Fv1 is the prototypic restriction factor that protects against infection by the murine leukemia virus (MLV). It was first identified in cells that were derived from laboratory mice and was found to be homologous to the gag gene of an endogenous retrovirus (ERV). To understand the evolution of the host restriction gene from its retroviral origins, Fv1s from wild mice were isolated and characterized. Most of these possess intact open reading frames but not all restricted N-, B-, NR-or NB-tropic MLVs, suggesting that other viruses could have played a role in the selection of the gene. The Fv1s from Mus spretus and Mus caroli were found to restrict equine infectious anemia virus (EIAV) and feline foamy virus (FFV) respectively, indicating that Fv1 could have a broader target range than previously thought, including activity against lentiviruses and spumaviruses. Analyses of the Fv1 sequences revealed a number of residues in the C-terminal region that had evolved under positive selection. Four of these selected residues were found to be involved in the novel restriction by mapping studies. These results strengthen the similarities between the two capsid binding restriction factors, Fv1 and TRIM5α, which support the hypothesis that Fv1 defended mice against waves of retroviral infection possibly including non-MLVs as well as MLVs.

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In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration.

The specificity determinants for susceptibility to resistance by the Fv1 n and b alleles map to amino acid 110 of the murine leukemia virus CA protein. To study the interaction between Fv1 and CA, we examined changes in CA resulting in the loss of susceptibility to Fv1 resistance in naturally occurring NB-and NR-tropic viruses. A variety of amino acid changes affecting Fv1 tropism were identified, at CA positions 82, 92 to 95, 105, 114, and 117, and they all were mapped to the apparent exterior of virion-associated CA. These amino acids may form a binding surface for Fv1.The Fv1 gene is one of a series of mouse genes, originally described in the early 1970s, that control the susceptibility of mice to murine leukemia virus (MLV) infection (32,34 nr , found in a few inbred strains of mice and apparently also in some wild mice, restricts B-tropic MLV and some, but not all, N-tropic viruses (28,48). In this paper, we will refer to N-tropic viruses that are not restricted by Fv1 nr as being NR tropic.Fv1 acts in a cell-autonomous manner to restrict virus replication (44), but the precise mechanism for restriction is unclear. It has been shown that viral replication is blocked at a stage after virus entry into the cell and before the integration of newly synthesized viral DNA into the host genome (22, 41). The block to infection is not absolute in vitro, but the number of infected cells is reduced by a factor of 50 to 1,000 (17). When expressed at natural levels, e.g., in mouse fibroblast lines, neither Fv1 n nor Fv1 b shows significant in vitro restriction of NB-tropic MLV.Genetic studies initially suggested that the target for Fv1 restriction is the MLV capsid (CA) protein (20, 43). Subsequent studies indicated that viral tropism is determined by a pair of adjacent amino acids, at positions 109 and 110, in CA (10, 40). A more recent study has shown that position 110 is the most important residue for N and B tropism (29). N-tropic MLV has an Arg residue at this position, and B-tropic MLV has a Glu residue. The determinants for NB and NR tropism have not been fully characterized.The Fv1 gene was cloned a few years ago (3) and was found to have sequence similarity to a family of endogenous retroviruses called HERV-L (60% identity over 1.3 kb) or MuERV-L (1, 3). Based on its position within the element and the presence of a major homology region (3), Fv1 apparently encodes a Gag-related protein. Gag proteins bind tightly to each other via interaction domains during virus assembly (35), which suggests a possible mechanism for Fv1's action on MLV CA (16). To date, however, there is no direct evidence for the binding of Fv1 to CA. Biochemical analyses are greatly complicated by the extremely low natural expression levels of Fv1 in vivo. We have therefore taken a genetic approach to analyzing the viral determinants of NB and NR tropism, using a rapid fluorescence-activated cell sorting (FACS)-based approach for Fv1 testing (5), in an attempt to delineate the region(s) of CA that interacts with Fv1. MATERIALS AND METHODS Cel...

Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE) and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific), EIAV-CMV-MYO7A (UshStat) or EIAV-CMV-Null (control) vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat) rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.