Oncogenes in Ras signalling pathway dictate host-cell permissiveness to herpes simplex virus 1.

The importance of herpes simplex viruses (HSV) as human pathogens and the emerging prospect of using mutant derivatives of HSV-1 as potential anti-cancer therapeutics have necessitated a thorough investigation into the molecular basis of host-cell permissiveness to HSV. Here we show that NIH-3T3 cells transformed with the oncogenes v-erbB, activated sos or activated ras become significantly more permissive to HSV-1. Inhibitors of the Ras signalling pathway, such as farnesyl transferase inhibitor 1 and PD98059, effectively suppressed HSV-1 infection of ras-transformed cells. Enhanced permissiveness of the transformed cells was linked to the inhibition of virus-induced activation (phosphorylation) of the double-stranded RNA-activated protein kinase (PKR), thereby allowing viral transcripts to be translated in these cells. An HSV-1-derived oncolytic mutant, R3616, was also found to infect preferentially both transformed cells and PKR-/- (but not PKR+/+) mouse embryo fibroblasts. These observations suggest that HSV-1 specifically targets cells with an activated Ras signalling pathway, and have important ramifications in the use of engineered HSV in cancer therapy, the development of strategies against HSV infections, and the controversial role of HSV in human cancers.

Dimerization of ERp29, a PDI-like protein, is essential for its diverse functions.

Protein disulfide isomerase (PDI)-like proteins act as oxido-reductases and chaperones in the endoplasmic reticulum (ER). How oligomerization of the PDI-like proteins control these activities is unknown. Here we show that dimerization of ERp29, a PDI-like protein, regulates its protein unfolding and escort activities. We have demonstrated previously that ERp29 induces the local unfolding of polyomavirus in the ER, a step required for viral infection. We now find that, in contrast to wild-type ERp29, a mutant ERp29 (D42A) that dimerizes inefficiently is unable to unfold polyomavirus or stimulate infection. A compensatory mutation that partially restores dimerization to the mutant ERp29 (G37D/D42A) rescues ERp29 activity. These results indicate that dimerization of ERp29 is crucial for its protein unfolding function. ERp29 was also suggested to act as an escort factor by binding to the secretory protein thyroglobulin (Tg) in the ER, thereby facilitating its secretion. We show that this escort function likewise depends on ERp29 dimerization. Thus our data demonstrate that dimerization of a PDI-like protein acts to regulate its diverse ER activities.

MAVS polymers smaller than 80 nm induce mitochondrial membrane remodeling and interferon signaling.

Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection and is sensed primarily by RIG-I-like receptors (RLRs). Oligomerization of RLRs following binding to cytosolic dsRNA activates and nucleates self-assembly of the mitochondrial antiviral-signaling protein (MAVS). In the current signaling model, the caspase recruitment domains of MAVS form helical fibrils that self-propagate like prions to promote signaling complex assembly. However, there is no conclusive evidence that MAVS forms fibrils in cells or with the transmembrane anchor present. We show here with super-resolution light microscopy that MAVS activation by dsRNA induces mitochondrial membrane remodeling. Quantitative image analysis at imaging resolutions as high as 32 nm shows that in the cellular context, MAVS signaling complexes and the fibrils within them are smaller than 80 nm. The transmembrane domain of MAVS is required for its membrane remodeling, interferon signaling, and proapoptotic activities. We conclude that membrane tethering of MAVS restrains its polymerization and contributes to mitochondrial remodeling and apoptosis upon dsRNA sensing.

Nerve growth factor somatic mosaicism produced by herpes virus-directed expression of cre recombinase.

Focal molecular genetic alteration of the intact mammalian brain will be required to elucidate gene product function in cells comprising synaptic networks. To this end, a somatic mosaic approach has been developed for the mouse whereby a dormant germline transgene is activated by the somatic delivery and expression of cre recombinase. Transgenic mice harboring a recombinational substrate, the germline-transmitted nerve growth factor excision activation transgene (NGF-XAT) were generated. Somatic delivery of virus vectors expressing cre recombinase into the brain of NGF-XAT mice resulted in regional recombination and activation of the transgene as demonstrated at the DNA level by PCR and at the protein level by both immunocytochemistry and ELISA. This approach has been used to evaluate a behavioral correlate of unilateral NGF mosaicism within the dorsal hippocampal formation. NGF-XAT mice activated by expression of cre recombinase manifest increased locomotor activity compared with NGF-XAT mice transduced by a control virus expressing Escherichia coli beta-galactosidase. These data indicate that focally increased expression of NGF in one part of a synaptic network can elicit changes in behavior presumably by altering the overall function of NGF-responsive neural circuitry. This approach should have broad application to other gene products and promises to provide the unprecedented ability to create and study discrete genetic modifications in the context of an intact adult mammal.

A specific point mutant at position 1 of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype.

We showed previously that substitution of the first residue of the influenza hemagglutinin (HA) fusion peptide Gly1 with Glu abolishes fusion activity. In the present study we asked whether this striking phenotype was due to the charge or side-chain volume of the substituted Glu. To do this we generated and characterized six mutants with substitutions at position 1: Gly1 to Ala, Ser, Val, Glu, Gln, or Lys. We found the following. All mutants were expressed at the cell surface, could be cleaved from the precursor (HA0) to the fusion permissive form (HA1-S-S-HA2), bound antibodies against the major antigenic site, bound red blood cells, and changed conformation at low pH. Only Gly, Ala, and Ser supported lipid mixing during fusion with red blood cells. Only Gly and Ala supported content mixing. Ser HA, therefore, displayed a hemifusion phenotype. The hemifusion phenotype of Ser HA was confirmed by electrophysiological studies. Our findings indicate that the first residue of the HA fusion peptide must be small (e.g., Gly, Ala, or Ser) to promote lipid mixing and must be small and apolar (e.g., Gly or Ala) to support both lipid and content mixing. The finding that Val HA displays no fusion activity underscores the idea that hydrophobicity is not the sole factor dictating fusion peptide function. The surprising finding that Ser HA displays hemifusion suggests that the HA ectodomain functions not only in the first stage of fusion, lipid mixing, but also, either directly or indirectly, in the second stage of fusion, content mixing.

Impairment of Moloney murine leukemia virus integration in a cell line underexpressing DNA topoisomerase II.

The possible intervention of nuclear proteins as cofactors of integrase-catalyzed integration of retroviral DNA into the host cell genome is not fully understood. Among various nuclear proteins, DNA topoisomerase II appears to be a plausible candidate. This hypothesis is supported by a series of evidence, including the fact that integration is markedly affected by the topology of the target DNA and mainly occurs in transcribed regions in which topoisomerase II is preferentially located. In an attempt to confirm the validity of this hypothesis, we have comparatively investigated the early stages of a recombinant Moloney murine leukemia virus (psi neo) in two related Chinese hamster cell lines (DC3F and R/DC3F) expressing different levels of both isoforms of topoisomerase II. R/DC3F is derived from the parental cell line DC3F and displays a resistant phenotype towards the usual anticancer topoisomerase II inhibitors (actinomycin D, doxorubicin, and taxol). Results show that the early stages of the retroviral cycle are markedly impaired in cells underexpressing topoisomerase II (R/DC3F). This alteration mimics Fv-1 restriction and is characterized by about a 6-fold decrease in viral DNA synthesis and total inhibition of viral genome integration. The specific impairment of integration in R/DC3F cells compared to DC3F cells is assessed by the absence of G418-resistant colonies upon viral infection and a lack of the viral genome in cellular nuclear DNA as detected by the PCR procedure. These features are observed in relevant infecting conditions leading, in both cell lines, to the same amount of linear viral DNA and to the occurrence of two long terminal repeats containing circular DNA in the nuclear fractions.

Induction of S phase and apoptosis by the human papillomavirus type 16 E7 protein are separable events in immortalized rodent fibroblasts.

The HPV16 E7 oncoprotein neutralizes several cell cycle checkpoints, favouring the entry of quiescent cells into S phase. This activity is mediated in part by association of E7 with the pocket proteins and consequent activation of E2F transcription factors. In addition, HPV16 E7 protein is able to promote apoptosis. In this study we demonstrate that the ability to induce apoptosis is a common property of E7s belonging to both benign and malignant HPV types. The E7-induced apoptosis is mediated by inactivation of pRb, whilst neutralization of the other two pRB-related proteins, p107 and 130, is not sufficient to trigger apoptosis. Moreover, we show that certain point mutations in the conserved region 1 (CR1) of HPV16 E7 abolish the induction of apoptosis without altering the ability to stimulate S phase. Thus, these two E7-mediated cellular events, apoptosis and S phase entry, can be separated in immortalized rodent fibroblasts. Our findings demonstrate that the E7-mediated pRb destabilization is not required for its ability to drive quiescent cells into S phase and to induce apoptosis. Finally, expression of E7 proteins in NIH3T3, which lack a functional p19ARF, does not lead to p53 accumulation, indicating that the E7 impacts upon additional cellular pathways to promote apoptosis.

Serum starved v-mos-transformed cells are unable to appropriately downregulate cyclins and CDKs.

Serum deprived v-mos-transformed NIH3T3 cells are unable to enter a true quiescent state, but instead, arrest in the early G1 phase of the cell cycle. We have analysed several cell cycle regulatory proteins in these G1 arrested cells and show altered regulation in the expression and activity of certain cyclins and cyclin-dependent kinases. In particular, p34cdc2, cyclin A, cyclin D and cyclin E are not appropriately down-regulated in serum starved, G1 arrested, v-mos-transformed cells as compared with quiescent NIH3T3 cells. Furthermore, serum starved v-mos-transformed cells have elevated histone H1 kinase activity associated with cyclin A, cyclin E, p33cdk2, and p34cdc2. Using a metallothionein-inducible c-mos(mu) expression system, we show that c-mos(mu) induction in quiescent NIH3T3 cells causes elevated expression of p34cdc2. However, this induction of c-mos(mu) and subsequent expression of p34cdc2 was not sufficient to promote significant entry of cells into S phase. Analysis of extracts from serum starved v-H-ras, v-src, and tpr-met transformed NIH3T3 cells demonstrates that these oncogene-transformed cells also contain elevated levels of p34cdc2. We propose that the altered regulation of these critical cell cycle regulatory molecules, and specifically the inability to fully downregulate their activity, contributes significantly to neoplastic transformation and subsequent unregulated growth of tumor cells.

Transactivation of the GATA-1 promoter by a myb-ets-containing mouse retrovirus is mediated by CACCC elements.

The myb-ets-containing ME26 virus causes erythroleukemia in mice by a novel mechanism involving the inappropriate activation of erythroid-specific genes in hematopoietic precursor cells. We have previously shown that the ME26 viral protein can transactivate the GATA-1 promoter in transient transactivation assays carried out in mouse fibroblasts. The mouse GATA-1 promoter, whose activity is regulated by the GATA-1 protein itself, contains a double GATA consensus sequence at its 5' end and two CACCC elements at its 3' end, both of which are crucial for promoter activity in erythroid cells, as well as a nonconsensus GATA sequence and several putative c-myb and c-ets binding sites. In order to determine which sequences in the GATA-1 promoter are crucial for activation by the ME26 viral protein, we made deletions of the promoter, cloned them into a luciferase expression vector and tested their activity in mouse fibroblasts, which do not express GATA-1. Our results indicate that sequences in the 3' end of the GATA-1 promoter, which include two CACCC elements, are essential for transactivation by ME26 virus, while other upstream sites contribute to full activation by the virus. Mutation of the CACCC sites abolishes ME26 viral transactivation. The interaction of cell extracts containing ME26 viral protein and the GATA-1 promoter fragment containing the two CACCC elements was examined by electrophoretic mobility shift analysis (EMSA) and the results showed no direct interaction between the two. However, we could detect the ubiquitous transcription factor Sp1 bound to this sequence. These data demonstrate that the CACCC element is necessary for GATA-1 promoter transactivation by ME26 virus and that the viral protein may indirectly transactivate the promoter by binding to Sp1.

Accumulation of p16INK4a in mouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status.

Viral transformation of mouse and human fibroblasts has very different effects on the composition of cyclin-dependent kinase (Cdk) complexes. In human cells transformed by the large T-antigen of simian virus 40 (SV40 T-Ag) and human tumour cell lines that lack a functional retinoblastoma gene product (pRb) no cyclin D1-Cdk4 complexes can be detected because all the available Cdk4 is associated with the Cdk-inhibitor p16INK4a. In contrast, SV40-transformed mouse cells and fibroblasts from Rh1-nullizygous mouse embryos contain normal levels of cyclin D1-Cdk4 complexes. To investigate this species difference, we have compared the biochemical properties and expression of mouse p16INK4a with that of its human counterpart. There is a marked increase in p16 RNA and protein levels as primary embryo fibroblasts approach their finite lifespan in culture, but mouse p16 expression does not appear to be influenced by the status of pRb. Transformed or spontaneously immortalized mouse cells therefore do not achieve the very high levels of p16 characteristic of pRb-negative human cell lines. We suggest that these differences may be related to the different frequencies with which mouse and human cells can be immortalized in culture.

Evidence for dysregulation of cell cycle by human polyomavirus, JCV, late auxiliary protein.

The late region of the human neurotropic JC virus encodes a 71 amino acid protein, named Agnoprotein, whose biological function remains elusive. Here we demonstrate that in the absence of other viral proteins, expression of Agnoprotein can inhibit cell growth by deregulating cell progression through the cell cycle stages. Cells with constitutive expression of Agnoprotein were largely accumulated at the G2/M stage and that decline in the activity of cyclins A and B is observed in these cells. Agnoprotein showed the ability to augment p21 promoter activity in transient transfection assay and a noticeable increase in the level of p21 is detected in cells continuously expressing Agnoprotein. Results from binding studies revealed the interaction of Agnoprotein with p53 through the N-terminal of the Agnoprotein spanning residues 1-36. Co-expression of p53 and Agnoprotein further stimulated transcription of the p21 promoter. Thus, the interaction of p53 and Agnoprotein can lead to a higher level of p21 expression and suppression of cell cycle progression during the cell cycle.

A new feature of Mpl receptor: ligand-induced transforming activity in FRE rat fibroblasts.

Mpl is the receptor for thrombopoietin, the primary regulator of platelet production by megakaryocytes. Upon stimulation by its ligand, Mpl receptor induces proliferation and differentiation of hematopoietic cell lines of various origins. In this paper, we show that Mpl is also able to transform FRE rat fibroblasts in the presence of MGDF (pegylated Megakaryocyte Growth and Development Factor), a modified form of its ligand. We also demonstrate that upon MGDF stimulation Mpl receptor activates the classical transduction pathways described for hematopoietic cell lines in FRE cells. Introduction of Mpl deletion mutants in FRE cells allowed us to demonstrate that the C-terminal region of the Mpl intracytoplasmic domain, which is involved in hematopoietic differentiation, is necessary for the transformation process. Within that region, site-directed mutagenesis showed that the Y112 residue, which is required for Shc phosphorylation, is essential for rat fibroblast transformation by Mpl/MGDF, suggesting the involvement of Shc in Mpl-mediated transformation. Interestingly, we showed that transformation correlated with strong and sustained MAPK activation. Neither Jak2, Stat3 nor Stat5 phosphorylation was sufficient to induce the transformation process. Taken altogether, our results suggest the oncogenicity of Mpl in fibroblastic cells in the presence of its ligand.

The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface.

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)-expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.

Comparison of transient and successful fusion pores connecting influenza hemagglutinin expressing cells to planar membranes.

Time-resolved admittance measurements were used to investigate the evolution of fusion pores formed between cells expressing influenza virus hemagglutinin (HA) and planar bilayer membranes. The majority of fusion pores opened in a stepwise fashion to semistable conductance levels of several nS. About 20% of the pores had measurable rise times to nS conductances; some of these opened to conductances of approximately 500 pS where they briefly lingered before opening further to semistable conductances. The fall times of closing were statistically similar to the rise times of opening. All fusion pores exhibited semistable values of conductance, varying from approximately 2-20 nS; they would then either close or fully open to conductances on the order of 1 microS. The majority of pores closed; approximately 10% fully opened. Once within the semistable stage, all fusion pores, even those that eventually closed, tended to grow. Statistically, however, before closing, transient fusion pores ceased to grow and reversed their conductance pattern: conductances decreased with a measurable time course until a final drop to closure. In contrast, pore enlargement to the fully open state tended to occur from the largest conductance values attained during a pore's semistable stage. This final enlargement was characterized by a stepwise increase in conductance. The density of HA on the cell surface did not strongly affect pore dynamics. But increased proteolytic treatment of cell surfaces did lead to faster growth within the semistable range. Transient pores and pores that fully opened had indistinguishable initial conductances and statistically identical time courses of early growth, suggesting they were the same upon formation. We suggest that transient and fully open pores evolved from common structures with stochastic factors determining their fate.

Induction of macrophage binding to virally infected cells by low levels of lipopolysaccharide.

Bone marrow culture-derived macrophages (BMCM) and vesicular stomatitis virus-infected BALB/c-3T3 cells (3T3-VSV) were used to determine whether macrophages could be activated to bind virally infected cells. Although lipopolysaccharide (LPS)-activated BMCM bound some uninfected BALB/c-3T3 cells, the number of targets that were bound increased with increasing times between infection and assay. Furthermore, LPS-activated BMCM bound more 3T3-VSV cells than did control macrophages. As more targets were added, the number of targets bound by the unactivated macrophages remained relatively level. However, the number of targets bound by the activated macrophages increased with increasing concentrations of added targets until they reached a plateau that was eight times greater than that bound by the unactivated BMCM. When BMCM were exposed to LPS for 24 h before assay, they lost both their ability to bind to 3T3-VSV and their cytolytic activity against those targets. However, as when using P815, a standard tumor target, the acquisition of binding of 3T3-VSV could be separated from macrophage cytolytic activity against those targets. The amount of LPS required to activated BMCM for increased binding of 3T3-VSV cells was 10-100 times lower than that needed to induce cytolytic activity for 3T3-VSV cells. Each of these values was approximately 100-fold lower than the amount of LPS required to induce the corresponding activity (binding or cytotoxicity) by using P815 targets.

Interaction with vesicular stomatitis virus-infected BALB/c3T3 cells inhibits the synthesis of nitric oxide in activated murine bone marrow culture-derived macrophages.

Bone marrow-culture-derived macrophages activated with interferon-gamma and lipopolysaccharide produced less nitric oxide (NO) when cultured with vesicular stomatitis virus (VSV)-infected BALB/c3T3 (3T3-VSV) than macrophages activated in an identical manner and cultured alone, with uninfected BALB/c3T3 (3T3), or with P815. However, all four groups of macrophages produced nearly the same amount of interleukin-6 (IL-6). Addition of VSV to activated macrophages did not change the amount of NO produced. The amount of NO generated by two non-macrophage sources of NO was not affected by the presence of either P815 or 3T3-VSV. Reverse transcriptase-polymerase chain reaction showed a decrease in the amount of inducible nitric oxide synthase (iNOS) but not IL-6 mRNA from macrophages cocultured with 3T3-VSV compared with macrophages cocultured with P815. The reduction in iNOS mRNA was confirmed by ribonuclease protection assay. When RAW 264.7 transfected with an iNOS regulatory construct were activated and incubated with 3T3-VSV there was a decrease in the expression of the reporter luciferase gene and NO production but not IL-6 production compared with cells incubated with either medium alone or with P815.

The vaccinia virus-stimulated mitogen-activated protein kinase (MAPK) pathway is required for virus multiplication.

Early events play a decisive role in virus multiplication. We have shown previously that activation of MAPK/ERK1/2 (mitogen-activated protein kinase/extracellular-signal-regulated kinase 1/2) and protein kinase A are pivotal for vaccinia virus (VV) multiplication [de Magalhães, Andrade, Silva, Sousa, Ropert, Ferreira, Kroon, Gazzinelli and Bonjardim (2001) J. Biol. Chem. 276, 38353-38360]. In the present study, we show that VV infection provoked a sustained activation of both ERK1/2 and RSK2 (ribosomal S6 kinase 2). Our results also provide evidence that this pattern of kinase activation depends on virus multiplication and ongoing protein synthesis and is maintained independently of virus DNA synthesis. It is noteworthy that the VGF (VV growth factor), although involved, is not essential for prolonged ERK1/2 activation. Furthermore, our findings suggest that the VV-stimulated ERK1/2 activation also seems to require actin dynamics, microtubule polymerization and tyrosine kinase phosphorylation. The VV-stimulated pathway MEK/ERK1/2/RSK2 (where MEK stands for MAPK/ERK kinase) leads to phosphorylation of the ternary complex factor Elk-1 and expression of the early growth response (egr-1) gene, which kinetically paralleled the kinase activation. The recruitment of this pathway is biologically relevant, since its disruption caused a profound effect on viral thymidine kinase gene expression, viral DNA replication and VV multiplication. This pattern of sustained kinase activation after VV infection is unique. In addition, by connecting upstream signals generated at the cytoskeleton and by tyrosine kinase, the MEK/ERK1/2/RSK2 cascade seems to play a decisive role not only at early stages of the infection, i.e. post-penetration, but is also crucial to define the fate of virus progeny.

Retroviral preparations derived from PA317 packaging cells contain inhibitors that copurify with viral particles and are devoid of viral vector RNA.

Obtaining high expression levels of a therapeutic gene in target cells could be achieved by integrating multiple copies of a recombinant retrovirus. However, we observed that cells retrovirally infected at high multiplicities of infection (MOIs) carried only single or double integrated proviral copies, suggesting that maximum retroviral transduction was achieved at relatively low MOIs. The same results were obtained when purified virus, free of most medium components, was used. Retroviral infection was shown to be inhibited by supernatants of other viral producer cell lines, and this inhibition could be removed by a centrifugation step that also removed more than 90% of infectious virus. Quantitative-competitive PCR of retroviral preparations showed that the amount of retroviral vector RNA present was similar to the amount expected on the basis of virus titers. Our data suggest that retroviral preparations derived from PA317 packaging cells contain inhibitors that copurify with retroviruses and do not contain viral vector RNA. We postulate that these inhibitor particles cannot achieve a productive infection but interfere with transduction of the target cells by infectious virions. This study might define an important criterion for the selection of more effective packaging cell lines.

Tricistronic and tetracistronic retroviral vectors for gene transfer.

We have combined the picornavirus foot-and-mouth disease virus (FMDV) 2A sequence and the internal ribosome entry sites (IRESes) from encephalomyocarditis virus (ECMV) and avian reticuloendotheliosis virus type A (REV-A) to construct tricistronic and tetracistronic vectors. All the polycistronic constructs show high titers and expression of the genes inserted. Clones have been obtained in which cells simultaneously express the three or four genes carried by the polycistronic vectors.

Correction of acid beta-galactosidase deficiency in GM1 gangliosidosis human fibroblasts by retrovirus vector-mediated gene transfer: higher efficiency of release and cross-correction by the murine enzyme.

Mutations in the lysosomal acid beta-galactosidase (EC 3.2.1.23) underlie two different disorders: GM1 gangliosidosis, which involves the nervous system and visceral organs to varying extents, and Morquio's syndrome type B (Morquio B disease), which is a skeletal-connective tissue disease without any CNS symptoms. This article shows that transduction of human GM1 gangliosidosis fibroblasts with retrovirus vectors encoding the human acid beta-galactosidase cDNA leads to complete correction of the enzymatic deficiency. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. Cross-correction experiments using retrovirus-modified cells as enzyme donors showed, however, that the human enzyme is transferred at low efficiencies. Experiments using a different retrovirus vector carrying the human cDNA confirmed this observation. Transduction of human GM1 fibroblasts and mouse NIH 3T3 cells with a retrovirus vector encoding the mouse beta-galactosidase cDNA resulted in high levels of enzymatic activity. Furthermore, the mouse enzyme was found to be transferred to human cells at high efficiency. Enzyme activity measurements in medium conditioned by genetically modified cells suggest that the human beta-galactosidase enzyme is less efficiently released to the extracellular space than its mouse counterpart. This study suggests that lysosomal enzymes, contrary to the generalized perception in the field of gene therapy, may differ significantly in their properties and provides insights for design of future gene therapy interventions in acid beta-galactosidase deficiency.