A single dominant locus restricts retrovirus replication in YBR/Ei mice

Abstract

Differential responses to viral infections are influenced by the genetic makeup of the host. Studies of resistance to retroviruses in human populations are complicated due to the inability to conduct proof-of-principle studies. Inbred mouse lines, which have a range of susceptible phenotypes to retroviruses, are an ideal tool to identify and characterize mechanisms of resistance and define their genetic underpinnings. YBR/Ei mice become infected with Mouse Mammary Tumor Virus, a mucosally transmitted murine retrovirus, but eliminate the virus from their pedigrees. Virus elimination correlates with a lack of virus-specific neonatal oral tolerance, which is a major mechanism for blocking the anti-virus response in susceptible mice. Virus control is unrelated to virus-neutralizing antibodies, cytotoxic CD8⁺ T cells, NK cells, and NK T cells, which are the best characterized mechanisms of resistance to retroviruses. We identified a single, dominant locus that controls the resistance mechanism, which we provisionally named attenuation of virus titers (Avt) and mapped to the distal region of chromosome 18. IMPORTANCE Elucidation of the mechanism that mediates resistance to retroviruses is of fundamental importance to human health, as it will ultimately lead to knowledge of the genetic differences among individuals in susceptibility to microbial infections.

Keywords: genetic mechanism; immune response; retroviruses.

Fig 1

Most known innate sensors and effectors and MHC class I responses are not required to control MMTV in YBR mice. (A) Experimental design to follow the virus fate in infected mouse pedigrees. Female mice with the specific genotype were fostered by MMTV(C3H)-infected C3H/HeN viremic mothers (Generation 0, G0) and bred to produce subsequent generations (G1-N). (B–D) Infection status of mice at each generation was evaluated by monitoring deletion of SAg-cognate T cells at 10 weeks of age and by PCR specific for integrated proviruses in the spleen. Lines deficient in MHC class I-specific responses (B), innate immune sensors and adaptors (C), and specific cytokines and caspase 1/11 (D) were evaluated. n represents number of individual pedigrees per genotype followed. Three-eight mice per family per generation were analyzed.

Fig 2

The cGAS-STING pathway is not involved in the anti-MMTV response in YBR mice. (A) YBR mice containing the disrupted Sting1-neo allele derived from 129 mice were fostered by viremic females and bred to follow the virus fate as in Fig. 1A. (B) Schematic of the Sting1-neo locus on chromosome 18 in “backcrossed” YBR.Sting^−/− mice shown in (A). Mb, megabase. The beginning and end of the region from 129 mice were not defined before the mouse line was lost due to the COVID-19 pandemic. (C) CRISPR/Cas9 targeting strategy for the generation of “clean” YBR.Sting-deficient mice. Two guides were used to target exon 2 and intron 2–3, resulting in a 126-bp deletion and the introduction of a premature stop codon. (D) Virus fate in YBR mice with “clean” STING or cGAS deficiency. n represents number of individual pedigrees per genotype followed. Three-eight mice per family per generation were analyzed. KO, knockout.

Fig 3

YBR resistance to MMTV is controlled by attenuation of virus titers (Avt), a single, dominant locus on chromosome 18. (A) MMTV(C3H) LTR-specific Taqman-based quantitative polymerase chain reaction (qPCR) was used to compare provirus load in splenic DNA of YBR and BALB/cJ G0 mice. Ct, Ct(MMTV) - Ct(beta-actin). Dotted line represents the limit of detection (average ΔCt from uninfected mice). Each dot shows individual mouse. G, generation. (B) Genetic crosses used to map Avt. (YBR × BALB/cJ)F1 females were fostered (F) by MMTV(C3H)-infected C3H/HeN mothers. Infected F1 females (G0)were bred to BALB/cJ males to generate N2 (G1) mice. A, the Avt dominant resistant YBR allele; a, the Avt recessive susceptible BALB/cJ allele. (C) MMTV(C3H) LTR-specific Taqman-based qPCR was used to compare provirus load in splenic DNA of N2 mice. Infected YBR and BALB/cJ G1 mice were used as controls. The ΔCt values for N2 mice ranged from 9.36 to 17.24 (mean ± SD = 11.89 ± 1.72). Arbitrary cut-offs were used to define N2 mice as resistant (ΔCt ≥11.89 + 1.72) and susceptible (ΔCt ≤11.89–1.72) based on the ΔCt mean and SD of total N2 mice. Res, resistant; Sus, susceptible. Each dot shows individual mouse. G, generation. (D) Linkage analysis across chromosome 18 in resistant and susceptible N2 mice. Proportion of mice heterozygous in each group at each single-nucleotide polymorphism (SNP) is plotted. Each dot shows individual mouse. Statistical significance was determined by two-tailed unpaired Welch’s t test (ns, P > 0.05; *, P ≤ 0.05; ****, P ≤ 0.0001). Mb, megabase.

Fig 4

Neonatal oral tolerance to MMTV is not induced in YBR mice, despite an intact subversion pathway. (A and B) Nucleated splenic cells from indicated mice were incubated with LPS and IL-6 (A) and IL-10 (B) were measured in cell supernatants by enzyme-linked immunosorbent assay (ELISA). C3H/HeJ mice lack the functional TLR4 (51) and were thus, used as a negative control. Each dot shows individual mouse. (C) Mice of indicated strains were or were not immunized at 8 weeks of age with Triton X-100-treated MMTV(C3H) virions in complete Freund’s adjuvant. Reactivity of serum samples against MMTV antigens was measured by ELISA after the boost. MMTV(C3H) (+), mice were fostered by viremic females. Each dot shows individual mouse. Statistical significance was determined by two-tailed unpaired Welch’s t test (ns, P > 0.05; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001).