expression of cell-intrinsic factors, such as those belonging to the A3 family, which restrict infection by a number of viruses (reviewed in reference 13). Included in this group of viruses is MMTV, which we previously showed was inhibited in vivo by mA3, since mA3−/− mice were more susceptible than mA3+/+ mice to MMTV infection (28). Here, we present evidence that allelic differences in the mA3 genes found in different inbred mouse strains also contribute to natural resistance to MMTV.

It has long been recognized that inbred mouse strains differ in their susceptibilities to MMTV infection (14, 26). Resistance is due in most cases to lack of infection or virus spread within the lymphoid compartment. The MMTV genome encodes a superantigen (Sag) protein which is presented by major histocompatibility complex (MHC) class II molecules on antigen-presenting cells, such as DCs, to T cells, thereby inducing their activation (34). Activated T cells in turn activate bystander antigen-presenting cells, ultimately resulting in virus amplification in the lymphoid compartment. Mouse strains like C57BL/6 that lack the MHC class II I-E gene are relatively resistant to infection by most MMTVs because of poor Sag presentation (4, 32); an exception to this is MMTV(RIII) (used in the current study), which is infectious in C57BL/6 mice and both activates Sag-cognate T cells (presumably through MHC class II I-A) and causes tumors (42). Other mechanisms of resistance to lymphocyte infection and virus spread include a hyperimmune antibody response that occurs in I/LnJ mice and increased viral clearance by T cells in YBr/Ei mice (8, 9, 33). The lymphoid tissues of mice lacking endogenous copies of the MMTV provirus are also resistant to MMTV infection, although the mechanism for this resistance is not known (5, 6).

Here, we examined 19 different inbred mouse strains and found that there are two alleles of the mA3 gene with regard to exon 5 and the surrounding introns. Mice derived from the C57BL lineage (C57BL/6 and B10.BR), both types of which are relatively resistant to MMTV infection, predominantly expressed the mA3 splice variant lacking exon 5, while BALB/c and C3H/HeN mice, which are highly sensitive to MMTV, have identical exon 5 genomic sequences and predominantly expressed a full-length mA3 gene that retains all exons. Interestingly, there is a 7-bp sequence (TACCAAC) 53 bp upstream from exon 5 in BALB/c and C3H/HeN mice that resembles the canonical branch-site selection sequence (TACTAAC) which in C57BL-derived mice has a single nucleotide change (TATCAAC). In addition, the C57BL allele has a 4-bp deletion 21 bp upstream of exon 5. Whether these sequence changes determine whether exon 5 is included or excluded from the RNA remains to be determined.

Recently, it has been suggested that the mA3 allele determines susceptibility to F-MLV (25, 37, 40). Earlier mapping studies had mapped the Rfv3 gene to the same region of chromosome 15 where mA3 is located (18, 25). Both the Miyazawa and Greene groups showed that mice with a targeted mutation of mA3 were more susceptible to infection by F-MLV than were mA3+/+ mice (37, 40). Moreover, (mA3−/− × BALB/c)F1 mice were more susceptible to infection than (mA3−/− × C57BL/6)F1 mice, while (BALB/c × C57BL/6)F1 mice showed levels of infection similar to those of their C57BL/6 parent; this outcome is similar to our results with MMTV infection, where we found that (BALB/c × C57BL/6)F1 animals more closely resembled their MMTV-resistant C57BL/6 parent (Fig. (Fig.5,5, ,6,6, and and7).7). Additionally, cloned versions of the C57BL/6 mA3−5 protein restricted F-MLV infection better than the 129 mA3+5 protein, which is identical in sequence to that reported for BALB/c mice (40).

In contrast to Santiago and colleagues (37), we did not find that the predominant RNA produced in BALB/c splenocytes lacked exon 2. Instead, this splice variant constituted only a minor fraction of the mA3 RNAs found in BALB/c splenocytes and DCs (Fig. (Fig.2B2B and data not shown). Indeed, we did not find any polymorphisms in exons 1, 2, and 3 or the surrounding intronic sequences of the BALB/c, 129/Ola, and A.BY genes. Moreover, by using PCR primers for a conserved region of mA3 to do quantitative analysis of RNA levels in different inbred strains, we found that the levels of mA3 in C57BL/6 and B10.BR splenocytes and DCs were five- to sevenfold higher than the levels of mA3 in C3H/HeN or BALB/c cells; Santiago and colleagues reported no difference in mA3 RNA levels in C57BL/6 and BALB/c splenocytes. We do not know the reason for the differences in our experiments, but Takeda et al. also showed that BALB/c mice expressed an exon 2-containing RNA and reported elevated mA3 RNA levels in the spleen and bone marrow of C57BL/6 mice compared to these levels in BALB/c mice (40).

We previously showed that B10.BR mice were relatively resistant to MMTV infection and that diminished MMTV spread in their lymphoid tissues was due in part to poor Sag-dependent activation of cognate T cells through an unknown mechanism (31). Here, we show that the mA3 allele found in B10.BR and C57BL/6 mice is also more restrictive than that found in MMTV-susceptible strains and that this probably contributes to resistance to infection. Indeed, MMTV infection of DCs isolated from C57BL/6 and B10.BR mice, both of which are derived from C57BL mice and contain the C57-mA3−5 allele, was at a lower level than infection in cells isolated from the MMTV-susceptible C3H/HeN and BALB/c strains, which apparently have the same mA3+5 allele. Since ex vivo DC infection is not affected by adaptive immunity and does not depend on Sag presentation, these data suggest that an intrinsic factor present in C57BL-derived DCs restricts infection. This is likely due to the C57-mA3−5 protein, since we also showed that packaging of this isoform into virions produced by MMTV-transfected 293T cells also resulted in a level of infectivity lower than that seen with virions containing the 129-mA+5 isoform. Whether the increased restriction of MMTV infection by the C57-mA3−5 protein is due to the absence of exon 5, the polymorphic amino acid differences in the two proteins, or the higher expression levels in C57BL-derived strains is currently not known. However, resistance to F-MLV mapped to amino acid polymorphisms residing in the N-terminal 192 amino acids of the A3 protein in the C57BL/6 allele product rather than to the proteins produced from the alternatively spliced RNAs, at least in tissue culture infection studies (40). Additionally, there is one report that the MLV protease cleaves mA3 in exon 5 and that the C57-mA3−5 protein is therefore more abundant than the mA3+5 protein, at least in tissue culture cells (1). It is not known whether MMTV protease also cleaves mA3.

It is also not clear for either MMTV or F-MLV whether expression levels or the allelic polymorphisms dictate the increased restriction seen in vivo or in primary cells. Our genetic analysis of both the F1 and G2 backcross progenies revealed that mA3−5 is inherited as a dominant autosomal allele in mice. However, both in the DC culture experiments (Fig. (Fig.55 and and6)6) and in vivo (Fig. (Fig.7),7), infection of mice that were heterozygous for the allele (mA3−5/mA3+5) was always slightly greater than that seen in their C57BL/6 (mA3−5/mA3−5) parent. This may be due to the lower level of expression of the mA3−5 protein in the F1 mice. Indeed, 1 μg/ml LPS added to DC cultures induced mA3 expression in F1 cells to levels similar to that seen in uninduced C57BL/6 DCs (Fig. (Fig.6A)6A) and concomitantly resulted in similar infection levels (Fig. (Fig.6B).6B). Although LPS induced mA3 RNA levels in BALB/c DCs, they never reached those of F1 or C57BL/6 mice, and while infection was reduced with LPS treatment, it was always higher at all concentrations in BALB/c DCs than in C57BL/6 or F1 DCs, suggesting that additional allelic differences in the transcriptional regulatory regions might also exist. The greater infection seen in mA3−5/mA3+5 mice and cells could be also due to other genetic differences in these inbred mice. There may be additional antiviral factors induced by LPS in BALB/c DCs, since at the highest level of LPS treatment, mA3 levels in BALB/c DCs were still lower than those seen in untreated C57BL/6 DCs, yet infection levels were similar. Indeed, there are clearly other genes involved in resistance to MMTV infection, since many strains inherit the “susceptible” mA3+5 allele and yet are resistant to MMTV infection (i.e., I/LnJ, YBR, and PERA mice; Fig. Fig.11).

Although we were not able to directly examine A3 packaging in mammary tissue in vivo, our data suggest that this does occur, since virus produced in mA3−/− mammary tissue was more infectious than that produced in mA3+/+ mice and virus isolated from C57BL/6 mice was less infectious than that from BALB/c animals. This predicts that in addition to playing a role in innate resistance to infection, the milk-borne transmitted virus produced in C57BL/6 mice will be less transmissible than that from BALB/c mice. Although it is believed that HIV-1 is also transmitted through milk, whether any human A3 proteins are made in mammary tissue is not known.

In contrast to the mouse genome, the human genome carries seven A3 genes, but little is known about alternative splicing of these genes. However, several recent studies have implicated genetic polymorphisms in human A3G (hA3G), the major HIV-1 restriction factor, in long-term nonprogression and resistance to AIDS (2, 20, 25). Although one of the A3G polymorphisms (H16R) associated with AIDS progression and declining CD4 T cell levels showed no resulting difference in in vitro antiviral activity from the allele found in nonprogressors (2), this may be the result of the experimental system, which relies on overexpression of cloned hA3G in tissue culture cells. Thus, the findings that mA3 polymorphisms affect infection by at least two endemic mouse retroviruses, MMTV and F-MLV, in vivo indicate that similar polymorphisms in hA3 genes may be relevant to HIV-1 infection, as well as the other human viruses believed to be restricted by hA3 proteins, such as human papillomavirus and hepatitis B virus (39, 41, 43).

Supplementary Material

[Supplemental material]


We thank David Derse for the mA3 plasmids and Troy Brady for the SV40-immortalized mA3−/− MEFs.

C.M.O. was supported by training grant PHS T32-CA-009140. This research was supported by a grant from the Penn Center for AIDS Research (CFAR), an NIH-funded program (P30 AI 045008).


[down-pointing small open triangle]Published ahead of print on 19 January 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.


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