The outermost protein of the retrovirus, termed the envelope, carries the primary determinant of viral tropism, by virtue of its interaction with a specific cell surface receptor. I am interested in characterizing the structural and functional aspects of the envelope-receptor interaction. The envelopes of two retroviruses which can infect human cells, gibbon ape leukemia virus (GaLV) and amphotropic murine leukemia virus (A-MuLV), have been used in retroviral vectors for the purposes of infecting human cells. The cDNAs for the cellular receptors for these viruses have recently been cloned and sequenced. Although the envelope sequences of GaLV and A-MuLV are not very related (sharing only about 35% amino acid identity), the receptors for these two viruses are similar in overall topology (10 transmembrane domains) and amino acid conservation (58% amino acid identity). We have now demonstrated that the cellular function of both of these receptors is to transport inorganic phosphate. Although both proteins appear to be ubiquitously expressed, suggesting a role in maintaining cellular phosphate homeostasis, certain functional parameters in phosphate uptake distinguish these two proteins (e.g., differences in binding affinity and capacity). A unique form of the A-MuLV receptor is expressed on a Chinese hamster cell line. We cloned, sequenced and functionally characterized this cDNA and its associated protein. We show that, in contrast to the human form of this receptor, which is only used by A-MuLV, and not by GaLV, the hamster form of the A-MuLV receptor can be used by both GaLV and A-MuLV. The hamster form of the A-MuLV receptor also functions as a sodium phosphate symporter. Further, we have now demonstrated that the 10A1 MuLV, a virus which shares 92.5% amino acid identity with A-MuLV in envelope sequence, but only 35% amino acid indentity with GaLV envelope sequence, can not only use the human form of the A-MuLV receptor, but surprisingly, can also use the human form of the GaLV receptor to infect cells. Study of these naturally occurring viruses and their receptors will help to elucidate 1) what region(s) of the GaLV and A-MuLV envelopes are critical for viral infection; 2) what region(s) of the receptors are used by each virus (i.e., do 10A1 MuLV and GaLV interact with the same or different regions of the GaLV receptor); and 3) regulation of the receptors/transporters in the presence or absence of viral infection. The results of these ongoing studies may allow for the development of novel retroviral vectors with altered tropisms and/or manipulation of their receptors to maximize in vivo gene transfer. This research is immediately relevant to regulatory issues regarding use of retroviral vectors for human gene therapy. GRANTZ01BM06004 (1) The trace element vanadium can act like insulin, a major regulator of lipid metabolism. We demonstrated that in vitro, vanadium at concentrations as low as 250 nM significantly reduces synthesis of the major atherogenic protein apolipoprotein B-100 in Caco-2 cells (an intestinal human cell line). In addition, another form of apoB, apoB- 48, considered nonatherogenic, is increased by vanadium. In a pilot in vivo study, a rabbit model of familial hypercholesterolemia (Watanabe breed) with elevated cholesterol, triglycerides and atherosclerosis was fed vanadate. A reduction was seen in cholesterol, triglyceride, and apolipoprotein B as well as an increase in HDL cholesterol, the so called """"""""good cholesterol"""""""". A followup study with 10 rabbits as well as rats will be done to further delineate the physiologic effects of vanadium.(2) We characterized the apoB RNA and DNA sequesnces in a patient with aberrant apoB metabolism. This patient was homozygous for a single base deletion in the apoB gene that introduced a frame shift and premature stop codon, yielding a truncated form of apoB (so called apoB-87). Surprisingly, the patient also synthesized an apoB virtually identical in size to full length apoB-100 called psuedo-B-100. We determined that a dinucleotide deletion (in the hepatic apoB mRNA transcript) located 40 base pairs from the original genomic deletion restores the translational reading frame. This AT deletion would be the first report of this novel RNA editing mechanism, and may represent a form of RNA repair. We have designed peptide antibodies to confirm this mechanism at the protein level.(3) We studied lipid profiles in 150 patients with systemic lupus erythematosus (SLE), an autoimmune disease often associated with hyperlipidemia. In the abscence of nephrosis, SLE patients have relatively normal lipid levels suggesting that the SLE disease itself is not, as heretofore accepted a cause of hyperlipidemia. We completed a trialin these patients showing the efficacy of the cholesterol lowering drug, mevacor, reducing cholesterol levels. We have begun a trial in these patients to examine the effect of niacin, another lipid lowering drug, on the markedly atherogenic lipoprotein Lp(a).
