Retroviruses can establish germline infection and become part of the host genome2,3. Most, if not all, ERVs have become inactive owing to mutations, or transcriptionally silenced through the action of diverse mechanisms2,3. However, RNA and protein expression of replication-defective ERVs are frequently increased in infection, autoimmunity and cancer2,3. Whether or not the immune system defends against potential threats posed by ERVs is unclear. To address the role of adaptive immunity in this process, we assessed ERV expression in B6 mice. We initially compared the transcriptional profiles of purified macrophages from B6 wild-type and T- and B-cell-deficient Rag1−/− mice. The two transcripts with the highest increase in expression levels in macrophages from Rag1−/− mice (Fig. 1a) correspond to the env and gag genes, respectively (Supplementary Table 1), of an endogenous ecotropic MLV (eMLV) locus, Emv2, a replication-defective single-copy ERV present in B6 mice5. Differential expression of eMLV was confirmed by quantitative reverse transcriptase PCR (qRT–PCR) for spliced env messenger RNA in macrophages (Fig. 1b) and in several tissues (Fig. 1c).

Figure 1: eMLV activation in antibody-deficient mice. a, Significantly upregulated (>4-fold) genes in CD11b+ MHC-IIhi B220− Gr1− macrophages from Rag1−/− mice compared with macrophages from wild-type (WT) mice. Triplicate microarrays from cells isolated from 40 mice are shown. b, eMLV spliced env mRNA expression in the same cells as in a. Each symbol represents macrophages from 20 mice (P = 0.024; paired Student’s t-test). c, eMLV spliced env mRNA expression in indicated organs from wild-type or Rag1−/− mice (spleen: P = 0.020; ileum: P = 0.032; colon: P = 0.004; lung: P = 0.001; muscle: P = 0.016; and kidney: P = 0.009; unpaired Student’s t-test). d, e, MLV SU expression (detected using the 83A25 monoclonal antibody; see Methods) in splenocytes (d) or indicated cell types (e) from wild-type or Rag1−/− mice. f, eMLV spliced env mRNA expression in the spleens of the indicated strains (P < 0.001 between wild-type and either Ighm−/− or Ighm−/− MD4 mice; one-way analysis of variance (ANOVA)). g, MLV SU expression in splenic lymphocytes from wild-type or Ighm−/− MD4 mice. h, eMLV spliced env mRNA expression in the spleens of the indicated strains (P < 0.001 between wild-type and either Myd88−/− or Tlr7−/− mice; one-way ANOVA). In c, f and h, each symbol is an individual mouse. In d, e and g, plots are representative of four mice per group. In f and h, values above 103 were considered high and are indicated by red-filled symbols. Full size image Download PowerPoint slide

Other ERV families were also differentially expressed in macrophages, albeit less strongly (1.7–2.1-fold; Supplementary Table 1). Not distinguishing between members of multicopy families, expression of polytropic MLVs (pMLVs), xenotropic MLVs (xMLVs) and the Mus musculus type D (MusD) retrovirus family of retrotransposons was also increased in the lungs of Rag1−/− mice (Supplementary Fig. 1). In line with increased MLV mRNA expression, 60–80% of total splenocytes and all haematopoietic lineages analysed from Rag1−/− but not wild-type mice, expressed MLV surface glycoprotein (SU) (Fig. 1d, e).

Tcra−/− or Tcrd−/− mice, lacking T-cell receptor (TCR)αβ and TCRγδ T cells, respectively, showed low eMLV expression (Fig. 1f). eMLV expression was similarly low in H2-A,E−/− mice (MGI allele H2dlAb1-Ea; lacking the region between the H2-Ab1 and H2-Ea genes), deficient in major histocompatibility complex (MHC) class II, MHC II-restricted T cells and T-cell-dependent antibodies (Fig. 1f). By contrast, mice lacking B cells (Ighm−/− mice) or mice unable to produce polyclonal antibodies (Ighm−/− MD4 mice) expressed substantially higher eMLV levels than did wild-type mice (Fig. 1f), demonstrating that high eMLV expression characterized mice lacking antigen-specific antibodies. In addition to splenocytes from Rag1−/− mice (Fig. 1e), T and B cells from Ighm−/− MD4 mice, but not from wild-type control mice, expressed MLV SU (Fig. 1g), indicating that eMLV can also be highly expressed in lymphocytes.

Toll-like receptors (TLRs) have been implicated in the control of B-cell responses, and Myd88−/− and Tlr7−/− mice have significantly reduced serum levels of natural antibodies and a defective antibody response to immunization or infection, including with retroviruses6,7,8,9. Notably, the expression of eMLV was markedly increased in Myd88−/− and Tlr7−/− mice in comparison with wild-type mice (Fig. 1h). Similarly increased eMLV expression was observed in Tlr7−/− mice, but not in Tlr9−/− mice housed in a different facility (Supplementary Fig. 2).

To investigate the mechanistic link between antibody deficiencies and increased MLV expression, we examined the origin of eMLV transcription. The B6 genome does not contain replication-competent eMLV proviruses, and, although the Emv2 locus can produce mRNA, it is unable to produce infectious virus owing to an inactivating G-to-C mutation at position 3576 of the pol region5,10. In addition, Emv2 gag encodes an N-tropic capsid, which would be restricted by the Fv1b restriction factor in B6 mice10. However, it was theoretically possible that recombination between replication-defective Emv2 and non-ecotropic MLVs resulted in an MLV with full infectivity11 that could spread in Rag1−/− mice. Remarkably, the plasmas of young and old Rag1−/− mice, but not of wild-type control mice, contained retroviruses that were capable of replicating in mouse cells in vitro (Fig. 2a), which we refer to as Rag1−/− mouse-associated retroviruses (RARVs). Sequencing of the pol region demonstrated repair of the Emv2-inactivating mutation in all RARV isolates (Supplementary Fig. 3). Functional in vitro assays (Fig. 2b) and sequencing of the gag region (Fig. 2c) showed that RARVs also exhibited B-tropism. Genome sequence comparisons between RARVs showed that young Rag1−/− mice contained highly similar viruses, which diverged substantially in old Rag1−/− mice (Fig. 2d). All RARVs were recombinants between Emv2 and endogenous non-ecotropic MLVs (Supplementary Fig. 4). The pol defect of Emv2 was probably restored in RARVs by recombination with Xmv43 (also known as Bxv1; Supplementary Fig. 4), an ERV that contains a functional pol region but is unable to infect mouse cells owing to polymorphisms in the mouse cellular receptor2. Recombination events involving Xmv43 have also been found to be responsible for the emergence of leukaemogenic MLVs in AKR mice12. However, the switch in capsid tropism resulted from recombination with other endogenous xMLVs (Supplementary Fig. 4). Notably, the divergence of RARVs isolated from old Rag1−/− mice was due to further recombination replacing the ecotropic env with polytropic env from Pmv1, Pmv5 or Pmv16 (Supplementary Fig. 4). Together, these findings indicate the emergence of infectious eMLVs that could have infected Rag1−/− mice and given further rise to infectious pMLVs. Supporting this notion, most gag/pol eMLV mRNA detected in Rag1−/− mice seemed to be transcribed from integrated RARVs, rather than the germline copy of Emv2 (Supplementary Fig. 5).

Figure 2: Retroviraemia and leukaemias/lymphomas in antibody-deficient mice. a, Detection of infectious MLV (RARV-5/XG7) from the plasma of a representative Rag1−/− mouse by restoring infectivity of the green fluorescent protein (GFP)-expressing XG7 retroviral vector in the indicated cell type. Numbers within the plots denote the percentage of retrovirally transduced (GFP+) cells. b, Fv1 tropism of RARVs isolated from 6 (R2–R4)- or 25 (R5–R8)-week-old healthy Rag1−/− mice, shown as the ratio of infectivity in B-3T3 to N-3T3 cells (B:N ratio). B- and N-tropic strains of Friend MLV (F-MLV) are shown for comparison. c, Amino acid residues of capsid positions 105–113 (CA105–113) deduced from the nucleotide sequence of Emv2 and the same RARVs as in b. Dots indicate identities. d, Phylogenetic tree of the same RARVs as in b. The scale indicates the probability of base substitution per site. e, eMLV spliced env mRNA expression in the spleens of Rag1−/− mice or vertically infected Rag1−/− Emv2−/− mice. Each dot is an individual mouse (P < 0.001; unpaired Student’s t-test). Values above 103 were considered high and are indicated by red-filled symbols. f, MLV SU expression in splenocytes from Rag1−/− or vertically infected Rag1−/− Emv2−/− mice (representative of nine mice per group). g, eMLV DNA copy numbers per haploid genome, determined by qPCR for the pol or ecotropic env gene, in DNA from the spleens of healthy Rag1−/− mice (right) or vertically infected Rag1−/− Emv2−/− mice (left). Symbols represent individual mice, grouped according to their age. The sensitivity limit of this PCR method was determined as a median of 0.0003 copies per haploid genome, using Emv2−/− mice. eMLV DNA copy numbers for Rag1−/− mice include Emv2 (1/N). h, Tumour (leukaemias/lymphomas) incidence in cohorts of wild-type (n = 37), Rag1−/− (n = 38) or vertically infected Rag1−/− Emv2−/− mice (n = 23) at the NIMR SPF facility (P < 0.000001 between wild-type and Rag1−/− mice; P = 0.00025 between wild-type and Rag1−/− Emv2−/− mice; log-rank survival analysis). Full size image Download PowerPoint slide

Emv2 and non-ecotropic MLV recombination events resulting in infectious eMLV generation might occur de novo in individual Rag1−/− mice. Alternatively, these RARVs might have been vertically transmitted through successive generations. To test the latter possibility directly and assess the capacity of RARVs to spread in immunodeficient mice, we established a colony of Rag1−/− Emv2−/− mice by first crossing an Emv2−/− male mouse13 with a Rag1−/− female mouse, and then intercrossing selected progeny to homozygosity. These mice lack the germline copy of Emv2, meaning any infectious eMLV present would have been vertically transmitted. Both eMLV spliced env mRNA (Fig. 2e) and MLV SU expression (Fig. 2f) were readily detected in the spleens of Rag1−/− Emv2−/− mice in this colony. Furthermore, analysis of eMLV env and pol DNA copies indicated extensive replication of vertically transmitted RARVs in Rag1−/− Emv2−/− mice (Fig. 2g). By contrast, sexual or in utero infection was not observed in separate crosses of either male or female virus-positive Rag1−/− Emv2−/− mice with virus-free Emv2−/− mice (Supplementary Fig. 6).

To examine the potential of RARVs to replicate in Rag1−/− mice further, we assessed the frequency of tumours characteristic of retroviral infection2,3 in cohorts of Rag1−/− mice. Notably, starting from 180 days and affecting 67% of the animals by 380 days, Rag1−/− mice, but not wild-type control mice, showed signs of morbidity (Fig. 2h). On examination, large tumours, often associated with anaemia, were observed in all morbid Rag1−/− mice (Fig. 2h and Supplementary Fig. 7). The pathogenic potential of infection with RARVs was established in aged cohorts of vertically infected Rag1−/− Emv2−/− mice, which developed tumours at a comparable incidence rate (Fig. 2h).

Thymic or splenic tumours in Rag1−/− mice consisted mainly of a single MLV SU-expressing cell type, which differed between animals, and had the histological appearance of lymphoblastic lymphosarcomas (Supplementary Fig. 8). Discrete chromosomal aberrations were found in most tumours analysed (Supplementary Fig. 8), suggestive of clonal origin. Consistent with MLV production by tumour cells, we observed an abundance of MLV-type particles in the extracellular space of tumour samples, but not in a spleen sample from a healthy Rag1−/− mouse (Supplementary Fig. 9). Furthermore, a substantial increase in both eMLV env and pol DNA copy numbers was detected in all tumour samples, with one exception in which only pol DNA copies were increased (Supplementary Fig. 9), indicating that RARVs had extensively infected the cells that gave rise to lymphomas. Together, these results support a model in which several recombination events restore Emv2 infectivity, leading to spontaneous retroviraemia and vertical transmission to progeny, and eventually drive an oncogenic process similar to that extensively described in mouse strains carrying fully infectious ERVs2,3.

Our results associated a lack of antibodies with establishment of infectious eMLVs in mouse colonies. Next, we investigated the potential mode of antibody action. Antiretroviral antibodies have a long-established role in limiting the spread of infectious endogenous retroviruses14, both within and between animals. However, it was also possible that antibodies were preventing a step before the emergence of infectious eMLV recombinants. Rescue of Emv2 infectivity by recombination with a non-ecotropic MLV necessitates co-expression of both proviruses in the same cell at sufficient levels for co-packaging into the same virion. Low expression of these proviruses in wild-type mice could be a rate-limiting factor in the emergence of infectious eMLVs. However, expression of certain endogenous MLVs in mouse cells is known to be inducible, notably, by microbial products2,3,15. For example, bacterial lipopolysaccharide (LPS) stimulation activates non-ecotropic MLVs, and Xmv43 in particular15,16,17,18,19. To examine the responsiveness of ERVs and other retroelements to microbial stimulation, we took advantage of probes or probe sets in standard microarray platforms that report ERV or retroelement expression. Analysis of a publicly available data set20 uncovered specific induction of non-ecotropic MLV transcripts by LPS and polyinosinic:polycytidylic acid (poly(I:C)), and suppression by Pam 3 CSK 4 (Fig. 3a, b). Early transposon transcripts were also induced by poly(I:C) (Fig. 3a). These findings were confirmed by LPS stimulation, which induced high MLV SU expression in wild-type and Emv2−/− mouse splenocytes, also in a MyD88-independent manner (Fig. 3c and Supplementary Fig. 10). These in vitro conditions did not significantly induce eMLV expression (Fig. 3a). However, ex vivo analysis showed higher expression of eMLV (Fig. 1c), as well as of other ERVs/retroelements (Supplementary Fig. 1), specifically in the colon, suggestive of microbial involvement.

Figure 3: Mouse ERV activation by microbial products. a, ERV/retroelement-reporting probe set (Supplementary Table 3) signals in a publicly available Affymetrix HT mouse genome 430A microarray data set20 (ArrayExpress accession E-GEOD-17721) of wild-type B6 bone marrow-derived dendritic cells after stimulation with microbial products. Black arrows indicate the probe sets that are significantly regulated (P < 0.05) more than twofold by at least one stimulus. Emv2-specific probe sets (annotated as Mela) are also indicated by grey arrows for comparison. ETn, early transposon. b, Mean log 2 fold change in the MLV-reporting probe set in the same data set. c, MLV SU expression in wild-type or Emv2−/− splenocytes before (open histograms) or after (filled histograms) stimulation with 10 μg ml−1 LPS for 48 h, according to forward scatter (Fsc) and CD19 expression. Numbers in the plots denote the percentage of cells within each gate and represent two donors each analysed in duplicate. Full size image Download PowerPoint slide

Antibodies have established roles in controlling intestinal bacteria and neutralizing their products, such as LPS, in the gut lumen or the systemic circulation, and antibody-deficient mice are known to display increased microbial trasnslocation1,9,21,22,23. It was, therefore, possible that antibody deficiency allowed microbial products to induce expression of MLV proviruses in Rag1−/− mice, including the parents of recombinant RARVs (Supplementary Fig. 1). In support of this notion, and in agreement with the established role of natural IgM in systemic clearance of bacterial LPS and protection from endotoxaemia9,21,22, production of non-hypermutated IgM alone was sufficient for eMLV control (Supplementary Fig. 11). If antibodies required for preventing MLV expression were, indeed, against several microbial products, then antibody deficiency should not result in increased MLV expression in the absence of microbial triggers (Supplementary Fig. 12).

To begin to examine the contribution of microbial triggers, we measured eMLV expression in specific pathogen-free (SPF) Rag1−/− mice from colonies that differed in intestinal microbiota. Importantly, the use of embryo transfer for the rederivation of these independent colonies removes adventitious organisms, including vertically transmitted eMLVs. Therefore, any RARVs found in these rederived Rag1−/− mouse colonies must be generated de novo in the life-history of each colony. In stark contrast to Rag1−/− mice that were maintained on neutral pH water at the National Institute for Medical Research (NIMR), colonies of Rag1−/− mice that were maintained on acidified water expressed minimal eMLV levels (Fig. 4a). Water acidification reduced overall bacterial diversity in the colons of Rag1−/− mice (Supplementary Fig. 13 and Supplementary Table 2) and is a common precautionary measure used in many animal facilities that reduces bacterial colonization within the intestinal tract and translocation into the circulation24. Lack of eMLV expression was noted in Rag1−/− mice obtained from the Jackson Laboratory (JAX), also maintained on acidified water (Fig. 4a). Furthermore, minimal levels of eMLV were detected in Rag1−/− mice on neutral pH water at the Rodent Center HCI (RCHCI; Fig. 4a), which contained distinct bacterial genera in comparison with Rag1−/− mice at NIMR (Supplementary Fig. 13 and Supplementary Table 2). Lastly, negligible levels of eMLV were expressed in Rag1−/− mice in germ-free facilities at the University of Michigan (UMICH) and offered neutral pH water (Fig. 4a). The latter two conditions also distinguished between effects of acidified water on intestinal flora and effects on other physiological processes. Thus, high eMLV expression in independently rederived colonies of Rag1−/− mice correlated with the presence of the normal SPF microbiota.

Figure 4: eMLV activation in antibody-deficiency depends on husbandry conditions. a, eMLV spliced env mRNA expression in the spleens of Rag1−/− mice on neutral pH (SPF) or acidified water (pH 2.5) at the NIMR, on acidified water (pH 2.8) at JAX, on neutral pH at RCHCI, or in germ-free (GF) facilities at UMICH (P < 0.016 between Rag1−/− mice at SPF NIMR and all other groups; one-way ANOVA). b, eMLV spliced env mRNA expression in the spleens of Ighm−/−, Myd88−/− or Tlr7−/− mice on neutral pH water (SPF) at the NIMR or on acidified water (pH 2.8) at JAX (P = 0.005 and P = 0.029 for Ighm−/− and Tlr7−/− mice, respectively; unpaired Student’s t-test). Each dot is an individual mouse and values above 103 were considered high and are indicated by red-filled symbols. Full size image Download PowerPoint slide

Husbandry conditions contributed to high eMLV expression also in independently rederived strains with distinct immunodeficiencies. Colonies of Ighm−/− and Tlr7−/− mice maintained on acidified water at JAX expressed minimal levels of eMLV (Fig. 4b). Variable eMLV expression levels were detected in Myd88−/− mice at JAX (Fig. 4b), probably because, once generated, vertical transmission of infectious eMLVs was unaffected by water acidification. Although defining the precise role of the microbiota on eMLV induction will require further investigation, collectively our results demonstrate that the high eMLV expression phenotype, in genotypes causing immunodeficiency, requires environmental interaction.

High eMLV expression in independently rederived immunodeficient colonies reveals de novo eMLV induction in each colony. It does not, however, indicate when or how often in the life-history of a colony infectious eMLVs may emerge. Adult RARV-free Rag1−/− mice, previously on acidified water, maintained low eMLV expression after a switch to neutral pH water, indicating a low probability of RARV emergence and spread in an individual mouse. To examine whether this probability is low in general or could be higher during early mouse development, we monitored successive generations of Ighm−/− mouse colonies from two independent rederivations, one of which was recent, into the NIMR SPF facility. This analysis suggested that eMLV induction occurred during the first few filial generations (F; Supplementary Fig. 14). Moreover, eMLV-expressing Myd88−/− mice at JAX (Fig. 4b), were at F 5 of homozygous breeding. Thus, although low in individual mice, there is high cumulative probability of infectious eMLV emergence, involving a sequence of recombination events similar to those seen in Rag1−/− mice (Supplementary Fig. 12), and subsequent establishment in an antibody-deficient colony over a few generations.

The B6 strain has historically dominated many research fields, partly owing to the resistance of this strain to retrovirally induced tumours. Our results demonstrate that this important attribute of B6 mice is conditional on their immune competence, with significant implications both for the design and interpretation of mouse studies. Furthermore, our results show that ERV activation is determined by husbandry conditions, thus accentuating potential differences in ERV expression between animal facilities.

Although well-established in the mouse2,3, the oncogenic potential of ERVs in humans has not been observed25,26. However, non-long terminal repeat (LTR) retroelement families have been documented to have caused human cancers by insertional mutagenesis at the somatic level25,26. Interestingly, we found that TLR stimulation of human cells induced expression of distinct ERVs and retroelements, including the mammalian apparent LTR retrotransposon (MaLR) family (Supplementary Fig. 15), previously implicated in the pathogenesis of human lymphomas27. Transcription of human ERVs and retroelements can also be induced by physiological activation of both adaptive and innate immune cells28. Moreover, increased risk of lymphomas in humans is linked to infection or inflammation29 and also to antibody deficiencies30. Thus, interactions between microbial symbionts, leading to ERV/retroelement activation, may provide a mechanistic link between cancer and stimulation of the immune system by microbiota or pathogenic infections.