Vesicular Stomatitis Virus Envelope Research Articles

Replication-Competent Recombinant Vesicular Stomatitis Virus Encoding Hepatitis C Virus Envelope Proteins

Although in vitro replication of the hepatitis C virus (HCV) JFH1 clone of genotype 2a (HCVcc) has been developed, a robust cell culture system for the 1a and 1b genotypes, which are the most prevalent viruses in the world and resistant to interferon therapy, has not yet been established. As a surrogate virus system, pseudotype viruses transiently bearing HCV envelope proteins based on the vesicular stomatitis virus (VSV) and retrovirus have been developed. Here, we have developed a replication-competent recombinant VSV with a genome encoding unmodified HCV E1 and E2 proteins in place of the VSV envelope protein (HCVrv) in human cell lines. HCVrv and a pseudotype VSV bearing the unmodified HCV envelope proteins (HCVpv) generated in 293T or Huh7 cells exhibited high infectivity in Huh7 cells. Generation of infectious HCVrv was limited in some cell lines examined. Furthermore, HCVrv but not HCVpv was able to propagate and form foci in Huh7 cells. The infection of Huh7 cells with HCVpv and HCVrv was neutralized by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. The infectivity of HCVrv was inhibited by an endoplasmic reticulum alpha-glucosidase inhibitor, N-(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin. Focus formation of HCVrv in Huh7 cells was impaired by Nn-DNJ treatment. These results indicate that the HCVrv developed in this study can be used to study HCV envelope proteins with respect to not only the biological functions in the entry process but also their maturation step.

Human immunodeficiency virus type 1 efficiently binds to human fetal astrocytes and induces neuroinflammatory responses independent of infection

BackgroundHIV-1 infects human astrocytes in vitro and in vivo but the frequency of infected cells is low and its biological significance is unknown. In studies in vitro, recombinant gp120 alone can induce profound effects on astrocyte biology, suggesting that HIV-1 interaction with astrocytes and its functional consequences extend beyond the limited levels of infection in these cells. Here we determined the relative efficiencies of HIV-1 binding and infection in human fetal astrocytes (HFA), mainly at the single cell level, using HIV-1 tagged with green fluorescence protein (GFP)-Vpr fusion proteins, termed HIV-GFP, to detect virus binding and HIV-1 expressing Rev and NefGFP fusion proteins to detect productive infection.ResultsEssentially all HFA in a population bound HIV-GFP specifically and independently of CCR5 and CXCR4. The dynamics of this binding at 37°C resembled binding of an HIV fusion mutant to CD4-positive cells, indicating that most of HIV-GFP arrested infection of HFA at the stage of virus-cell fusion. Despite extensive binding, only about 1% of HFA were detectably infected by HIV-RevGFP or HIV-NefGFP, but this proportion increased to the majority of HFA when the viruses were pseudotyped with vesicular stomatitis virus envelope glycoprotein G, confirming that HFA impose a restriction upon HIV-1 entry. Exposure of HFA to HIV-1 through its native proteins rapidly induced synthesis of interleukin-6 and interleukin-8 with increased mRNA detected within 3 h and increased protein detected within 18 h of exposure.ConclusionOur results indicate that HIV-1 binding to human astrocytes, although extensive, is not generally followed by virus entry and replication. Astrocytes respond to HIV-1 binding by rapidly increased cytokine production suggesting a role of this virus-brain cell interaction in HIV-1 neuropathogenesis.

Targeted transgene expression in muller glia of normal and diseased retinas using lentiviral vectors.

Müller glia play crucial roles in retinal homeostasis and function. Genetic modification of Müller cells by viral gene delivery would be valuable for studies of their normal physiology and roles in retinal disease states. However, stable and efficient transgene expression in Müller cells after delivery of gene transfer vectors has remained elusive. Transcriptional and transductional targeting approaches were used to engineer recombinant HIV-1-based lentiviral (LV) vectors capable of highly efficient and sustained Müller cell transgene expression in healthy and diseased rodent retinas. Expression cassettes containing glia-specific promoters (CD44, glial fibrillary acidic protein, and vimentin) and an enhanced green fluorescent protein (eGFP) cDNA were cloned into LV backbones, which were packaged into infectious vector particles displaying either the vesicular stomatitis virus (VSV) or Ross River virus (RRV) envelope surface glycoproteins. Vectors were injected by intravitreal and subretinal approaches in wild type Sprague-Dawley (SD) and retinal degenerate S334Ter(+/-) transgenic rats aged 1 to 180 days. In vivo fluorescent fundus imaging and immunofluorescent confocal microscopy were used for comparison of expression efficiency, cell type specificity, and temporal expression characteristics. The choice of viral pseudotype, regulatory promoter, and surgical delivery site each had a measurable effect on the level of eGFP transgene expression in Müller cells. The highest expression levels in SD retinas were attained with subretinal injection of VSV-G pseudotyped LV vectors containing the CD44 promoter. With these vectors, persistent eGFP expression in Müller glia was observed for more than 6 months, covering 25% to 30% of the retinal surface area after a single subretinal injection. Immunohistochemistry (alpha-glutamine synthetase) revealed that approximately 95% of the Müller cells were transduced in the region near the injection site. Delivery of these viral vectors and subsequent Müller cell eGFP expression had no negative impact on visual function, as assessed by electroretinography (ERG). Pseudotyped LV vectors containing glia-specific promoters efficiently transduce and direct sustained transgene expression in retinal Müller glia. Vectors of this type will be useful for experimental treatment of retinal disease, as well as for physiological and developmental investigations of the retina.

Enhanced Lentiviral Transduction of Monocyte-Derived Dendritic Cells in The Presence of Conditioned Medium from Dying Monocytes

Lentiviral vectors (LVs) are attractive vehicles for the transduction of human dendritic cells (DCs) in order to mobilize their endogenous antigen presentation pathways. We analyzed here how to improve the efficiency of LV transduction, which we performed at the initial stages of the differentiation of purified monocytes into dendritic cells (Mo-DCs). Using LVs pseudotyped with the vesicular stomatitis virus envelope G glycoprotein (VSV-G), we found that a conditioned medium derived from dying monocytes (MCM) improved by 2- to 10- fold the proportion of transduced Mo-DCs. This enhanced transduction efficiency requires the presence of MCM during the initial stage of LV transduction and does not affect the phenotype and antigen presentation function of terminally differentiated Mo-DCs. Importantly, we found that MCM derived from a human acute monocytic leukemia cell line, THP-1, was equally effective. The MCM activity was heat stable (56 degrees C) and was present in the soluble fraction after high-speed centrifugation. Altogether our results show that a soluble factor present in dying monocyte cultures can replace advantageously facilitating agents such as Polybrene, to achieve high LV transductions levels. This protocol can be performed with autologous monocytes and is therefore applicable in clinical settings.

Inhibition of HIV-1 replication by vesicular stomatitis virus envelope glycoprotein pseudotyped baculovirus vector-transduced ribozyme in mammalian cells

The baculovirus has recently emerged as a promising vector for in vivo gene therapy. To investigate its potential as a delivery vector for an anti-virus ribozyme targeting HIV-1, we constructed recombinant baculovirus vectors bearing a ribozyme-synthesizing cassette driven by the tRNA i Met promoter with enhanced transduction efficiency by displaying vesicular stomatitis virus glycoprotein (VSV-G) on the viral envelope. Transduction of HeLa CD4 + cells with a recombinant baculovirus delivering the HIV-1 U5 gene-specific ribozyme dramatically suppressed HIV-1 expression in this cell line. The VSV-G pseudotyped baculovirus vector-transduced ribozyme potently inhibited HIV-1 replication compared to a recombinant baculovirus vector-transduced ribozyme lacking VSV-G. The use of a baculovirus vector might be beneficial for application in gene therapy.

Cell Death Induced by the Herpes Simplex Virus-1 Thymidine Kinase Delivered by Human Immunodeficiency Virus-1-Based Virus-like Particles

HIV-1 Nef incorporates into virions at low levels, likely about 10 molecules per viral particle. Here, we describe a Nef mutant (Nef7) apparently showing more than 100-fold higher efficiency of virion incorporation. Interestingly, Nef7 can act as a cargo molecule for protein delivery into the cells, as its virion incorporation appeared conserved even upon C-terminal fusion with proteins of up to 30 kDa. This was demonstrated first by assessing the intracellular fluorescence of cells challenged with lentivirus-based virus-like particles (VLPs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) and incorporating Nef7 fused with the green fluorescent protein. Furthermore, the biologic activity of products delivered by Nef7-based VLPs was demonstrated by tagging Nef7 with the herpes simplex virus-1 thymidine kinase (HSV-1 TK). In fact, we observed that both cell lines and primary human macrophages challenged with (VSV-G) Nef7/TK VLPs died after 5 to 7 days of treatment with ganciclovir (GCV). In sum, our findings support the notion that Nef7-based VLPs can be considered platforms for original systems of protein delivery. In particular, the here- described Nef7/TK VLPs represent a first applicative example opening the way toward new HSV-1 TK/GCV-based cell suicide therapies circumventing cell gene engineering procedures.

Histidylated Lipid-modified Sendai Viral Envelopes Mediate Enhanced Membrane Fusion and Potentiate Targeted Gene Delivery

Recent studies have demonstrated that covalent grafting of a single histidine residue into a twin-chain aliphatic hydrocarbon compound enhances its endosome-disrupting properties and thereby generates an excellent DNA transfection system. Significant increase in gene delivery efficiencies has thus been obtained by using endosome-disrupting multiple histidine functionalities in the molecular architecture of various cationic polymers. To take advantage of this unique feature, we have incorporated L-histidine (N,N-di-n-hexadecylamine) ethylamide (L(H)) in the membrane of hepatocyte-specific Sendai virosomes containing only the fusion protein (F-virosomes (Process for Producing a Targeted Gene (Sarkar, D. P., Ramani, K., Bora, R. S., Kumar, M., and Tyagi, S. K. (November 4, 1997) U. S. Patent 5,683,866))). Such L(H)-modified virosomal envelopes were four times more (p < 0.001) active in terms of fusion with its target cell membrane. On the other hand, the presence of L(H) in reconstituted influenza and vesicular stomatitis virus envelopes failed to enhance spike glycoprotein-induced membrane fusion with host cell membrane. Circular dichroism and limited proteolysis experiments with F-virosomes indicated that the presence of L(H) leads to conformational changes in the F protein. The molecular mechanism associated with the increased membrane fusion induced by L(H) has been addressed in the light of fusion-competent conformational change in F protein. Such enhancement of fusion resulted in a highly efficient gene delivery system specific for liver cells in culture and in whole animals.

Pseudotypes of vesicular stomatitis virus with CD4 formed by clustering of membrane microdomains during budding.

Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

Retinal cell type expression specificity of HIV-1-derived gene transfer vectors upon subretinal injection in the adult rat: influence of pseudotyping and promoter

Gene therapy, and particularly gene restoration, is currently a great hope for non-curable hereditary retinal degeneration. Clinical applications require a gene transfer vector capable of accurately targeting particular cell types in the retina. To develop such a vector, we compared the expression of a reporter gene after subretinal injections of lentiviral constructs of various pseudotypes and with the transgene expression driven by various promoters. Lentiviral vectors expressing the green fluorescent protein (GFP) under the transcriptional control of cytomegalovirus (CMV), mouse phosphoglycerate kinase (PGK), human elongation factor 1-alpha (EF1alpha), or human rhodopsin (RHO) promoters were pseudotyped by vesicular stomatitis virus (VSV) or Mokola virus envelope proteins. These constructs were injected into the subretinal space of adult rdy rats. GFP expression was analyzed in vivo 1 and 4 weeks after injection by fundus examination. The precise location of transgene expression was then determined by immunohistochemistry and in situ hybridization. Constructs of both vesicular stomatitis virus and Mokola pseudotypes with ubiquitous promoters led to a strong expression of GFP in vivo. Histological studies confirmed the production of GFP in the retinal pigment epithelium (RPE) in most cases. However, only the combination of the VSV pseudotype with the RHO promoter led to GFP production in photoreceptors, and did so in a sporadic manner. Mokola-pseudotyped lentiviral vectors are effective for specific gene transfer to the RPE. Neither VSV- nor Mokola-pseudotyped lentiviral vectors are adequate for efficient gene transfer to photoreceptors of adult rats.

Lentiviral Delivery of Suicide Gene and Selection Molecules to Non-Dividng T Cells.

T cell suicide gene therapy is designed to permit the use of donor T cells for their powerful anti-leukaemic and anti-viral effects following haematopoietic stem cell transplantation. In the event of graft versus host disease (GVHD) such cells can be eliminated through the administration of suicide-gene dependent prodrugs. Though the feasibility of the strategy has been demonstrated in clinical trials, there has been a lower incidence of GVHD than anticipated and evidence of impaired anti-viral immunity. Current vectors under clinical investigation are based on retroviral delivery systems and require T cells to be actively dividing at the time of vector exposure to for successful transduction. The process of ex-vivo activation to induce mitogenesis has been shown to impair subsequent functional potential, and even under optimized conditions using combinations of anti-CD3 and anti-CD28 antibodies, T cells become prone to exhaustion. It has been previously reported that lentiviral vectors permit T cell transduction without the need for mitogenic stimulation if used in combination with certain cytokines such as Interleukin-2 (IL-2) or Interleukin-7 (IL-7). Thus we have adapted a self-inactivating lentiviral vector system based on HIV-1 and encoding strong promoter elements derived from the long terminal repeat (LTR) regions of Spleen Focus forming virus (SFFV) for the delivery of suicide genes to T cells. The Woodchuck hepatitis virus post-transcriptional regulatory element and the central polypurine tract element of HIV-1 have been incorporated to enhance transduction efficiency and increase transgene expression. T cells cultured in the presence of either IL-2 or IL-7 alone, or in combination could be transduced at between 20–30% efficiency following a single round of exposure to virus pseudotyped with the vesicular stomatitis virus envelope or the feline endogenous leukaemia virus envelope. By the end of the procedure there were minimal changes in T cell surface phenotype, preservation of niave/memory subsets and retention of virus specific populations as quantified by tetramer staining of Cytomegalovirus (CMV) specific T cells. Functional analysis indicated preservation of proliferative responses to autologous dendritic cells pulsed with CMV antigen. In addition, strong responses in mixed lymphocyte culture indicated intact allo-reactive responses. Cells transduced to express a fusion construct encoding a variant herpes simplex thymidine kinase fused to the truncated CD34 selection marker could be enriched to >95% purity by a single round of anti-CD34 magnetic bead selection. The functional integrity of the suicide gene was confirmed by elimination of enriched cells following exposure to the prodrugs Gancilcovir and Aciclovir. In conclusion, non-dividing T cells transduced with suicide gene/selection transgenes using lentiviral constructs, retain phenotypic characteristics and functional responsiveness.

Phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus.

The envelope protein from vesicular stomatitis virus (VSV) has become an important tool for gene transfer and gene therapy. It is widely used mainly because of its ability to mediate virus entry into all cell types tested to date. Consistent with the broad tropism of the virus, the receptor for VSV is thought to be a ubiquitous membrane lipid, phosphatidylserine (PS). However, the evidence for this hypothesis is indirect and incomplete. Here, we have examined the potential interaction of VSV and PS at the plasma membrane in more detail. Measurements of cell surface levels of PS show a wide range across cell types from different organisms. We demonstrate that there is no correlation between the cell surface PS levels and VSV infection or binding. We also demonstrate that an excess of annexin V, which binds specifically and tightly to PS, does not inhibit infection or binding by VSV. While the addition of PS to cells does allow increased virus entry, we show that this effect is not specific to the VSV envelope. We conclude that PS is not the cell surface receptor for VSV, although it may be involved in a postbinding step of virus entry.

413. Development and Characterization of Lentiviral Vectors for Hemophilia Gene Therapy

Monogenic inherited diseases can potentially be cured by gene therapy, which has already proven to be successful in a number of preclinical and clinical studies. A major target for gene therapy is the liver, since many proteins are synthesized in hepatocytes and secreted proteins can readily be delivered into the bloodstream. Lentiviral vectors are attractive vectors for hepatic gene delivery since they stably integrate into the target cell genome and thus can potentially give rise to life-long expression of the therapeutic protein (VandenDriessche et al., Blood 100:813–822, 2002). Lentiviral vectors do not express any potentially immunogenic viral genes and induce only minimal and transient liver toxicity and no pro-inflammatory cytokines (IL-6). However, intravenous delivery of a lentiviral vector pseudotyped with the commonly used vesicular stomatitis virus envelope glycoprotein (VSV-G), resulted in efficient transduction of antigen presenting cells (APCs), primarily in liver and spleen, and transgene expression in APCs when a ubiquitously expressed cytomegalovirus (CMV) immediate early promoter was used. This increased the risk of developing a humoral immune response directed against the transgene product. Obviously, to obtain long-term expression, such an immune reaction has to be avoided. Different strategies are being pursued to direct expression of the transgene exclusively in hepatocytes and to prevent gene expression in APCs. We have therefore generated lentiviral vectors containing different hepatocyte-specific expression cassettes, which were titered by an optimized Q-PCR approach. Expression of coagulation factor IX using a human alpha1-antitrypsin promoter in conjunction with an ApoE enhancer repeat and a matrix attachment region (MAR) resulted in therapeutic FIX expression levels in vivo that were as high as when a CMV promoter were used. An alternative strategy to redirect expression to hepatocytes is to increase hepatocyte-specific transduction, while reducing vector uptake by APCs. This could potentially be achieved by pseudotyping the lentiviral vectors with alternative envelope proteins. Another potential advantage of reducing vector uptake by APCs, is that it may reduce the total vector dose necessary to reach a certain expression level. In a separate study with high-capacity adenoviral (HC-Ad) vectors, 10-fold higher expression levels were obtained after temporary depletion of macrophages (Chuah et al., Blood 101:1734–1743, 2003). However, the effect of transient APC depletion on transgene expression levels was not as dramatic following lentiviral gene transfer, suggesting that lentiviral vectors result in reduced APC transduction compared to when HC-Ad vectors are used. To further improve the safety profile of lentiviral vectors, purification of vector preparations is being optimized by ion-exchange chromatography. In summary, lentiviral vectors for hepatic gene delivery are being improved further at multiple levels by (i) designing more potent and hepatocyte-specific expression cassettes; (ii) developing alternative pseudotypes and (iii) implementing improved vector purification methods.

In vivo gene marking of rhesus macaque long-term repopulating hematopoietic cells using a VSV-G pseudotyped versus amphotropic oncoretroviral vector.

Gene transfer efficiency into primitive hematopoietic cells may be limited by their expression of surface receptors allowing vector entry. Vectors pseudotyped with the vesicular stomatitis virus (VSV-G) envelope do not need receptors to enter cells, and therefore may provide superior transduction efficiency. Using a competitive repopulation model in the rhesus macaque, we examined in vivo gene marking levels of blood cells transduced with two vectors: (i) a VSV-G pseudotyped retrovirus and (ii) a conventional amphotropic retrovirus. The VSV-G vector, containing the human glucose-6-phosphate dehydrogenase (G6PD) gene, was constructed for treatment of severe hemolytic anemia caused by G6PD deficiency. Three myeloablated animals were transplanted with peripheral blood CD34+ cells, half of which were transduced with the VSV-G vector and the other half with the amphotropic vector. In all animals post-transplantation, levels of in vivo marking in circulating granulocytes and mononuclear cells were similar: 1% or less with both vectors. In one animal, the human G6PD enzyme transferred by the VSV-G vector was expressed in erythrocytes, early after transplantation, at a level of 45% of the endogenous rhesus G6PD protein. In a clinically relevant animal model, we found similar in vivo marking with a VSV-G pseudotyped and a standard amphotropic oncoretroviral vector. Amphotropic receptor expression may not be a limiting factor in transduction efficiency, but VSV-G pseudotypes possess other practical advantages that may make them advantageous for clinical use.

A novel method for analysis of membrane microdomains: vesicular stomatitis virus glycoprotein microdomains change in size during infection, and those outside of budding sites resemble sites of virus budding

Membrane proteins, including viral envelope glycoproteins, may be organized into areas of locally high concentration, commonly referred to as membrane microdomains. Some viruses bud from detergent-resistant microdomains referred to as lipid rafts. However, vesicular stomatitis virus (VSV) serves as a prototype for viruses that bud from areas of plasma membrane that are not detergent resistant. We developed a new analytical method for immunoelectron microscopy data to determine whether the VSV envelope glycoprotein (G protein) is organized into plasma membrane microdomains. This method was used to quantify the distribution of the G protein in microdomains in areas of plasma membrane that did not contain budding sites. These microdomains were compared to budding virus envelopes to address the question of whether G protein-containing microdomains were formed only at the sites of budding. At early times postinfection, most of the G protein was organized into membrane microdomains outside of virus budding sites that were approximately 100–150 nm, with smaller amounts distributed into larger microdomains. In contrast to early times postinfection, the increased level of G protein in the host plasma membrane at later times postinfection led to distribution of G protein among membrane microdomains of a wider variety of sizes, rather than a higher G protein concentration in the 100- to 150-nm microdomains. VSV budding occurred in G protein-containing microdomains with a range of sizes, some of which were smaller than the virus envelope. These microdomains extended in size to a maximum of 300–400 nm from the tip of the budding virion. The data support a model for virus assembly in which G protein organizes into membrane microdomains that resemble virus envelopes prior to formation of budding sites, and these microdomains serve as the sites of assembly of internal virion components.

Efficiency of onco-retroviral and lentiviral gene transfer into primary mouse and human B-lymphocytes is pseudotype dependent.

B lymphocytes are attractive targets for gene therapy of genetic diseases associated with B-cell dysfunction and for immunotherapy. Transduction of B lymphocytes was evaluated using green fluorescent protein (GFP)-encoding onco-retroviral and HIV-derived lentiviral vectors which were pseudotyped with ecotropic, amphotropic or vesicular stomatitis virus (VSV-G) envelopes. Transduction of mouse B lymphocytes activated with lipopolysaccharides (LPS) or by cross-linking CD40 in conjunction with interleukin-4 (IL-4) was significantly more efficient (p < 0.003) with ecotropic (11%) than with VSV-G pseudotyped onco-retroviral vectors (1%). Using high-titer cell-free ecotropic viral supernatant or by coculture with ecotropic onco-retroviral vector-producing cells, transduction efficiency increased significantly (p < 0.001) to approximately 50%, whereas transduction efficiency by coculture with VSV-G pseudotyped vector-producing cells remained low (< 2%). Similarly, transduction of mouse B lymphocytes was significantly more efficient (twofold, p < 0.01) with the ecotropic (7%) than with the VSV-G pseudotyped lentiviral vectors although gene transfer efficiency remained low because of dose-limiting toxicity of the concentrated vector preparations on the LPS-activated murine B cells. Consistent with murine B-cell transduction, human B cells activated with CD40L and IL-4 were also found to be relatively refractory to VSV-G pseudotyped onco-retroviral vectors (< 1%). However, higher transduction efficiencies could be achieved in activated primary human B lymphocytes using VSV-G pseudotyped lentiviral vectors instead (5%-6%). Contrary to the significant increase in mouse B-cell transduction efficiency with ecotropic vectors, the use of amphotropic onco-retroviral or lentiviral vectors did not increase transduction efficiency in primary human B cells. The present study shows that the transduction efficiency of onco-retroviral and lentiviral vectors in human and mouse B lymphocytes is pseudotype-dependent and challenges the widely held assumption that VSV-G pseudotyping facilitates gene transfer into all cell types.

Role for Human Immunodeficiency Virus Type 1 Membrane Cholesterol in Viral Internalization

The membrane of human immunodeficiency virus type 1 (HIV-1) virions contains high levels of cholesterol and sphingomyelin, an enrichment that is explained by the preferential budding of the virus through raft microdomains of the plasma membrane. Upon depletion of cholesterol from HIV-1 virions with methyl-beta-cyclodextrin, infectivity was almost completely abolished. In contrast, this treatment had only a mild effect on the infectiousness of particles pseudotyped with the G envelope of vesicular stomatitis virus. The cholesterol-chelating compound nystatin had a similar effect. Cholesterol-depleted HIV-1 virions exhibited wild-type patterns of viral proteins and contained normal levels of cyclophilin A and glycosylphosphatidylinositol-anchored proteins. Nevertheless, and although they could still bind target cells, these virions were markedly defective for internalization. These results indicate that the cholesterol present in the HIV-1 membrane plays a prominent role in the fusion process that is key to viral entry and suggest that drugs capable of disturbing the lipid composition of virions could serve as a basis for the development of microbicides.

Disruption of Golgi Morphology and Trafficking in Cells Expressing Mutant Prenylated Rab Acceptor-1

Prenylated Rab acceptor (PRA1) is a protein that binds Rab GTPases and the v-SNARE VAMP2. The protein is localized to the Golgi complex and post-Golgi vesicles. To determine its functional role, we generated a number of point mutations and divided them into three classes based on cellular localization. Class A mutants were retained in the endoplasmic reticulum (ER) and exerted an inhibitory effect on transport of vesicular stomatitis virus envelope glycoprotein (VSVG) from the ER to Golgi as well as to the plasma membrane. Class B mutants exhibited a highly condensed Golgi complex and inhibited exit of anterograde cargo from this organelle. Class C mutants exhibited an intermediate phenotype with Golgi and ER localization along with extensive tubular structures emanating from the Golgi complex. There was a direct correlation between the cellular phenotype and binding to Rab and VAMP2. Class A and C mutants showed a significant decrease in Rab and VAMP2 binding, whereas an increase in binding was observed in the class B mutants. Thus, PRA1 is required for vesicle formation from the Golgi complex and might be involved in recruitment of Rab effectors and SNARE proteins during cargo sequestration.

Human immunodeficiency virus type 1 uses lipid raft-colocalized CD4 and chemokine receptors for productive entry into CD4(+) T cells.

In this report, we describe a crucial role of lipid raft-colocalized receptors in the entry of human immunodeficiency virus type 1 (HIV-1) into CD4(+) T cells. We show that biochemically isolated detergent-resistant fractions have characteristics of lipid rafts. Lipid raft integrity was required for productive HIV-1 entry as determined by (i) semiquantitative PCR analysis and (ii) single-cycle infectivity assay using HIV-1 expressing the luciferase reporter gene and pseudotyped with HIV-1 HXB2 envelope or vesicular stomatitis virus envelope glycoprotein (VSV-G). Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) relocalized raft-resident markers to a nonraft environment but did not significantly change the surface expression of HIV-1 receptors. MbetaCD treatment inhibited productive infection of HIV-1 by 95% as determined by luciferase activity in cells infected with HXB2 envelope-pseudotyped virus. In contrast, infection with VSV-G-pseudotyped virus, which enters the cells through an endocytic pathway, was not suppressed. Biochemical fractionation and confocal imaging of HIV-1 receptor distribution in live cells demonstrated that CD4, CCR5, and CXCR4 colocalized with raft-resident markers, ganglioside GM1, and glycosylphosphatidylinositol-anchored CD48. While confocal microscopy analysis revealed that HIV-1 receptors localized most likely to the same lipid microdomains, sucrose gradient analysis of the receptor localization showed that, in contrast to CD4 and CCR5, CXCR4 was associated preferentially with the nonraft membrane fraction. The binding of HIV-1 envelope gp120 to lipid rafts in the presence, but not in the absence, of cholesterol strongly supports our hypothesis that raft-colocalized receptors are directly involved in virus entry. Dramatic changes in lipid raft and HIV-1 receptor redistribution were observed upon binding of HIV-1 NL4-3 to PM1 T cells. Colocalization of CCR5 with GM1 and gp120 upon engagement of CD4 and CXCR4 by HIV-1 further supports our observation that HIV-1 receptors localize to the same lipid rafts in PM1 T cells.

Simian foamy virus vectors. Preparation and use.

The life cycle of retroviruses involves stable integration of viral genetic material into the host genome; expression of viral genes is, in part, regulated by host cell factors (). These features make retroviruses a widely used efficient means for introducing foreign DNA into the cell genome. Most retroviral vector systems used in clinical gene transfer are based on murine leukemia virus (MuLV) (,). MuLV replication is cell cycle dependent, and these vectors are inadequate to transduce nondividing and/or terminally differentiated cells stably, which severely restricts their potential utility for clinical gene transfer (, , ). In contrast, vectors based on the human immunodeficiency virus (HIV) can deliver genes into nondividing cells as efficiently as into proliferating cells (,). HIV vectors, then, serve as vehicles to deliver a gene of interest into clinically important terminally differentiated, quiescent, and nondividing cells. The use of vectors derived from a pathogenic virus for human gene therapy, however, remains a major safety concern and health risk issue. Recently, it was shown that animal lentivirus vectors pseudotyped with vesicular stomatitis virus envelope glycoprotein G (VSV-G) can transduce human cells, overcoming the restricted host tropism (). These vectors deliver genes into nondividing cells as efficiently as HIV. However, the comparative safety and efficacy of vectors based on nonprimate lentiviruses in humans remain to be determined.

Hepatocyte-Specific Gene Expression by Baculovirus Pseudotyped with Vesicular Stomatitis Virus Envelope Glycoprotein

We have developed the recombinant baculovirus pseudotyped with vesicular stomatitis virus (VSV) G protein. The VSV-G gene was under the control of the polyhedrin promoter so that it was expressed at high levels in infected insect cells but not in mammalian cells. The presence of VSV-G protein in purified baculovirus preparations was confirmed by Western analysis. This recombinant baculovirus also carried human AFP (α-fetoprotein) promoter for hepatocyte-specific gene expression. After an in vitro infection by a recombinant baculovirus carrying the luciferase gene under the control of human AFP promoter/enhancer (BacG-AFP-Luc+), the luciferase gene was expressed in AFP-producing Huh7, Hep3B, and HepG2 cell lines, but not in AFP-nonproducing cell lines. BacG-AFP-Luc+ transduced with human hepatoma cells in vitro at an efficiency about fivefold greater than the recombinant baculovirus lacking VSV-G (the virus Bac-AFP-Luc+). The utilization of the AFP promoter/enhancer in a baculovirus vector could provide benefits in gene therapy applications.