Moloney Murine Leukemia Retrovirus Research Articles

Comparative study of HIV-1 inhibition efficiency by carrageenans from red seaweeds family gigartinaceae, Tichocarpaceae and Phyllophoraceae

The efficiency of human immunodeficiency virus-1 (HIV-1) inhibition by sulfated polysaccharides isolated from the various families of red algae of the Far East Pacific coast were studied. The anti-HIV-1 activity of kappa and lambda-carrageenans from Chondrus armatus, original highly sulfated X-carrageenan with low content of 3,6-anhydrogalactose from Tichocarpus crinitus and i/κ–carrageenan with hybrid structure isolated from Ahnfeltiopsis flabelliformis was found. The antiviral action of these polysaccharides and its low-weight oligosaccharide was compared with commercial κ-carrageenan. Here we used the HIV-1-based lentiviral particles and evaluated that these carrageenans in non-toxic concentrations significantly suppress the transduction potential of lentiviral particles pseudotyped with different envelope proteins, targeting cells of neuronal or T-cell origin. The antiviral action of these carrageenans was confirmed using the chimeric replication competent Mo-MuLV (Moloney murine leukemia retrovirus) encoding marker eGFP protein. We found that X-carrageenans from T. crinitus and its low weight derivative and λ-carrageenan from C. armatus effectively suppress the infection caused by retrovirus. The obtained data suggest that the differences in the suppressive effect of carrageenans on the transduction efficiency of HIV-1 based lentiviral particles may be related to the structural features of the studied polysaccharides.

ROS upregulation during the early phase of retroviral infection plays an important role in viral establishment in the host cell

Recent studies suggest that low levels of reactive oxygen species (ROS) often modulate normal intracellular signalling pathways, determine cell fates and control cell proliferation. We found that infection of astrocytes with the neuropathogenic retrovirus ts1, a mutant of Moloney murine leukemia retrovirus, upregulated ROS at low levels during the early phase of infection. This upregulation of intracellular ROS with downregulation of NADPH levels during the early phase of ts1 infection was a separate event from the upregulation of ROS during the late phase while ts1-mediated cell death occurred. The treatment of apocynin, a potential inhibitor of NADPH oxidase (NOX), inhibited establishment of the ts1 virus in the host cell. These results suggested that ROS generated as a consequence of the activation of NOX may play an important role in the early events of the virus life cycle leading to the establishment of the virus in the host cell. The in vitro results were further supported by an in vivo experiment which showed that the treatment of apocynin decreased viral titre in the ts1-infected mouse brain and increased the lifespan of infected mice. This study provides the first in vitro and in vivo evidence on a mechanism for how ROS are involved in ts1 retrovirus infection and ts1-mediated neurodegenerative disease. Our findings focusing on the early phase of the ts1 retrovirus life cycle could provide a better understanding of retroviral life cycle, which may offer specific therapeutic targets for suppressing viral replication and alleviating neurodegenerative symptoms in a mouse model.

Persistent retroviral infection with MoMuLV influences neuropathological signature and phenotype of prion disease

A fundamental step in pathophysiology of prion diseases is the conversion of the host encoded prion protein (PrP(C)) into a misfolded isoform (PrP(Sc)) that accumulates mainly in neuronal but also non-neuronal tissues. Prion diseases are transmissible within and between species. In a subset of prion diseases, peripheral prion uptake and subsequent transport to the central nervous system are key to disease initiation. The involvement of retroviruses in this process has been postulated based on the findings that retroviral infections enhance the spread of prion infectivity and PrP(Sc) from cell to cell in vitro. To study whether retroviral infection influences the phenotype of prion disease or the spread of prion infectivity and PrP(Sc) in vivo, we developed a murine model with persistent Moloney murine leukemia retrovirus (MoMuLV) infection with and without additional prion infection. We investigated the pathophysiology of prion disease in MoMuLV and prion-infected mice, monitoring temporal kinetics of PrP(Sc) spread and prion infectivity, as well as clinical presentation. Unexpectedly, infection of MoMuLV challenged mice with prions did not change incubation time to clinical prion disease. However, clinical presentation of prion disease was altered in mice infected with both pathogens. This was paralleled by remarkably enhanced astrogliosis and pathognomonic astrocyte morphology in the brain of these mice. Therefore, we conclude that persistent viral infection might act as a disease modifier in prion disease.

Making Sense of Viral RNA Sensing

Gene therapy vectors have commonly been derived from the Moloney murine leukemia retrovirus virus (MuLV). Recent clinical trials of severe combined immunodeficiency support the potential of retroviruses as therapeutic agents.1 However, our understanding of how such nonreplicating viruses interact with the immune system is tentative and incomplete. Two articles published in recent issues of Immunity add to our understanding of the complexities of immune sensing of viral nucleic acids,2,3 suggesting that nonreplicative retroviruses derived from MuLV can induce a prolonged antibody antiviral response by virtue of their ability to activate innate immune sensors.3 Given the growing number of such innate immune sensors,2 these studies highlight the need for careful monitoring of immune responses when using viruses for gene therapy.

A novel strategy to derive iPS cells from porcine fibroblasts

Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors. This technology has created an interest in deriving iPS cells from domesticated animals such as pigs, sheep and cattle. Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells. However, this retrovirus system infects only mouse and rat cells, which limits its use in establishing iPS cells from other mammals. In our study, we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts. We transfected four human reprogramming factors (Oct4, Sox2, Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells. We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF. Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies. Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.

Integration of reporter transgenes intoSchistosoma mansonichromosomes mediated by pseudotyped murine leukemia virus

The recent release of draft genome sequences of two of the major human schistosomes has underscored the pressing need to develop functional genomics approaches for these significant pathogens. The sequence information also makes feasible genome-scale investigation of transgene integration into schistosome chromosomes. Retrovirus-mediated transduction offers a means to establish transgenic lines of schistosomes, to elucidate schistosome gene function and expression, and to advance functional genomics approaches for these parasites. We investigated the utility of the Moloney murine leukemia retrovirus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) for the transduction of Schistosoma mansoni and delivery of reporter transgenes into schistosome chromosomes. Schistosomula were exposed to virions of VSVG-pseudotyped MLV, after which genomic DNA was extracted from the transduced schistosomes. Southern hybridization analysis indicated the presence of proviral MLV retrovirus in the transduced schistosomes. Fragments of the MLV transgene and flanking schistosome sequences recovered using an anchored PCR-based approach demonstrated definitively that somatic transgenesis of schistosome chromosomes had taken place and, moreover, revealed widespread retrovirus integration into schistosome chromosomes. More specifically, MLV transgenes had inserted in the vicinity of genes encoding immunophilin, zinc finger protein Sma-Zic, and others, as well as near the endogenous schistosome retrotransposons, the fugitive and SR1. Proviral integration of the MLV transgene appeared to exhibit primary sequence site specificity, targeting a gGATcc-like motif. Reporter luciferase transgene activity driven by the schistosome actin gene promoter was expressed in the tissues of transduced schistosomula and adult schistosomes. Luciferase activity appeared to be developmentally expressed in schistosomula with increased activity observed after 1 to 2 wk in culture. These findings indicate the utility of VSVG-pseudotyped MLV for transgenesis of S. mansoni, herald a tractable pathway forward toward germline transgenesis and functional genomics of parasitic helminths, and provide the basis for comparative molecular pathogenesis studies of chromosomal lesions arising from retroviral integration into human compared with schistosome chromosomes.

Identifying candidate genes involved in brain tumor formation

Malignant primary brain tumors, gliomas, often overexpress both platelet-derived growth factor (PDGF) ligands and receptors providing an autocrine and/or paracrine boost to tumor growth. Glioblastoma multiforme (GBM) is the most frequent glioma. Its aggressive and infiltrative growth renders it extremely difficult to treat. Median survival after diagnosis is currently only 12-14 months. The present review describes the use of retroviral tagging to identify candidate cancer-causing genes that cooperate with PDGF in brain tumor formation.Newborn mice injected intracerebrally with a Moloney murine leukemia retrovirus carrying the sis/PDGF-B oncogene and a replication competent helper virus developed brain tumors with many characteristics of human gliomas. Analysis of proviral integrations in the brain tumors identified almost 70 common insertion sites (CISs). These CISs were named brain tumor loci and harbored known but also putative novel cancer-causing genes.Microarray analysis identified differentially expressed genes in the mouse brain tumors compared to normal brain. Known tumor genes and markers of immature cells were upregulated in the tumors. Tumors developed 13-42 weeks after injection and short latency tumors were further distinguished as fast growing and GBM-like. Long latency tumors resembled slow-growing oligodendrogliomas and contained significantly less integrations as compared to short latency tumors.Several candidate genes tagged in this retroviral screen have known functions in neoplastic transformation and oncogenesis. Some candidates with a previously unknown function in tumorigenesis were found and their putative role in brain tumor formation will be discussed in this review. The results show that proviral tagging may be a useful tool in the search for candidate glioma genes.

Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo

Single-cell genetic manipulation in an intact brain environment is an informative approach to study molecular mechanism of adult neurogenesis. Here, we describe a protocol for retrovirus-mediated single-cell gene knockout in adult new neurons in vivo. A gene of interest is disrupted in adult floxed mice by a vector based on the Moloney murine leukemia retrovirus, expressing Cre recombinase. High-titer retrovirus is prepared by transfecting plasmids into the HEK293T cells and by concentrating the supernatant containing virus. The retrovirus is stereotaxically injected into the dentate gyrus. Cre recombinase is transduced and expressed in a small fraction of adult new neurons in an intact environment, and the gene knockout is highly efficient within the transduced neurons. Virus preparation takes 7 days, but virus injections take less than 1 h per mouse. By changing the survival time of the mice after the injection, one can analyze the effects on new neurons at different ages.

694. Involvement of Glycosaminoglycans in VSV-G Pseudotyped Lentiviral Vector Mediated Gene Transfer into Airway Epithelial Cells

HIV-1-derived lentiviral (LV) vectors are considered promising vehicles for Cystic Fibrosis (CF) gene therapy since they integrate in the host genome, transduce non-dividing cells and ensure long-term expression of the transgene. Although some knowledge on the infection of respiratory epithelial cells has been accumulated, the receptors involved in the transduction of these cells by VSV-G-pseudotyped LV vectors are not well known. Previous studies have shown that heparin inhibited infection of HT1080 cells by Moloney Murine Leukemia retrovirus pseudotyped with VSV-G (Guibinga, 2002), indicating that interaction with glycosaminoglycans (GAGs) of cellular membrane plays a role in infectivity of viral particles with VSV-G envelope. Our study investigated the involvement of heparan sulfate (HS) and chondroitin sulfate B in gene transfer mediated by a VSV-G pseudotyped LV vector in a respiratory epithelial cell line of tracheal origin (CFT1-C2). The HIV-1 derived LV vector PPT-GFP carries the Green Fluorescence Protein (GFP) reporter gene under the control of the CMV promoter (Follenzi, 2000). To evaluate the interaction between GAGs expressed on the apical surface of respiratory epithelial cells and lentiviral particles, the LV vector (3.2|[times]|10E8 TU, corresponding to MOI 2000) was pre-incubated with heparin or chondroitin sulfate B at different concentrations (10|[ndash]|250 micrograms/ml) for 60 minutes in binding buffer and then added to polarized CFT1-C2 cells (polarization was evaluated by the measure of transepithelial resistance and apical expression of ZO-1). The virus-containing solution was incubated with cells for 24 hours and GFP expression was analyzed by flow cytometry 72 hours after. Heparin and chondroitin sulfate B determined a dose-dependent inhibition of transduction efficiency up to 86% and 83% of untreated vector, respectively. HS expression in CFT1-C2 cells was analyzed by flow cytometry and confocal microscopy. HS was expressed by 85|[ndash]|88% of cells, the expression being much stronger on the basolateral side of polarized cells. Cells were incubated with 12 mM EGTA before LV administration to disrupt the epithelial tight junctions and allow the lentiviral particles to access the basolateral side of the epithelium. In the absence of EGTA, 60% of cells resulted GFP positive, whereas the pre-treatment with EGTA increased the percentage of GFP-expressing cells up to 80.5%. Our data strongly indicate that the little amount of GAGs restricted to the apical surface might be sufficient to mediate transduction of respiratory epithelial cells by a VSV-G pseudotyped LV vector. However, we can not exclude that other receptor(s) could be involved.

Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography

We used cryo-electron tomography in conjunction with single-particle averaging techniques to study the structures of frozen-hydrated envelope glycoprotein (Env) complexes on intact Moloney murine leukemia retrovirus particles. Cryo-electron tomography allows 3D imaging of viruses in toto at a resolution sufficient to locate individual macromolecules, and local averaging of abundant complexes substantially improves the resolution. The averaging of repetitive features in electron tomograms is hampered by a low signal-to-noise ratio and anisotropic resolution, which results from the "missing-wedge" effect. We developed an iterative 3D averaging algorithm that compensates for this effect and used it to determine the trimeric structure of Env to a resolution of 2.7 nm, at which individual domains can be resolved. Strikingly, the 3D reconstruction is shaped like a tripod in which the trimer penetrates the membrane at three distinct locations approximately 4.5 nm apart from one another. The Env reconstruction allows tentative docking of the x-ray crystal structure of the receptor-binding domain. This study thus provides 3D structural information regarding the prefusion conformation of an intact unstained retrovirus surface protein.

Increased probability of expression from modified retroviral vectors in embryonal stem cells and embryonal carcinoma cells.

Gene expression from the Moloney murine leukemia retrovirus (Mo-MuLV) is highly restricted in embryonic carcinoma (EC) and embryonic stem (ES) cells. We compared levels of expression in PA317 fibroblasts, F9 (EC) cells, and CCE (ES) cells by Mo-MuLV-based vectors and vectors based on our previously reported MND backbone, which has alterations to address three viral elements implicated as repressors of expression by Mo-MuLV: the enhancer, the primer binding site, and the negative-control region. Expression was evaluated with three reporter genes, the chloramphenicol acetyltransferase (CAT) gene, whose expression was measured by enzymatic assay and by Northern blotting; a truncated nerve growth factor receptor (tNGFR), whose expression was measured by fluorescence-activated cell sorting (FACS) as a cell surface protein; and the enhanced green fluorescent protein (EGFP), whose expression was measured intracellularly by flow cytometry. We found significantly higher levels of CAT activity (5- to 300-fold) and greater quantities of vector-specific transcripts in ES and EC cells transduced with the modified MND-CAT-SN vector than in those transduced with L-CAT-SN. Northern blot analysis indicated that long terminal repeat transcripts from MND-CAT-SN are >80 times more abundant than the L-CAT-SN transcripts. FACS analysis of tNGFR expression from a pair of vectors, L-tNGFR-SN and MND-tNGFR-SN, indicated that only 1.04% of the CCE cells containing the L-tNGFR-SN vector expressed the cell surface reporter, while the MND-tNGFR-SN vector drove expression in 99.54% of the CCE cells. Of the F9 cells containing the L-tNGFR-SN vector, 13.32% expressed tNGFR, while 99.89% of the F9 cells transduced with MND-tNGFR-SN showed expression. Essentially identical results were produced with an analogous pair of vectors encoding EGFP. In unselected pools of F9 cells 48 h posttransduction, the L-EGFP-SN vector drove expression in only 5% of the population while the MND-EGFP-SN vector drove expression in 88% of the cells. After more than 3 weeks in culture without selection, the proportion of cells showing expression from L-EGFP-SN decreased slightly to 3% while expression from the MND-EGFP-SN vector persisted in 80% of the cells. Interestingly, in the few ES and EC cells which did show expression from the L-tNGFR-SN or L-EGFP-SN vectors, the magnitude of reporter expression was similar to that from the MND-tNGFR-SN or MND-EGFP-SN vector in nearly all cells, suggesting that the MND vectors are far less susceptible to position-dependent variegation of expression than are the Mo-MuLV-based vectors. Therefore, the modified retroviral vector, MND, achieves higher net levels of expression due to a greater frequency of expression, which may be useful for the expression of exogenous genes in EC and ES cells.

Tissue- and stage-specific activation of an endogenous provirus after transcription through its integration site in the opposite orientation.

Endogenous proviruses of the Moloney murine leukemia retrovirus (Mo-MuLV) are transcriptionally blocked in early embryos and in general remain silent even when the tissues have become permissive to the expression of newly integrated copies. Eventually, activation in presumably very few cells initiates rapid superinfection leading to viremia and leukemia, but the processes leading to provirus activation are unknown. Differences in the onset and development of viremia between several mouse strains carrying an endogenous Mo-MuLV (Mov lines) are attributed to a chromosomal position effect, but neither cell type nor stage of provirus activation is known for any strain. We have now monitored the appearance of viral transcripts and particles in the Mov13 strain, which carries the Mo-MuLV provirus in inverted orientation in the first intron of a collagen gene (Col1a1) with well-characterized transcriptional activity. We report obligatory tissue- and stage-specific virus activation in osteoblasts and odontoblasts. The significance of this activation pattern is indicated by the fact that of the great variety of cells expressing the wild-type collagen gene, only these two cell types can also transcribe the mutant allele including its viral insert. We propose that this transcription of the proviral genome, albeit in the opposite direction, leads to the activation of the viral promoter.

A Model for Mixed Virus Disease: Co-infection with Moloney Murine Leukemia Virus Potentiates Runting Induced by Polyomavirus (A2 Strain) in Balb/c and NIH Swiss Mice

Polyomavirus was originally isolated by Ludwick Gross from a mixture that also contained a murine retrovirus. A possible pathogenic interaction between polyomavirus and an endogenous mouse retrovirus locus (mtv-7) in polyomavirus-induced cancer has also been reported. To study potential interactive effects of polyomavirus (Py) and Moloney murine leukemia retrovirus (M-MuLV), newborn Balb/c and NIH Swiss mice were infected with high titer wild-type Py (A2 strain) and M-MuLV. Dramatically stunted growth (runting) occurred in 100% of the doubly inoculated mice, while much lower frequency of runting occurred in animals infected with Py alone and not at all with M-MuLV-infected mice. In situ hybridization for Py DNA showed ongoing Py replication and inflammation in kidneys (atypical of most mice singly infected by Py) of runted doubly inoculated mice. In addition, high Py viral replication continued well past the usual acute stage termination. M-MuLV replication was also initially inhibited in bone marrow by simultaneous Py infection. No M-MuLV replication was seen in singly or doubly infected mouse kidneys. Runting was very rapid, observable within 2 days after co-infection, arguing against an adaptive or antigen-specific immunological mechanism. One possibility was that a cytokine-driven acute response mechanism was involved. Supporting this view, RNAse protection assays for various cytokine RNAs showed that several were specifically elevated in kidneys of doubly infected mice. Three patterns were observed: (1) IL-6 was elevated in doubly infected mice early after infection (7 days), but it declined at later times (19 days); (2) IFN-γ, IL-1β, and IL-10 were elevated at both early and late times; and (3) TNF-α, IL-12p40, and possibly TNF-β were elevated only at late times. While the cytokines in the third category might be indicative of infiltrating inflammatory cells, it seems possible that cytokines in the first or second categories might be involved in establishing runting and ongoing polyoma DNA replication in the doubly infected mice.

Binding kinetics of ecotropic (Moloney) murine leukemia retrovirus with NIH 3T3 cells

A quantitative analysis of the binding kinetics of intact Moloney murine leukemia retrovirus (MoMuLV) particles with NIH 3T3 cells was performed with an immunofluorescence flow cytometry assay. The virus-cell binding equilibrium dissociation constant (KD), expressed in terms of virus particle concentration, was measured to be 8.5 (+/- 6.4) x 10(-12) M at 4 degrees C and was three- to sixfold lower at temperatures above 15 degrees C. The KD of virus binding is about 1,000-fold lower than the KD of purified MoMuLV envelope. The association rate constant was determined to be 2.5 (+/- 0.9) x 10(9) M-1 min-1 at 4 degrees C and was 5- to 10-fold higher at temperatures above 15 degrees C. The apparent dissociation rate constant at 4 degrees C was 1.1 (+/- 0.4) x 10(-3) min-1 and was doubled for every 10 degrees C increase in temperature over the range tested (15 to 37 degrees C).

Isolation of mammalian cell mutants that are X-ray sensitive, impaired in DNA double-strand break repair and defective for V(D)J recombination

The Chinese hamster lung V79-4 cell line was infected with a Moloney murine leukemia retrovirus and the infected cells were subsequently screened for mutants that were sensitive to X-rays using a toothpicking/96-well replica plating technique. Four independent mutants that were sensitive to X-irradiation (sxi-1 to sxi-4) were isolated from 9000 retrovirally infected colonies. A pulse-field gel electrophoresis (PFGE) assay demonstrated that all of the sxi mutants were impaired in DNA double-strand break (DSB) repair, thus providing a molecular explanation for the observed X-ray sensitivity. Interestingly, additional PFGE experiments demonstrated that for any given X-ray dose all of the mutants incurred more DNA DSBs than the parental V79-4 cell line indicating there may be some inherent fragility to sxi chromosomes. Cross-sensitivity to other DNA-damaging agents including bleomycin, mitomycin C and methyl methanesulfonate indicated that sxi-2, sxi-3 and sxi-4 appear to be specifically hypersensitive to genotoxic agents that cause DNA DSBs, whereas sxi-1 appeared to be hypersensitive to multiple types of DNA lesions. Lastly, in preliminary experiments all of the sxi mutants demonstrated an inability to carry out V(D)J recombination, a somatic DNA rearrangement process required for the assembly of lymphoid antigen receptor genes. Thus, the sxi cell lines have interesting phenotypes which should make them valuable tools for unraveling the mechanism(s) of DNA DSB repair and recombination in mammalian cells.

The dimerization/packaging sequence is dispensable for both the formation of high-molecular-weight RNA complexes within retroviral particles and the synthesis of proviruses of normal structure

Retroviral particles contain a dimer of two genomic RNA molecules, linked by noncovalent intermolecular bonds. Studies by electron microscopy of viral RNA extracted from virions as well as in vitro studies have implicated a sequence, designated the dimer linkage sequence (DLS), in the dimerization process. The DLS has been localized within a short region encompassing the psi packaging sequence, between nucleotides 212 and 563 for the Moloney murine leukemia retrovirus (MoMLV) RNA. In this report, we show that viral RNAs lacking both the DLS and psi packaging sequences--and even an RNA lacking the first 6,537 nucleotides of MoMLV--can assemble within retroviral particles as high-molecular-weight, slow-migrating, heat-sensitive complexes closely related to those observed for wild-type viral RNAs. Furthermore, we show that proviruses of normal structure are generated upon infection of test cells with retroviral particles which contain the DLS/psi-deleted viral RNAs. These observations demonstrate that the DLS and psi packaging sequences are not essential in cis to form a functional RNA complex for reverse transcription and integration.

A Liquid Chromatographic Preparation of Retroviral RNA

Liquid chromatography was used to prepare native dimer RNA from Moloney murine leukemia retrovirus by gel filtration on a TSK 6000PW column. Three RNA peaks were separated from viral lysate. RNA from the first eluted peak migrated in gel electrophoresis as a native dimer prepared by phenolic extraction and saccharose gradient separation. The last eluted RNA peak likely represents tRNA for proline. HPLC preparation was twice as fast and 20 times more productive than the other method, considering the quantity of pure RNA obtained for the same volume of viral lysate. Using several natural RNAs, it was verified that the dispersion coefficient was inversely proportional to RNA size, at least between 3.6 and 16.6 kb. Within the range of laboratory use, peak surface was in a direct ratio to the injected quantity of a given RNA species. Thus size exclusion chromatography could represent a valuable tool for preparation, analysis, and quantitation of large RNAs.

Analysis of mammalian cell genetic regulation in situ by using retrovirus-derived "portable exons" carrying the Escherichia coli lacZ gene.

Self-inactivating derivatives of Moloney murine leukemia retrovirus containing the Escherichia coli lacZ gene were used to detect and study the regulation of transcription initiated at chromosomally located promoters in mouse fibroblasts. The introduction of splice acceptor sites in all three translational reading frames relative to lacZ and the inclusion of an in-frame ATG translation start codon in one construct allowed synthesis of beta-galactosidase fusion proteins upon insertion of retrovirus vectors containing lacZ into introns 3' to either protein-coding or noncoding exons. Selection of lacZ-expressing cells by fluorescence-activated cell sorting and the analysis of beta-galactosidase production after serum deprivation has yielded lines in which lacZ was fused to genes induced by growth arrest in the G0 state.