Search PubMed⌕ Search

Biomedical subjects

W F Anderson

Publications and source records attributed to W F Anderson.

At least 55 records · Page 3Linked to original sources
PubMed1999

High efficiency in vitro gene transfer into vascular tissues using a pseudotyped retroviral vector without pseudotransduction.

Murine leukemia virus (MuLV)-derived retroviral vectors have had limited application in vascular gene therapy because of low transduction efficiency of vascular tissues, both in vitro and in vivo. In this study, we compared the gene transfer efficiency of two retroviral vectors: amphotropic MuLV and a MuLV vector pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) envelope. Target vascular tissues included human endothelial cells (EC), smooth muscle cells (SMC) and saphenous veins (SV). Transduction efficiency of human EC and SMC was significantly higher for VSV-G pseudotyped MuLV vector (90%) than for Amphotropic MuLV (20%). Luminal surface en face analysis of transduced cultured SV showed a six- to 10-fold greater transduction efficiency with VSV-G pseudotyped MuLV. The tissue plasminogen activator (tPA) gene was transduced into EC using each vector. Four days following transduction, a 12-fold higher tPA antigen concentration and a 38-fold higher tPA enzymatic activity was measured from cells transduced with the VSV-G pseudotyped vectors as compared with the amphotropic MuLV. There was no detectable pseudotransduction (protein transfer) associated with the VSV-G MuLV vector. Both AZT inhibition of reverse transcriptase and cell division arrest by gamma irradiation inhibited transduction, indicating that viral transduction correlated with RNA reverse transcription and cell proliferation. MuLV pseudotyped with the VSV-G envelope glycoprotein is an effective retroviral vector for vascular gene therapy.

Cell Division↗
PubMed1999

Structure of the fibrinogen gamma-chain integrin binding and factor XIIIa cross-linking sites obtained through carrier protein driven crystallization.

The human fibrinogen gamma-chain C-terminal segment functions as the platelet integrin binding site as well as the Factor XIIIa cross-linking substrate and thus plays an important role in blood clot formation and stabilization. The three-dimensional structure of this segment has been determined using carrier protein driven crystallization. The C-terminal segment, gamma-(398-411), was attached to a linker sequence at the C-terminus of glutathione S-transferase and the structure of this fusion protein determined at 1.8 A resolution. Functional studies of the chimeric protein demonstrate that the fibrinogen sequence in the presence of the carrier protein retains its specific functions as ligand for platelet integrin alpha(IIb)beta3 (gpIIb/IIIa) and as a cross-linking substrate for Factor XIIIa. The structure obtained for the fibrinogen gamma-chain segment is not affected by crystal packing and can provide the missing links to the recently reported model of cross-linked fibrin.

Animals↗
PubMed1999

Receptor-mediated Moloney murine leukemia virus entry can occur independently of the clathrin-coated-pit-mediated endocytic pathway.

To investigate receptor-mediated Moloney murine leukemia virus (MoMuLV) entry, the green fluorescent protein (GFP)-tagged ecotropic receptor designated murine cationic amino acid transporter (MCAT-1) (MCAT-1-GFP) was constructed and expressed in 293 cells (293/MCAT-1-GFP). 293/MCAT-1-GFP cells displayed green fluorescence primarily at the cell membrane and supported wild-type levels of MoMuLV vector binding and transduction. Using immunofluorescence labeling and confocal microscopy, it was demonstrated that the surface envelope protein (SU) gp70 of MoMuLV virions began to appear inside cells 5 min after virus binding and was colocalized with MCAT-1-GFP. However, clathrin was not colocalized with MCAT-1-GFP, suggesting that MoMuLV entry, mediated by MCAT-1, does not involve clathrin. Double immunofluorescence labeling of SU and clathrin in 293 cells expressing untagged receptor (293/MCAT-1) gave the same results, i.e., SU and clathrin did not colocalize. In addition, we examined the transduction ability of MoMuLV vector on HeLa cells overexpressing the dominant-negative GTPase mutant of dynamin (K44A). HeLa cells overexpressing mutant dynamin have a severe block in endocytosis by the clathrin-coated-pit pathway. No significant titer difference was observed when MoMuLV vector was tranduced into HeLa cells overexpressing either wild-type or mutant dynamin, while the transduction ability of vesicular stomatitis virus glycoprotein pseudotyped vector into HeLa cells overexpressing mutant dynamin was decreased significantly. Taken together, these data suggest that MoMuLV entry does not occur through the clathrin-coated-pit-mediated endocytic pathway.

3T3 Cells↗
PubMed1998

In utero gene therapy: transfer and long-term expression of the bacterial neo(r) gene in sheep after direct injection of retroviral vectors into preimmune fetuses.

We investigated whether directly injecting retroviral vectors into preimmune fetuses could result in the transfer and long-term expression of exogenous genes. Twenty-nine preimmune sheep fetuses were injected with helper-free retroviral vector preparations. Twenty-two fetuses survived to term, 4 of which were sacrificed at birth. Of the remaining 18 animals, 3 were controls and 15 had received vector preparations. Twelve of these 15 animals demonstrated transduction of hematopoietic cells when blood and marrow were analyzed by neo(r)-specific PCR. Eight experimental sheep have been followed for 5 years, during which time we have consistently observed proviral DNA and G418-resistant hematopoetic progenitors. The G418-resistant colonies were positive when analyzed by neo(r)-specific PCR. neo(r) gene expression was also demonstrated using several immunological and biochemical methods. The transduction of hematopoietic stem cells was confirmed when lambs transplanted with bone marrow from in utero-transduced sheep exhibited neo(r) activity in marrow and blood. Vector distribution was widespread in primary animals without pathology. PCR analysis indicates that the germ line was not altered. These studies demonstrate that direct injection of an engineered retrovirus is a feasible means of safely delivering a foreign gene to a developing fetus and achieving long-term expression without modifying the germ line of the recipient.

Animals↗
PubMed1998

Targeted inactivation of the coagulation factor IX gene causes hemophilia B in mice.

Hemophilia B is a leading target for gene therapy because current therapy is not optimal. Hence, a murine model of factor IX (F. IX) deficiency was generated to develop gene therapy strategies for hemophilia B. A targeting vector was created by replacing a 3.2-kb segment of the gene encompassing the catalytic domain with a phosphoglycerokinase promoter-driven neomycin resistant (neor) gene cassette. The transfected embryonic stem cell clones generated chimeric male mice, and germ line transmission of the inactivated F. IX gene was observed in their offsprings. Southern analysis confirmed the mutant genotype in hemizygous male and carrier female mice. F. IX transcripts were not detected in liver RNA isolated from hemizygous mice, and lower levels of F. IX mRNA were noted in carrier female mice when compared with those of normal litter mates. As expected, the mean F. IX coagulant titer of affected male mice was 2.8 U/dL (n = 10), while the mean F. IX titer of carrier female mice was 35 U/dL (n = 14), compared with 69 U/dL (n = 9) for the normal female mice and 92 U/dL (n = 22) for normal male and female litter mates. Further, the tail bleeding time of hemizygous mice was markedly prolonged (>3 hours) compared with those of normal and carrier female litter mates (15 to 20 minutes). Seven of 19 affected male mice died of exsanguination after tail snipping, and two affected mice died of umbilical cord bleeding. Currently, there are 10 affected mice surviving at 4 months of age. Aside from the factor IX defect, the carrier female and hemizygous male mice had no liver pathology by histologic examination, were fertile, and transmitted the F. IX gene mutation in the expected Mendelian frequency. Taken together, we have generated a F. IX knockout mouse for evaluation of novel gene therapy strategies for hemophilia B.

Animals↗
PubMed1998

The influence of patient-related factors on ex vivo retroviral-mediated gene transfer into mobilized peripheral blood myeloid progenitors.

Mobilized human peripheral blood progenitor cells are potential alternatives to bone marrow cells as targets for ex vivo gene transfer. We report the transduction efficiency of retroviral-mediated gene transfer into myeloid progenitors of peripheral blood progenitor cell (PBPC) harvests, mobilized by high-dose cyclophosphamide and GM-CSF, compared with nonmobilized bone marrow (BM). Eleven PBPC samples were enriched for CD34+ cells, preincubated with IL-3 (10 ng/ml), IL-6 (50 ng/ml), and 10% autologous plasma for 42 hr, and transduced over a 6-hr incubation with IL-3 + IL-6 and a retroviral vector carrying the NeoR gene. NeoR-specific sequences were detected by polymerase chain reaction in 10 cell pellets (91%). Gene expression in CFU-GM colonies was found in nine transduced samples (82%), with a mean transduction efficiency of 5.2% (95% CI, 1.3-11.8%) CFU-GM per PBPC sample. In univariate analysis, a higher transduction efficiency into CFU-GM correlated significantly with a higher CFU-GM concentration in the CD34+-enriched sample (p = 0.009), a shorter interval from diagnosis (p < 0.001), and fewer months of prior cytotoxic treatment (p = 0.001); correlation with younger age was of borderline statistical significance (p = 0.077). In multivariate analysis a shorter interval from diagnosis and, to a lesser degree, a higher CFU-GM concentration in the CD34+-enriched sample were independent predictors of higher transduction efficiency. Twelve BM samples were similarly transduced; 11 pellets were PCR positive. CFU-GM NeoR gene expression was 4.2% (95% CI, 2.0-7.2%) CFU-GM per BM sample, which was not significantly different from the transduction efficiency into PBPC cells. No correlation was found between the transduction efficiency of CFU-GM in BM samples and CFU-GM concentration in the CD34+-enriched sample, time from diagnosis, months of prior cytotoxic treatment, and/or patient age. Our data suggest that the transduction efficiency ex vivo may be influenced by time from diagnosis, CFU-GM concentration in the sample, and possibly by the extent of prior cytotoxic administration.

Adult↗
PubMed1998

Intravenous RMP-7 increases delivery of ganciclovir into rat brain tumors and enhances the effects of herpes simplex virus thymidine kinase gene therapy.

Herpes simplex virus thymidine kinase (HSV-tk) gene therapy for brain tumors depends on ganciclovir (GCV) and its transport across the blood-brain tumor barrier (BBTB). We examined whether RMP-7, the bradykinin analog and potent BBTB permeabilizer, could enhance the efficacy of GCV treatment of brain tumors by increasing the BBTB delivery of GCV. In vitro, a significant bystander cytocidal effect of GCV was shown in mixed HSV-tk-transduced (HSV-tk+) and control vector-transduced (HSV-tk-) C6 glioma cultures. A dose-dependent cytotoxic effect of GCV on untransformed C6 cells was also shown. In vivo, rats with 100% HSV-tk+ or 100% HSV-tk- intracerebral C6 gliomas were treated for 7 days with intravenous infusions of GCV alone or with GCV and RMP-7 (2.5 microg/kg/day). The growth of HSV-tk+ and HSV-tk- gliomas decreased with increasing doses of GCV. A high dosage (100 mg of GCV/kg/day) eradicated all HSV-tk- and HSV-tk+ tumors. An intermediate dosage (5 mg of GCV/kg/day) reduced the growth of HSV-tk- gliomas by 42% if given alone, and by 88% in combination with RMP-7. A low dosage (0.5 mg of GCV/kg/day) in combination with RMP-7 enhanced the regression of HSV-tk+ gliomas by 87% compared with GCV alone. Low-dose GCV was ineffective in HSV-tk- tumors. RMP-7 increased [3H] GCV tumoral uptake by 2.6- and 1.7-fold in the tumor center and periphery, respectively. We conclude that RMP-7 could be an important adjunctive treatment for suicide gene therapy of brain tumors, while an RMP-7/GCV combination may also have a significant antitumor effect in untransfected gliomas.

3T3 Cells↗
PubMed1998

Quantitation of MoMuLV envelope protein on the cell surface.

The envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMuLV) is proteolytically processed and transported to the cell surface where it can be incorporated into budding virions. Cell surface Env is frequently detected using an indirect immunofluorescence assay and fluorescence-activated cell sorting (FACS). We found that the detection of Env in this manner requires the expression of the MoMuLV receptor (ATRC-1) on the cell surface, and the level of envelope protein detected correlates with the level of receptors expressed on the cell. In addition, Env detection corresponds to the Env protein's ability to bind to its receptor and can be competed out by the addition of a truncated form of the Env protein. These data suggest that Env detected on the cell surface by the FACS assay is protein that has rebound to its receptor after being secreted or shed, rather than actual surface-expressed protein. In contrast, a combined immunoprecipitation and biotinylation assay detected equal amounts of Env on the surface of both receptor-lacking and receptor-expressing cell lines. The immunoprecipitation-biotinylation assay is therefore a more appropriate method for detecting surface expression of the MoMuLV envelope protein.

3T3 Cells↗
PubMed1998

Intercellular communication mediates the bystander effect during herpes simplex thymidine kinase/ganciclovir-based gene therapy of human gastrointestinal tumor cells.

A bystander effect is described when nontransduced or genetically unmodified cells are killed during death of genetically modified tumor cells transduced with a suicide gene. The "bystander effect" greatly enhances the efficacy of the herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy approach for cancer. The mechanism of the bystander effect is controversial. In this study, we examined the role of intercellular gap junction communication (GJIC) for the bystander effect in human gastrointestinal tumor cells. Our results show that the extent of the bystander effect varied amongst the tumor cell lines; pancreatic cancer cells BXPC-3 exhibited excellent bystander effects in vitro and in vivo studies whereas other gastrointestinal tumor cell lines such as pancreatic cancer cells MIAPACA-2, and colon cancer cells HT-29 showed poor bystander effects. Bystander effects were only found in the presence of cell-to-cell contact. The extent of the bystander effect was independent of the level of HSV-TK activity in the transduced tumor cells and was correlated with GJIC as demonstrated by an in vitro dye-transfer assay. Expression of the mRNA levels of gap junction protein connexin 43 was 8- to 26-fold or greater and connexin 26 gene expression was 2- to 229-fold greater in BXPC-3 cells compared to HT-29, MIAPACA-2, and PANC3 cells. Our results suggest that intercellular communication is essential for the bystander effect. The correlation between GJIC and the extent of the bystander effect suggest a role for GJIC in mediating the bystander effect. Analysis of tumors for GJIC or expression of gap junction proteins may identify the subset of patients suitable for gene therapy with the HSV-TK/GCV approach.

Animals↗
PubMed1998

New member of aldose reductase family proteins overexpressed in human hepatocellular carcinoma.

The multistep process of liver carcinogenesis involves various genetic and phenotypic alterations. To identify genes whose expression is increased during hepatocarcinogenesis, differential-display polymerase chain reaction (DD-PCR) was used to examine differences in the mRNA composition of hepatocellular carcinoma (HCC) versus normal liver (nontumor) tissues. This approach identified 67 cDNAs that were preferentially expressed in HCC tissue. When these cDNAs were analyzed by reverse-Northern analysis, five were reproducibly expressed at high levels in HCC. Interestingly, Northern blot analysis revealed that one of the genes showed significantly increased mRNA levels in all five tested tumor samples, while its mRNA level in the nontumor samples was minimal. BLAST analysis revealed that this gene has high sequence identity with the genes from aldo-keto reductase family of proteins including the mouse fibroblast growth factor-induced gene (FR-1) (80% identity), mouse vas deferens protein (MVDP) (76%), and human aldose reductase (AR) (62%). Expression of this novel AR-related protein in all five tested HCCs suggests that this protein may play an important role in liver carcinogenesis.

Aldehyde Reductase↗
PubMed1998

Retrovirus-mediated gene transfer targeted to retinal photocoagulation sites.

Diabetic retinopathy is a major cause of acquired blindness due to the development of retinal neovascularization and associated traction retinal detachment. It is commonly treated with retinal photocoagulation therapy; however, progression to blindness remains a significant problem. To determine the feasibility of adjunctive anti-angiogenic gene therapy, we evaluated the capability of retroviral vectors, which transfer exogenous genes only into dividing cells, to transfer and express a beta-galactosidase gene selectively into photocoagulation sites. Thirty-five rabbits received 30 retinal photocoagulation burns in the right eye followed 2 days later by beta-galactosidase (G1nBgSvNa) or control (G1XSvNa) vector injection into the subretinal space. Beta-galactosidase expression was observed in the photocoagulation sites from 5 days after vector administration (31.7+/-7.0%) to 12 weeks (6.7+/-3.4%). Immunohistochemical studies of the treated retinas using antibody Ber-MAC3 and anti-cytokeratin antibodies revealed that transduced cells were macrophages and retinal pigment epithelial cells. To determine feasibility in a primate, two monkeys received 10 laser burns in the macula superior to the fovea followed 2 days later by G1nBgSvNa vector. beta-galactosidase expression was found in photocoagulation sites and foveal retina was well preserved. We conclude that gene transfer to retinal photocoagulation sites provides stable expression of the transduced gene with relatively high efficiency. This feasibility study suggests the possibility of transferring genes encoding for anti-angiogenic factors into photocoagulation sites to improve the efficacy of laser photocoagulation therapy.

Angiography↗
PubMed1998

Ability of retroviral transduction to modify the angiogenic characteristics of RPE cells.

BACKGROUND: Retinal pigment epithelial (RPE) cells play an important role in the modulation of ocular angiogenesis. Transduction of RPE cells with retroviral vectors bearing modulating genes can result in long-term transgene expression and may alter the angiogenic characteristics of RPE cells. This study was designed to determine whether changes in angiogenic characteristics of RPE cells result from transduction with retroviral vectors bearing modulating genes, using in vitro angiogenic assays, including analysis of endothelial proliferation and wound healing. METHODS: Human RPE cells were transduced with retroviral vectors bearing either a urokinase-type plasminogen activator (u-PA) or a tissue-type plasminogen activator (t-PA) cDNA. Ten weeks after gene transfer, RPE cells transduced with the u-PA (u-PA-RPE cells) or the t-PA cDNA (t-PA-RPE cells), or untransduced (control) RPE cells, were cocultured with human umbilical vein endothelial cells (HUVECs) by contacting and non-contacting coculture methods. The effects of these cells on proliferation and in vitro "wound healing" of HUVECs were evaluated. RESULTS: Over 18 weeks, u-PA-RPE cells released large amounts of biologically active u-PA (total amount, 50.2 +/- 9.7 ng/10(6) cells/24 h), while t-PA-RPE cells released large amounts of functional t-PA (15.4 +/- 3.2 ng/10(6) cells/24 h). Control RPE cells did not release any detectable t-PA or u-PA. In the proliferation assay, u-PA-RPE cells stimulated HUVEC proliferation in contacting cell cultures, but not in non-contacting cell cultures. In contrast, t-PA-RPE cells, normal RPE cells or exogenous u-PA had no effect on HUVEC proliferation. In the wound healing assay, u-PA-RPE cells in contacting coculture and exogenous u-PA stimulated wound healing of HUVECs, while non-contacting u-PA-RPE cells, t-PA-RPE cells and normal RPE cells had no effect on HUVEC wound healing. RPE cells transduced with u-PA secreted large amounts of u-PA for as long as 18 weeks, and these cells stimulate HUVEC proliferation and in vitro wound healing. As a result, the angiogenic characteristics of RPE cells can undergo long-term changes. CONCLUSIONS: These results suggest that genetically modified RPE cells can be used to modulate ocular angiogenesis and may have potential for gene therapy of ocular diseases.

Cell Division↗
PubMed1998

Retroviral vector-mediated gene transfer into keratocytes: in vitro effects of polybrene and protamine sulfate.

BACKGROUND: To determine the potential of somatic gene transfer as a novel technique for modulating corneal wound healing on a cellular level, the successful transduction of human keratocytes should be ascertained in vitro. In addition, the ability of different polycations to increase the transduction efficiency and their antiproliferative and cytotoxic effects should be assessed. METHODS: To test transduction efficiency (X-Gal staining), cultured human keratocytes were incubated for 2 h with a retroviral vector bearing the beta-galactosidase gene, with and without the addition of polybrene or protamine sulfate. To test the antiproliferative and cytotoxic effects, cultured human keratocytes were incubated with various concentrations of polybrene and protamine sulfate (0.08 to 800 micrograms/ml) for 2, 24 and 72 h, and evaluations were performed by means of an XTT-based colorimetric assay and phase-contrast microscopy. RESULTS: Human keratocytes in vitro were transduced successfully with the beta-galactosidase gene (3.5 +/- 1.0%). Transduction efficiency was significantly (P < or = 0.01) improved by addition of a polycation (from 12.3 +/- 1.7% to 18.6 +/- 2.3%), but there was no significant difference between the effects of polybrene and those of protamine sulfate. Both drugs induced a highly significant dose-dependent inhibition of proliferation (P < 0.001). ID50 ranged from 11 to 22 micrograms/ml with polybrene and from 15 to 244 micrograms/ml with protamine sulfate. Only with doses of 80 and 800 micrograms/ml did protamine sulfate produce less antiproliferative effects than polybrene (P < or = 0.04). The lowest concentrations induced no morphological signs of cytotoxicity, whereas these signs were mild at 8 micrograms/ml and moderate to severe at the highest concentrations. CONCLUSIONS: Both polybrene and protamine sulfate can significantly improve the in vitro efficiency of successful retroviral vector-mediated gene transfer into keratocytes. Mild cytotoxic and moderate antiproliferative effects are to be expected in cultured keratocytes with a standard transduction procedure (8 micrograms/ml for 2 h).

Cell Division↗
PubMed1998

Retroviral vector-mediated gene transfer into keratocytes in vitro and in vivo.

PURPOSE: To determine the potential of somatic gene transfer as a technique for modulating corneal wound healing after superficial keratectomy. METHODS: The transduction of human and rabbit keratocytes with beta-galactosidase and herpes simplex virus thymidine kinase genes was performed. In vitro, human and rabbit keratocytes were transduced with retroviral vectors bearing beta-galactosidase or HStk (herpes simplex virus thymidine kinase) genes. In vivo, rabbit keratocytes were transduced by topical application of vector supernatant after a superficial keratectomy. In vitro and in vivo, expression of the beta-galactosidase gene was examined with histochemical staining. In vitro, ganciclovir cytotoxicity in HStk gene-transduced keratocytes and bystander effect in co-cultures of HStk(+) and HStk(-) keratocytes were measured by determining the degree of confluency of cells in 6-well plates after 10 days of incubation. Corneal haze in rabbits was measured after transduction with Hstk and subsequent treatment with topical ganciclovir. RESULTS: In vitro, both human and rabbit keratocytes were transduced successfully with both beta-galactosidase and HStk genes. Transduction efficiency was greater with human (22%) than with rabbit (16%) cells, and both HStk-transduced cell lines showed dose-dependent ganciclovir cytotoxicity and a significant bystander effect. In vivo, expression of beta-galactosidase within vimentin-positive corneal stromal cells confirmed transduction of keratocytes in the rabbit after superficial stromal keratectomy with an efficiency of 25% to 40%. Postoperative application of topical ganciclovir reduced corneal stromal haze in rabbits. CONCLUSIONS: The ability to genetically transduce stromal keratocytes provides a new strategy for understanding the important cellular and molecular events that influence corneal wound healing, thus offering a potential approach to decrease or prevent corneal haze and scarring after superficial keratectomy.

Animals↗
PubMed1998

Role of variable regions A and B in receptor binding domain of amphotropic murine leukemia virus envelope protein.

For the amphotropic murine leukemia virus (MuLV), a 208-amino-acid amino-terminal fragment of the surface unit (SU) of the envelope glycoprotein is sufficient to bind to its receptor, Pit2. Within this binding domain, two hypervariable regions, VRA and VRB, have been proposed to be important for receptor recognition. In order to specifically locate residues that are important for the interaction with Pit2, we generated a number of site-specific mutations in both VRA and VRB and analyzed the resulting envelope proteins when expressed on retroviral vectors. Concurrently, we substituted portions of the amphotropic SU with homologous regions from the polytropic MuLV envelope protein. The amphotropic SU was unaffected by most of the point mutations we introduced. In addition, the deletion of eight residues in a region of VRA that was previously suggested to be essential for Pit2 utilization only decreased titer on NIH 3T3 cells by 1 order of magnitude. Although the replacement of the amino-terminal two-thirds of VRA with the polytropic sequence abolished receptor binding, smaller nonoverlapping substitutions did not affect the function of the protein. We were not able to identify a single critical receptor contact point within VRA, and we suggest that the amphotropic receptor binding domain probably makes multiple contacts with the receptor and that the loss of some of these contacts can be tolerated.

3T3 Cells↗
PubMed1998

Mutational analysis of the fusion peptide of Moloney murine leukemia virus transmembrane protein p15E.

Fusion peptides are hydrophobic sequences located at the N terminus of the transmembrane (TM) envelope proteins of the orthomyxoviruses and paramyxoviruses and several retroviruses. The Moloney murine leukemia virus TM envelope protein, p15E, contains a hydrophobic stretch of amino acids at its N terminus followed by a region rich in glycine and threonine residues. A series of single amino acid substitutions were introduced into this region, and the resulting proteins were examined for their abilities to be properly processed and transported to the cell surface and to induce syncytia in cells expressing the ecotropic receptor. One substitution in the hydrophobic core and several substitutions in the glycine/threonine-rich region that prevented both cell-cell fusion and the transduction of NIH 3T3 cells when incorporated into retroviral vector particles were identified. In addition, one mutation that enhanced the fusogenicity of the resulting envelope protein was identified. The fusion-defective mutants trans dominantly interfered with the ability of the wild-type envelope protein to cause syncytium formation in a cell-cell fusion assay, although no trans-dominant inhibition of transduction was observed. Certain substitutions in the hydrophobic core that prevented envelope protein processing were also found. These data indicate that the N-terminal region of p15E is important both for viral fusion and for the correct processing and cell surface expression of the viral envelope protein.

3T3 Cells↗
PubMed1998

Modulation of activity of Moloney murine leukemia virus preintegration complexes by host factors in vitro.

We have explored the requirements for host proteins in the integration of Moloney murine leukemia virus (MoMuLV) cDNA in vitro. Following infection, it is possible to lyse cells and obtain preintegration complexes (PICs) capable of integrating the MoMuLV cDNA into an added target DNA in vitro (intermolecular integration). PICs can be stripped of required proteins by gel filtration in high-salt buffers (600 mM KCI), allowing the nature of the removed factors to be investigated by in vitro reconstitution. In a previous study of human immunodeficiency virus type 1 (HIV-1) PICs, the host protein HMG I(Y) was found to be able to restore activity to salt-stripped PICs. In contrast, salt stripping and reconstitution of MoMuLV PICs led to the proposal that a host factor is important for a different activity, blocking integration into the cDNA itself (autointegration). In this report, we investigated reconstitution of salt-stripped MoMuLV PICs and found that addition of cellular extract from uninfected NIH 3T3 cells could block autointegration and also restore intermolecular integration. Isolation of the intermolecular integration-complementing activity yielded HMG I(Y), as in the HIV-1 case. However, HMG I(Y) could not block autointegration, implicating a different host factor in this process. Additionally, when MoMuLV PICs were partially purified but not salt stripped, the intermolecular integration activity was reduced but could be stimulated by the addition of any of several purified DNA binding proteins. In summary, three activities were detected: (i) the intermolecular integration cofactor HMG I(Y), (ii) an autointegration barrier protein, and (iii) stimulatory DNA binding proteins.

3T3 Cells↗