Retrovirus-associated heparan sulfate mediates immobilization and gene transfer on recombinant fibronectin

Recombinant retroviruses have been shown to bind to fibronectin (FN) and increase the efficiency of gene transfer to a variety of cell types. Despite recent work to optimize gene transfer on recombinant FN, the mechanism of retrovirus binding to FN and the interactions of target cells with the bound virus remain elusive. We investigated the roles of virus surface glycoprotein (gp70), cell-conditioned medium, and proteoglycans in mediating retrovirus binding to FN. We also examined the role of Polybrene (PB) in these interactions. We found that gp70 is not involved in retrovirus binding to FN. Immobilization of the virus, however, does not overcome its receptor requirement, and gp70 is still needed for successful gene transfer. Our results clearly show that retrovirus binds FN through virus-associated heparan sulfate (HS) and that binding is necessary for transduction without PB. Two distinct modes of gene transfer occur depending on PB: (i) in the presence of PB, retrovirus interacts directly with the target cells; and (ii) in the absence of PB, retrovirus binds to FN and target cells interact with the immobilized virus. PB may promote the former mode by interacting with the virus HS and reducing the negative charge of the viral particles. Interestingly, the latter mode is more efficient, leading to significantly enhanced gene transfer. A better understanding of these interactions may provide insight into virus-cell interactions and lead to a more rational design of transduction protocols.     

Differential inhibition of retrovirus transduction by proteoglycans and free glycosaminoglycans.

We have previously shown that the efficiency of retrovirus-mediated gene transfer is limited in part due to the presence of chondroitin sulfate proteoglycans in virus stocks. In this study, we have used a model recombinant retrovirus encoding the Escherichia coli lacZ gene, bovine aorta chondroitin sulfate proteoglycan (CSPG), various free glycosaminoglycan chains (GAGs), and quantitative assays for retrovirus transduction to explore the mechanism by which proteoglycans and glycosaminoglycans inhibit retroviruses. We found that CSPG and GAGs block an early step in virus-cell interactions but do not act by inactivating viruses or by reducing the growth rate of the target cells. CSPG and most of the GAGs tested (chondroitin sulfate A, chondroitin sulfate B, heparin, heparan sulfate, and hyaluronic acid) inhibited transduction, but with widely varying degrees of activity. The chemical structure of GAGs was found to be an important determinant of their inhibitory activity, which suggests that GAGs do not inhibit transduction simply because they are highly negatively charged polymers. When GAGs were used in combination with a cationic polymer (Polybrene), however, their inhibitory activity was neutralized, and interestingly, at optimal doses of GAG and Polybrene, transduction efficiency was actually enhanced by as much as 72%. In contrast, the inhibitory activity of CSPG, due to the influence of its core protein, was not substantially reduced by Polybrene. The importance of these findings to our understanding of retrovirus-cell interactions and to the development of more efficient retrovirus gene transfer protocols is discussed.     

High efficiencies of gene transfer with immobilized recombinant retrovirus: kinetics and optimization

We used a combination of mathematical modeling and experiments to investigate the rate-limiting steps of retroviral transduction on surface-bound fibronectin (FN) and identify the conditions that maximize the efficiency of gene transfer. Our results show that fibronectin-assisted gene transfer (FAGT) is a strong function of the time and temperature of virus incubation in FN-coated plates. Gene transfer increases sharply at short times, reaches a maximum at intermediate times, and eventually declines as a result of loss of retroviral activity. The maximum transduction efficiency and the time at which this is attained increase with decreasing temperature of virus incubation. Depending on the temperature and the type of target cells, the initial rate of gene transfer increases by 3- to 10-fold and the maximum transduction efficiency increases by 2- to 4-fold as compared to traditional transduction (TT). Interestingly, Polybrene (PB) inhibits FAGT in a dose-dependent manner by inhibiting binding of retrovirus to FN. In contrast to traditional transduction, FAGT yields higher than 10-fold transduction efficiencies with concentrated retrovirus stocks. Gene transfer is directly proportional to the concentration of the virus-containing medium with no sign of saturation for the range of concentrations tested. These results suggest that immobilization of recombinant retrovirus can be rationally optimized to yield high efficiency of gene transfer to primary cells and improve the prospect of gene therapy for the treatment of human disease.     

New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors.

We prepared retrovirus packaging cell lines containing gag-pol genes from spleen necrosis virus (expressed from a cytomegalovirus promoter and the simian virus 40 (SV40) polyadenylation sequences) and, on a separate vector, either the env gene from spleen necrosis virus (expressed from the Rous sarcoma virus promoter and the SV40 polyadenylation sequences) or the env gene from amphotropic murine leukemia virus (expressed from a cytomegalovirus promoter and the SV40 polyadenylation sequences). The nucleotide sequences in these packaging cell lines have almost no homology to the retrovirus vectors we used. Retrovirus vectors were produced from these new helper cell lines without any genetic interactions between the vectors and sequences in the helper cells and without transfer of the packaging sequences.     

A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection

Murine type C ecotropic retrovirus infection is initiated by virus envelope binding to a membrane receptor expressed on mouse cells. We have identified a cDNA clone that may encode for this receptor through a strategy combining gene transfer of mouse NIH 3T3 DNA into nonpermissive human EJ cells, selection of EJ clones that have acquired susceptibility to infection by retrovirus vectors containing drug resistance genes, and identification of the putative receptor cDNA clone through linkage to a mouse repetitive DNA sequence. Human EJ cells that express the cDNA acquire a million-fold increase in MuLV infectivity. The predicted 622 amino acid sequence of the putative receptor protein is extremely hydrophobic; 14 potential membrane-spanning domains have been identified. A computer-based search of sequence data banks did not identify a protein with significant similarity to the putative receptor. We conclude that a novel membrane protein determines susceptibility to ecotropic MuLV infection by binding and/or fusion with the virus envelope.     

Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes

Cationic polymers, such as polybrene and protamine sulfate, are typically used to increase the efficiency of retrovirus-mediated gene transfer, however, the mechanism of their enhancement of transduction has remained unclear. As retrovirus transduction is fundamentally limited by the slow diffusion of virus to the target cell surface, we investigated the ability of polybrene to modulate this initial transport step. We compared the ability of both envelope (gp70) and capsid (p30) protein based assays to quantitate virus adsorption and found that p30 based assays were more reliable due to their ability to distinguish virus binding from free gp70 binding. Using the p30 based assay, we established that polybrene concentrations, which yielded 10-fold increases in transduction also, yielded a significant increase in virus adsorption rates on murine fibroblasts. Surprisingly, this enhancement, and adsorption in general, were receptor and envelope independent, as adsorption occurred equivalently on receptor positive and negative Chinese hamster ovary cells, as well as with envelope positive and negative virus particles. These findings suggest that the currently accepted physical model for early steps in retrovirus transduction may need to be reformulated to accommodate an initial adsorption step whose driving force does not include the retrovirus concentration, and the reclassification of currently designated 'receptor' molecules as fusion triggers. The implication of these findings with respect to the development of targeted retrovirus-mediated gene therapy protocols is discussed.     

Envelope gene of the human endogenous retrovirus HERV-W encodes a functional retrovirus envelope

A member of the human endogenous retrovirus (HERV) family termed HERV-W encodes a highly fusogenic membrane glycoprotein that appears to be expressed specifically in the placenta. It is unclear whether the glycoproteins of the HERVs can serve as functional retrovirus envelope proteins to confer infectivity on retrovirus particles. We found that the HERV-W envelope glycoprotein can form pseudotypes with human immunodeficiency virus type 1 virions and confers tropism for CD4-negative cells. Thus, the HERV-W env gene represents the first HERV env gene demonstrated to encode the functional properties of a retrovirus envelope glycoprotein.     

Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection.

Previous reports have shown that retrovirus infection is inhibited in nonreplicating (stationary-phase [hereafter called stationary]) cells. Infection of stationary cells was shown to occur when the cells were allowed to replicate at times up to a week after infection, suggesting that an unintegrated retrovirus could persist in a form that was competent to integrate after release of the block to replication. However, those studies were complicated by the use of replication-competent virus, which can spread in the infected cells. We have used a replication-defective retrovirus vector to compare the efficiency of gene transfer in stationary and replicating rat embryo fibroblasts. In agreement with previous results, gene transfer was inhibited 100-fold in stationary versus replicating cells. In contrast to previously reported results, the block to infection could not be relieved by stimulating stationary cells to divide at times from 6 h to 10 days after infection. Thus, for successful retroviral infection, the infected cells must be replicating at the time of infection. These results have important implications for the use of retroviral vectors for gene transfer.     

A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography

Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000.     

Rapid concentration and purification of retrovirus by flocculation with Polybrene

We have previously shown that the combined addition of Polybrene (PB) and chondroitin sulfate C (CSC) to retrovirus stocks leads to the formation of retrovirus-polymer complexes (i.e., flocs) that rapidly sediment onto cells, increases the efficiency of gene transfer, and can be used to rapidly concentrate and purify retrovirus stocks. The viruses remain associated with the polyelectrolyte complexes, however, which may complicate their use in downstream applications. In this study we determined if retrovirus could be flocculated using only one polymer (PB). We found that when retrovirus stocks were incubated with 320 microg/ml of PB, more than 70% of the viruses, and fewer than 0.3% of all other proteins, were pelleted by low-speed centrifugation. In contrast to retrovirus complexes formed with two polymers, retrovirus flocculated with PB disaggregated when they were resuspended in fresh medium. We conclude that flocculation of retroviruses with a single cationic polymer (PB) is a useful method for rapidly concentrating and purifying retroviruses, and may prove particularly useful when it is desirable to generate purified virus that is not part of a polymer complex.     

Survival of early preimplantation porcine embryos after co-culture with cells producing an avian retrovirus

To determine the relative survival of porcine embryos after co-culture with cells producing an avian retrovirus, four-cell stage embryos were obtained from sows following synchronization with altrenogest and superovulation with gonadotropins. These embryos were randomly assigned to the following treatments: no manipulation (zona-intact); zona removed with acidified Tyrode's solution (zona-free); and zona removed followed by co-culture with D-17 canine cells producing an avian retrovirus vector derived from spleen necrosis virus (zona-free + co-culture). The survival rates of four-cell stage embryos to morulae or early blastocysts during a 48-h culture period were 93.3, 80.0 and 57.7% in zona-intact, zona-free and zona-free + co-culture groups, respectively. Following embryo transfer, the development of embryos to fetuses at six weeks of gestation was 37.5, 30.0 and 11.7% in zona-intact, zona-free and zona-free + co-culture groups. These results indicate that early preimplantation porcine embryos can develop to apparently normal fetuses following co-culture with cells producing a retrovirus, and the feasibility of this method for retrovirus-mediated gene transfer in pigs was demonstrated.     

Gene transfer in swine embryos by injection of cells infected with retrovirus vectors

Key components for gene transfer to swine embryos using an avian retrovirus are described. A replication-defective reticuloendotheliosis (REV) viral vector can infect and be expressed in pig embryo fibroblasts (PEF). Infection with a replication-competent vector (REV-A) indicates a presumptive block to viral replication in PEF. Swine embryos obtained at the morula stage can be cultured in vitro to the blastocyst stage, injected with retrovirus helper cells or quail embryo fibroblasts producing REV, and transferred to recipient swine with survival to at least 6 wk of gestation.     

Exploration of critical parameters for transient retrovirus production

The pCL system was developed to aid in the production of retrovirus that encodes cytotoxic or cytostatic cDNA's. A principal feature of this system is the transient production of virus after co-transfection of the viral and packaging vectors in the 293T cell line. This approach obviates the need for selection of the producer cells, thus minimizing potential affects of the encoded genes. However, the transient nature of this system also creates a number of experimental variables. In this study we have examined and optimized elements related to the production of the pCL retrovirus. For example, co-transfection of the packaging sequence along with the viral vector has been optimized in terms of both the total amount of DNA transfected and the relative proportion of each plasmid. We have also tested the affect of increased synthesis of viral proteins in the producer cells and the kinetics of virus accumulation in the supernatant. These findings may be of interest to those who use pCL or any transient packaging system in their gene transfer studies. In addition, these studies may aid in the validation and development of transient retrovirus production systems for clinical applications.     

Complexation with chondroitin sulfate C and Polybrene rapidly purifies retrovirus from inhibitors of transduction and substantially enhances gene transfer

Using amphotropic retrovirus stocks produced by TELCeB6-A cells that encode the Escherichia coli lacZ gene, we found that complexation with chondroitin sulfate C (CSC) and Polybrene (PB) is an effective means to purify retrovirus. Virus stocks contained high levels of inhibitory activity that blocked amphotropic, but not ecotropic, retrovirus transduction. When virus stocks were brought to 80 microg/mL each of CSC and PB, complexes of CSC and PB formed. These complexes incorporated more than 70% of the virus particles but less than 0.4% of all other proteins and no detectable inhibitory activity. Purified virus transduced NIH 3T3 murine fibroblasts 21 to 186-fold more efficiently than virus that was not purified. In addition, virus purification significantly altered the dose response of transduction. When virus that had not been purified was used to transduce cells, the relationship between transduction and virus concentration was highly non-linear. In contrast, when purified virus was used, transduction increased monotonically and was linearly proportional to virus concentration, except when high doses of virus were used. Interestingly, when high doses of virus were used gene transfer reached a maximum plateau level, most likely because particle-associated amphotropic envelope proteins had saturated the cellular receptors for the virus. Our findings illustrate that retrovirus purification increases the maximum number of genes that can be transferred, reduces the amount of virus required to achieve a given level of gene transfer, and reduces uncertainties about the relationship between the amount of virus used and the number of genes transferred.     

Kinetics of retrovirus production and decay.

There has been only limited success in using recombinant retroviruses to transfer genes for the purposes of human gene therapy, in part because the average number of genes delivered to the target cells (transduction efficiency) is often too low to achieve the desired therapeutic effect [Miller, AD. 1990. Blood 76:271-278; Mulligan RC. 1993. Science 260:926-932; Orkin SH, Motulsky AG. 1995. Report and recommendations of the panel to assess the NIH investment in research on gene therapy. Bethesda, MD: National Institutes of Health.]. One strategy to improve transduction efficiency is to focus on understanding and improving the processes used to produce recombinant retroviruses. In this report, we characterized the dynamics of retrovirus production and decay in batch cultures of virus producer cells using a simple mathematical model, a recombinant retrovirus encoding the Escherichia coli lacZ gene, and quantitative assays for virus activity and number. We found that the rate at which recombinant retroviruses spontaneously lose their activity (decay) is a strong function of temperature, decreasing roughly 2-fold for every 5 degrees C reduction in temperature, whereas the rate at which retroviruses are produced is only weakly affected by temperature, decreasing about 10% for every 5 degrees C reduction in temperature. In addition, we developed a simple mathematical model of virus production and decay that predicted that the virus titer in batch cultures of virus producer cells would reach a maximum steady-state at a rate that is inversely proportional to the virus decay rate and to a level that is proportional to the ratio of the virus production rate to the virus decay rate. Consistent with the model, we observed that the steady-state levels of virus titer increased more than 3-fold when the cell culture temperature was reduced from 37 to 28 degrees C. Despite their higher titers, virus stocks produced at 28 degrees C, when used in undiluted form so as to mimic human gene transfer protocols, did not transduce substantially more cells than virus stocks produced at 37 degrees C. The implications of our findings on the production of retroviruses for use in human gene therapy protocols are discussed.     

Acylation of gelatin-agarose and the enhancement by heparin of fibronectin binding

The enhancement of the binding of plasma fibronectin to collagen or gelatin by heparin was previously thought to be due primarily to interaction of heparin with fibronectin. We observed, however, that the elution of purified human plasma fibronectin from heparin-treated gelatin-agarose required the same high urea concentrations regardless of whether heparin treatment preceded or followed fibronectin adsorption. Acylation of gelatin-agarose with acetic anhydride or succinic anhydride had little effect upon fibronectin binding, yet the heparin enhancement of fibronectin binding was abolished by either acylation reaction. When heparin binding to gelatin-agarose was investigated with dansyl heparin, gelatin-agarose bound substantial quantities of labeled heparin which could be readily dissociated from the matrix with 2 M NaCl. Acetylated gelatin-agarose did not bind detectable amounts of dansyl heparin. We interpret these results as evidence that the stronger binding of fibronectin to gelatin-agarose in the presence of heparin is due to heparin itself binding to gelatin, thus allowing fibronectin to bind simultaneously to both immobilized ligands through appropriate domains of the glycoprotein.     

GGA and Arf proteins modulate retrovirus assembly and release

The Gag protein is the major structural determinant of retrovirus assembly. Although a number of cellular factors have been reported to facilitate retrovirus release, little is known about the cellular machinery that directs Gag to the site of virus assembly. Here, we report roles for the Golgi-localized gamma-ear containing Arf-binding (GGA) and ADP ribosylation factor (Arf) proteins in retrovirus particle assembly and release. Whereas siRNA-mediated depletion of GGA2 and GGA3 led to a significant increase in particle release in a late domain-dependent manner, GGA overexpression severely reduced retrovirus particle production by impairing Gag trafficking to the membrane. GGA overexpression inhibited retroviral assembly and release by disrupting Arf protein activity. Furthermore, disruption of endogenous Arf activity inhibited particle production by decreasing Gag-membrane binding. These findings identify the GGA proteins as modulators of HIV-1 release and the Arf proteins as critical cellular cofactors in retroviral Gag trafficking to the plasma membrane.