The mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B) can use the same receptor, Pit1, to infect human cells. A highly polymorphic nine-residue sequence within Pit1, designated region A, has been proposed as the virus binding site, because mutations in this region abolish Pit1-mediated cellular infection by GALV and FeLV-B. However, a direct correlation between region A mutations deleterious for infection and loss of virus binding has not been established. We report that cells expressing a Pit1 protein harboring mutations in region A that abolish receptor function retain the ability to bind virus, indicating that Pit1 region A is not the virus binding site. Furthermore, we have now identified a second region in Pit1, comprising residues 232 to 260 ( Pit1 is the human ortholog of a ubiquitous multiple-membrane-spanning protein that functions as the type III sodium phosphate cotransporter (14, 19) and as the receptor for feline leukemia virus type B (FeLV-B) (33), woolly monkey virus (33), gibbon ape leukemia virus (GALV) (18), and 10A1 murine leukemia virus (10A1 MLV) (17, 38). Pit2, another type III sodium phosphate cotransporter, is the human ortholog of a highly related protein (approximately 60% amino acid identity), which functions as a receptor for amphotropic murine leukemia virus (17, 34), 10A1 MLV, and FeLV-B molecular recombinants (30), but not for GALV or naturally occurring FeLV-B isolates (21). The ability of Pit1 and Pit2 to function as discrete viral receptors with unique properties presumably is reflected in critical residue differences between these two proteins. Early efforts to identify regions within Pit1 important for virus receptor function implicated residues 550 to 558 for both GALV and FeLV-B entry. It has been proposed that this nine-Pit1-residue stretch, designated region A, is the binding site for both of these viruses (6, 11, 35) based on the following observations: (i) mutations in Pit1 region A render Pit1 nonfunctional for GALV or FeLV-B entry (3, 9, 22, 27, 31, 32); (ii) Pit2, MusPit1 (the murine ortholog of Pit1), and Pho-4 (a distantly related Neurospora crassa phosphate transporter), which are not GALV or FeLV-B receptors, become functional receptors when region A residues are substituted for the corresponding residues of these proteins (12, 13, 33); and (iii) region A is predicted by Kyte-Doolittle hydropathy plots to reside in an extracellular domain (12). It should be noted, however, that all published reports supporting the role of region A as a binding site have been based on functional assays of viral entry rather than actual receptor-binding studies (reviewed in reference 20). Therefore we sought to determine if mutations in region A that abolish virus receptor function do so by abolishing virus binding.We have used data derived from the analyses of epitopetagged Pit proteins, glycosylation studies, and transmembrane (TM) structure predictions performed by using the PHD PredictProtein algorithm (23, 24) to derive a...

Human PiT2 (PiT2) is a multiple-membrane-spanning protein that functions as a type III sodium phosphate cotransporter and as the receptor for amphotropic murine leukemia virus (A-MuLV). Human PiT1 (PiT1), another type III sodium phosphate cotransporter, is a highly related protein that functions as a receptor for gibbon ape leukemia virus but not for A-MuLV. The ability of PiT1 and PiT2 to function as discrete viral receptors with unique properties presumably is reflected in critical residue differences between these two proteins. Early efforts to map the region(s) within PiT2 that is important for virus binding and/or entry relied on infection results obtained with PiT1-PiT2 chimeric cDNAs expressed in Chinese hamster ovary (CHOK1) cells. These attempts to localize the PiT2 virus-binding site were hampered because they were based on infectivity, not binding, assays, and therefore, receptors that bound but failed to facilitate virus entry could not be distinguished from receptors that did not bind virus. Using a more accurate topological model for PiT2 as well as an A-MuLV receptor-binding assay, we have identified extracellular domain one (ECD1) of the human PiT2 receptor as being important for A-MuLV binding and infection.PiT1 and PiT2 are type III sodium-dependent phosphate transporters that also function as receptors for the mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and amphotropic murine leukemia virus (A-MuLV), respectively (10,18,19,34,36). While these receptors have similar cellular functions and structures, they do not overlap in their virus receptor functions; this has been attributed to critical amino acid differences between PiT1 and PiT2.Early structural predictions for the arrangement of the PiT receptors in the plasma membrane were based on KyteDoolittle hydropathy analyses (8). Both proteins were initially predicted to be nearly identical in structure, each comprising 10 transmembrane (TM) domains. Additionally, the observed absence of a signal peptide for both proteins was used to assign cytoplasmic locations for the N and C termini; both were initially predicted to contain five extracellular domains (ECDs) and four intracellular domains, with all potential N-linked glycosylation sites being positioned within intracellular domains (8).In order to understand how differences in amino acid composition between PiT1 and PiT2 affect receptor function, researchers have used chimeric PiT1-PiT2 proteins to map regions that are critical for GALV (2,5,9,21,22,27,13,14,21,28,30) entry. Previous studies based on Kyte-Doolittle hydropathy models of PiT1 and PiT2 have demonstrated that replacement of the second ECD (ECD2) of PiT1 with the corresponding region of PiT2 results in a chimeric protein which functions as an A-MuLV receptor (12). This result was supported by studies by Lundorf et al. that showed that substitution of PiT2 residues from ECD2 and flanking regions for the corresponding residues of Pho-4, a sodium-dependent phosphate transporter from the filamentous fungus Neurospora...

The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.

Therapeutic gene delivery mediated by retroviral vectors has the advantage of stable integration into the host genome. A major safety concern for gene delivery achieved by murine leukemia virus (MLV)-based retroviral vectors is the activation of adjacent cellular genes including oncogenes following integration into the host genome. Self-inactivating (SIN) vectors lacking viral enhancers/promoters in their 3' long terminal repeat (LTR) have been proposed as a means of overcoming this safety concern. However the MLV-based SIN vectors currently used by laboratories to assess insertional mutagenesis, integration site selection, and the potency of transgene expression are not uniform in the composition of their 3' LTRs. We constructed a series of SIN vectors representative of the currently employed vectors, but lacking an internal promoter. Green fluorescent protein (GFP) was used as a reporter gene. Target cells exposed to these vectors were evaluated for number of integrants and GFP expression at the messenger RNA (mRNA) level and protein level. We found that viral promoter activity in the 3' LTR is not attenuated in many currently employed SIN vectors. These results suggest that the influence of strong residual promoter activity should be taken into consideration when interpreting experimental results obtained using SIN vectors in gene therapy research.