Genetic conflicts between retroviruses and their receptors result in the evolution of novel host entry restrictions and novel virus envelopes, and such variants can influence trans-species transmission. We screened rodents and other mammals for sequence variation in the Xpr1 receptor for the mouse xenotropic or polytropic mouse leukemia viruses (X-MLVs or P-MLVs, respectively) of the gammaretrovirus family and for susceptibility to mouse-derived X/P-MLVs and to XMRV (xenotropic murine leukemia virus-related virus), an X-MLV-like virus isolated from humans with prostate cancer and chronic fatigue syndrome. We identified multiple distinct susceptibility phenotypes; these include the four known Xpr1 variants in Mus and a novel fifth Xpr1 gene found in Mus molossinus and Mus musculus. We describe the geographic and species distribution of the Mus Xpr1 variants but failed to find the X-MLV-restrictive laboratory mouse allele in any wild mouse. We used mutagenesis and phylogenetic analysis to evaluate the functional contributions made by constrained, variable, and deleted residues. Rodent Xpr1 is under positive selection, indicating a history of host-pathogen conflicts; several codons under selection have known roles in virus entry. All non-Mus mammals are susceptible to mouse X-MLVs, but some restrict other members of the X/P-MLV family, and the resistance of hamster and gerbil cells to XMRV indicates that XMRV has unique receptor requirements. We show that the hypervariable fourth extracellular XPR1 loop (ECL4) contains three evolutionarily constrained residues that do not contribute to receptor function, we identify two novel residues important for virus entry (I579 and T583), and we describe a unique pattern of ECL4 variation in the three virus-restrictive Xpr1 variants found in MLV-infected house mice; these mice carry different deletions in ECL4, suggesting either that these sites or loop size affects receptor function.The XPR1 receptor mediates entry for the mouse leukemia viruses (MLVs) with xenotropic and polytropic host ranges (X-MLVs and P-MLVs, respectively). X-MLVs and P-MLVs can be isolated from laboratory mice and are capable of infecting cells of nonrodent species; these viruses are distinguished by the ability of P-MLVs, but not X-MLVs, to infect cells of the laboratory mouse and by the cytopathic and leukemogenic properties of P-MLVs, also termed MCF MLVs (mink cell focus-inducing MLVs) (11,16,24). XPR1 is also the receptor for several wild mouse isolates with an atypical host range (6, 48, 49) and for the recently described virus XMRV (xenotropic murine leukemia virus-related virus) (8), isolated from human patients with prostate cancer or chronic fatigue syndrome (27,37,43). Studies on the XPR1 receptor have identified residues critical for virus entry and described functionally distinct variants of XPR1 in human and rodent species that differ in their abilities to mediate entry of various virus isolates (18,29,31,48,49).In Mus, four receptor variants of Xpr1 are found in different taxonomic groups...

Mouse APOBEC3 (mA3) is a cytidine deaminase with antiviral activity. mA3 is linked to the Rfv3 virus resistance factor, a gene responsible for recovery from infection by Friend murine leukemia virus, and mA3 allelic variants differ in their ability to restrict mouse mammary tumor virus. We sequenced mA3 genes from 38 inbred strains and wild mouse species, and compared the mouse sequence and predicted structure with human APOBEC3G (hA3G). An inserted sequence was identified in the virus restrictive C57BL strain allele that disrupts a splice donor site. This insertion represents the long terminal repeat of the xenotropic mouse gammaretrovirus, and was acquired in Eurasian mice that harbor xenotropic retrovirus. This viral regulatory sequence does not alter splicing but is associated with elevated mA3 expression levels in spleens of laboratory and wild-derived mice. Analysis of Mus mA3 coding sequences produced evidence of positive selection and identified 10 codons with very high posterior probabilities of having evolved under positive selection. Six of these codons lie in two clusters in the N-terminal catalytically active cytidine deaminase domain (CDA), and 5 of those 6 codons are polymorphic in Rfv3 virus restrictive and nonrestrictive mice and align with hA3G CDA codons that are critical for deaminase activity. Homology models of mA3 indicate that the two selected codon clusters specify residues that are opposite each other along the predicted CDA active site groove, and that one cluster corresponds to an hAPOBEC substrate recognition loop. Substitutions at these clustered mA3 codons alter antiviral activity. This analysis suggests that mA3 has been under positive selection throughout Mus evolution, and identified an inserted retroviral regulatory sequence associated with enhanced expression in virus resistant mice and specific residues that modulate antiviral activity.

Mutations in fukutin-related protein (FKRP) gene cause a wide spectrum of disease phenotypes including the mild limb-girdle muscular dystrophy 2I (LGMD2I), the severe Walker-Warburg syndrome, and muscle-eye-brain disease. FKRP deficiency results in α-dystroglycan (α-DG) hypoglycosylation in the muscle and heart, which is a biochemical hallmark of dystroglycanopathies. To study gene replacement therapy, we generated and characterized a new mouse model of LGMD2I harboring the human mutation leucine 276 to isoleucine (L276I) in the mouse alleles. The homozygous knock-in mice (L276I(KI)) mimic the classic late onset phenotype of LGMD2I in both skeletal and cardiac muscles. Systemic delivery of human FKRP gene by AAV9 vector in the L276I(KI) mice, at either neonatal age or at the age of 9 months, rendered body wide FKRP expression and restored glycosylation of α-DG in both skeletal and cardiac muscles. FKRP gene therapy ameliorated dystrophic pathology and cardiomyopathy such as muscle degeneration, fibrosis, and myofiber membrane leakage, resulting in restoration of muscle and heart contractile functions. Thus, these results demonstrated that the treatment based on FKRP gene replacement was effective.

Diabetes poses a substantial burden to society as it can lead to serious complications and premature death. The number of cases continues to increase worldwide. Two major causes of diabetes are insulin resistance and insulin insufficiency. Currently, there are few antidiabetic drugs available that can preserve or protect β-cell function to overcome insulin insufficiency in diabetes. We describe a therapeutic strategy to preserve β-cell function by overexpression of follistatin (FST) using an AAV vector (AAV8-Ins-FST) in diabetic mouse model. Overexpression of FST in the pancreas of db/db mouse increased β-cell islet mass, decreased fasting glucose level, alleviated diabetic symptoms, and essentially doubled lifespan of the treated mice. The observed islet enlargement was attributed to β-cell proliferation as a result of bioneutralization of myostatin and activin by FST. Overall, our study indicates overexpression of FST in the diabetic pancreas preserves β-cell function by promoting β-cell proliferation, opening up a new therapeutic avenue for the treatment of diabetes.

Xenotropic mouse leukemia viruses (X-MLVs) are broadly infectious for mammals except most of the classical strains of laboratory mice. These gammaretroviruses rely on the XPR1 receptor for entry, and the unique resistance of laboratory mice is due to two mutations in different putative XPR1 extracellular loops. Cells from avian species differ in susceptibility to X-MLVs, and 2 replacement mutations in the virus-resistant chicken XPR1 (K496Q and Q579E) distinguish it from the more permissive duck and quail receptors. These substitutions align with the two mutations that disable the laboratory mouse XPR1. Mutagenesis of the chicken and duck genes confirms that residues at both sites are critical for virus entry. Among 32 avian species, the 2 disabling XPR1 mutations are found together only in the chicken, an omnivorous, ground-dwelling fowl that was domesticated in India and/or Southeast Asia, which is also where X-MLV-infected house mice evolved. The receptor-disabling mutations are also present separately in 5 additional fowl and raptor species, all of which are native to areas of Asia populated by the virus-infected subspecies Mus musculus castaneus. Phylogenetic analysis showed that the avian XPR1 gene is under positive selection at sites implicated in receptor function, suggesting a defensive role for XPR1 in the avian lineage. Contact between bird species and virus-infected mice may thus have favored selection of mouse virus-resistant receptor orthologs in the birds, and our data suggest that similar receptor-disabling mutations were fixed in mammalian and avian species exposed to similar virus challenges.T he transmission of retroviruses to new hosts can result in the emergence of new infectious diseases and can alter the host genomic architecture and gene-regulatory networks, but the factors that determine whether a pathogen can successfully infect a novel host are poorly understood (1). The xenotropic mouse leukemia viruses (X-MLVs) are gammaretroviruses originally isolated from laboratory mice that are unable to infect the cells of these mice (2). These viruses rely on the XPR1 cell surface receptor for entry (3-5), and receptor orthologs from other mammals, like humans and cats, as well as the various species of wild mice, all permit X-MLV entry (6, 7). The resistance of laboratory mouse cells to infection is due to mutations that alter two XPR1 residues, K500E in the putative third extracellular loop (ECL3) and T582⌬ in the fourth loop (ECL4) (8).There is substantial sequence variation in the receptor-determining regions of the mammalian XPR1 orthologs, and some of these receptor variants show altered ability to mediate entry of one or more of the 6 known host range variants in the XP-MLV family of xenotropic/polytropic viruses (6, 9). In addition to X-MLV, this family includes viruses first described as broadly polytropic (PMLVs) because of their ability to infect mouse cells, as well as cells of other species (10, 11). Other variants, like xenotropic murine leukemia virus-related virus (XMRV) (12) an...