Actin depolymerization is associated with meiotic acceleration in cycloheximide-treated ovine oocytes.

Oocytes treated with the protein synthesis inhibitor cycloheximide (CHX) arrest at the germinal vesicle (GV) stage and undergo accelerated GV breakdown (GVBD) after CHX is removed. However, little is known about the underlying mechanism of accelerated meiotic maturation. Here, we investigated this mechanism and found that oocytes released from CHX arrest have higher amounts of cyclin B1 (CCNB1) and phosphorylated mitogen-activated protein kinase (pMAPK) proteins. Increased levels of these factors were not associated with mRNA polyadenylation or increased transcription rates of CCNB1 and MOS (Moloney murine sarcoma viral oncogene homolog) during CHX arrest. We found that treatment of CHX-arrested oocytes with the actin filament-stabilizing agent Jasplakinolide (Jasp) delayed GVBD following release from CHX arrest and that this was correlated with reduced maturation-promoting factor (MPF) activity. These results suggest that CCNB1 mRNAs released from actin filaments during CHX arrest increase CCNB1 transcripts available for translation after release from CHX arrest, leading to the precocious activation of MPF and accelerated meiotic progression.

Significant differences in integration sites of Moloney murine leukemia virus/Moloney murine sarcoma virus retroviral vector carrying recombinant coagulation factor IX in two human cell lines.

Ligation-mediated-PCR was performed followed by the mapping of 177 and 150 integration sites from HepG2 and Hek293 transduced with chimera vector carrying recombinant human Factor IX (rhFIX) cDNA, respectively. The sequences were analyzed for chromosome preference, CpG, transcription start site (TSS), repetitive elements, fragile sites and target genes. In HepG2, rhFIX was had an increased preference for chromosomes 6 and 17; the median distance to the nearest CpG islands was 15,240 base pairs and 37 % of the integrations occurred in RefSeq genes. In Hek293, rhFIX had an increased preference for chromosome 5; the median distance to the nearest CpG islands was 209,100 base pairs and 74 % of the integrations occurred in RefSeq genes. The integrations in both cell lines were distant from the TSS. The integration patterns associated with this vector are different in each cell line.

Architecture of a gamma retroviral genomic RNA dimer.

Retroviral genomes contain two sense-strand RNAs that are noncovalently linked at their 5' ends, forming a dimer. Establishing a structure for this dimer is an obligatory first step toward understanding the fundamental role of the dimeric RNA in retroviral biology. We developed a secondary structure model for the minimal dimerization active sequence (MiDAS) for the Moloney murine sarcoma virus in the final dimer state using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). In this model, two self-complementary, or palindromic, sequences (PAL1 and PAL2) form extended intermolecular duplexes of 10 and 16 base pairs, respectively. The monomeric starting state was shown previously to contain a flexible domain in which nucleotides do not form stable interactions with other parts of the RNA. In the final dimer state, portions of this initial flexible domain form stable base pairs, while previously base-paired elements lie in a new flexible domain. Thus, partially overlapping and structurally well-defined flexible domains are prominent features of both monomer and dimer states. We then used hydroxyl radical cleavage experiments to characterize the global architecture of the dimer state. Extensive regions, including portions of both PAL1 and PAL2, are occluded from solvent-based cleavage indicating that the MiDAS domain does not function simply as a collection of autonomous secondary structure elements. Instead, the retroviral dimerization domain adopts a compact architecture characterized by close packing of its constituent helices.

The SL1-SL2 (stem-loop) domain is the primary determinant for stability of the gamma retroviral genomic RNA dimer.

Retroviral genomes are assembled from two sense-strand RNAs by noncovalent interactions at their 5' ends, forming a dimer. The RNA dimerization domain is a potential target for antiretroviral therapy and represents a compelling RNA folding problem. The fundamental dimerization unit for the Moloney murine sarcoma gamma retrovirus spans a 170-nucleotide minimal dimerization active sequence. In the dimer, two self-complementary sequences, PAL1 and PAL2, form intermolecular duplexes, and an SL1-SL2 (stem-loop) domain forms loop-loop base pairs, mediated by GACG tetraloops, and extensive tertiary interactions. To develop a framework for assembly of the retroviral RNA dimer, we quantified the stability of and established nucleotide resolution secondary structure models for sequence variants in which each motif was compromised. Base pairing and tertiary interactions between SL1-SL2 domains contribute a large free energy increment of -10 kcal/mol. In contrast, even though the PAL1 and PAL2 intermolecular duplexes span 10 and 16 bp in the dimer, respectively, they contribute only -2.5 kcal/mol to stability, roughly equal to a single new base pair. First, these results emphasize that the energetic costs for disrupting interactions in the monomer state nearly balance the PAL1 and PAL2 base pairing interactions that form in the dimer. Second, intermolecular duplex formation plays a biological role distinct from simply stabilizing the structure of the retroviral genomic RNA dimer.

Construction of retroviral vectors with enhanced efficiency of transgene expression.

Retroviral vectors have been widely used in gene therapy due to their simple genomic structure and high transduction efficiency. We report a construction of Moloney murine sarcoma virus (MoMSV) and Moloney murine leukemia virus (MoMLV) hybrid-based retroviral vectors with significantly improved efficiency of transgene expression after stable incorporation into the host genome. In these vectors, the residual gag gene coding sequence located in the extended region of packaging signal was removed. These vectors, therefore, contain no coding sequence for the gag, pol, or env gene that can be used for homologous recombination with sequences introduced in the packaging system for a recombinant competent retrovirus (RCR) generation. A strong splice acceptor site obtained from the exon/intron junction of either the chimpanzee EF1-alpha gene or the human CMV major immediate early gene was placed downstream of the MoMSV packaging signal (Psi), significantly improving the efficiency of transgene expression. The 5' LTR U3 sequence was replaced with an extended human CMV major immediate early gene enhancer/promoter for a strong expression of full-length messages from the viral backbone, helping to maintain high levels of viral titer. These newly developed retroviral vectors should facilitate RCR-free gene transfer with significantly improved efficacy in clinical gene therapy trials.

Establishment of modified retroviral vector targeting X-linked severe combined immunodeficiency.

Gene therapy targeting hematopoietic stem cells has been proposed as a potential therapy for numerous genetic disorders affecting hematopoiesis. Moloney murine leukemia retroviral vectors are now widely used for clinical gene transfer into hematopoietic progenitors and progeny. However, maintaining expression of therapeutic genes inserted via moloney murine leukemia virus (MoMLV)-based vectors has proven to be more difficult than previously expected. In this study, an MND-IL-2R vector containing IL-2Rc gamma cDNA to treat X-linked severe combined immunodeficiency (X-SCID) was constructed from an MND vector that was modified by substituting the myeloproliferative sarcoma virus (MPSV) enhancer for that of MoMLV, deleting the negative control region located in the long terminal repeat (LTR) as an enhancer, and replacing the primer binding site (PBS) of MoMLV with the PBS of the endogenous murine retrovirus dl587rev. This vector was transduced into human CD34 + progenitor cells with comparable efficiency to that of the MoMLV-based vector. The use of this newly created vector may be advantageous for gene therapy of X-SCID.

Quantitative imaging of the T cell antitumor response by positron-emission tomography.

We describe a noninvasive, quantitative, and tomographic method to visualize lymphocytes within the whole animal. We used positron-emission tomography (PET) to follow the localization of adoptively transferred immune T lymphocytes. Splenic T cells from animals that had rejected a Moloney murine sarcoma virus/Moloney murine leukemia virus (M-MSV/M-MuLV)-induced tumor were marked with a PET reporter gene, injected into tumor-bearing mice, and imaged in a microPET by using a substrate specific for the reporter. Specific localization of immune T cells to the antigen-positive tumor was detected over time, by sequential imaging of the same animals. Naive T cells did not localize to the tumor site, indicating that preimmunization was required. Autoradiography and immunohistochemistry analysis corroborated the microPET data. The method we have developed can be used to assess the effects of immunomodulatory agents intended to potentiate the immune response to cancer, and can also be useful for the study of other cell-mediated immune responses, including autoimmunity.

A single Glu(62)-to-Lys(62) mutation in the Mos residues of the R7Delta447Gag-tMos protein causes the mutant virus to induce brain lesions.

We previously reported that R7Delta447, a 2954-base-pair (bp) laboratory-generated Moloney murine sarcoma virus, induced subcutaneous tumors in about 14% of infected mice but did not induce brain lesions. We now report that R7Delta447K, a spontaneous mutant of R7Delta447, induced brain lesions as well as subcutaneous tumors in all injected mice. The genomes of the two viruses differ in a single base pair: the deduced Glu(62) of the Mos residue of the R7Delta447 Gag-tMos protein is changed to Lys(62). More R7Delta447 than R7Delta447K focus-forming units were detected in both NIH3T3 and mouse cerebral vascular endothelial (MCVE) cells. However, R7Delta447K transformed NIH3T3 and MCVE cells more acutely than did R7Delta447. A distinctive feature that distinguished the morphologic transformation of R7Delta447- and R7Delta447K-infected MCVE cells is the markedly prolonged spindle-shaped phase exhibited by R7Delta447-infected MCVE cells. In addition, R7Delta447K was more efficient in inducing the phosphorylation of ERK1/2 than R7Delta447 in both MCVE and NIH3T3 cells. Moreover morphologic transformation was inhibited, and levels of phosphorylated ERK1/2 were reduced when R7Delta447- or R7Delta447K-infected NIH3T3 or MCVE cells were grown in the presence of the MEK1/2-specific inhibitor PD98095. Thus, we have identified a key residue in the Gag-tMos protein that profoundly affects activation of the Mos/MEK/ERK pathway, virus and cell replication, morphologic transformation in vitro and pathogenicity in vivo.

Effects of homology length in the repeat region on minus-strand DNA transfer and retroviral replication.

Homology between the two repeat (R) regions in the retroviral genome mediates minus-strand DNA transfer during reverse transcription. We sought to define the effects of R homology lengths on minus-strand DNA transfer. We generated five murine leukemia virus (MLV)-based vectors that contained identical sequences but different lengths of the 3' R (3, 6, 12, 24 and 69 nucleotides [nt]); 69 nt is the full-length MLV R. After one round of replication, viral titers from the vector with a full-length downstream R were compared with viral titers generated from the other four vectors with reduced R lengths. Viral titers generated from vectors with R lengths reduced to one-third (24 nt) or one-sixth (12 nt) that of the wild type were not significantly affected; however, viral titers generated from vectors with only 3- or 6-nt homology in the R region were significantly lower. Because expression and packaging of the RNA were similar among all the vectors, the differences in the viral titers most likely reflected the impact of the homology lengths on the efficiency of minus-strand DNA transfer. The molecular nature of minus-strand DNA transfer was characterized in 63 proviruses. Precise R-to-R transfer was observed in most proviruses generated from vectors with 12-, 24-, or 69-nt homology in R, whereas aberrant transfers were predominantly used to generate proviruses from vectors with 3- or 6-nt homology. Reverse transcription using RNA transcribed from an upstream promoter, termed read-in RNA transcripts, resulted in most of the aberrant transfers. These data demonstrate that minus-strand DNA transfer is homology driven and a minimum homology length is required for accurate and efficient minus-strand DNA transfer.

Changes in the N- and C-terminal sequences of the murine R7 Gag-tMos protein affect brain lesion induction.

Our preliminary studies suggested that the novel gag-truncated mos (tmos) open reading frame (ORF) of R7, a spontaneous deletion mutant of Moloney murine sarcoma virus 124 (MoMuSV124), may be responsible for R7's unique ability to induce brain lesions in all R7-injected mice. However, when we replaced the gag-tmos ORF with either the MoMuSV124 or the homologous myeloproliferative sarcoma virus env-mos gene, we found that both recombinant viruses also induced brain lesions in all injected mice. Although these studies suggested that the critical determinants for brain lesion induction may reside in the tmos sequence common to all three viruses, they did not demonstrate if the N-terminus of Mos was dispensable for this activity. By inserting the FLAG sequence at the 3' end of the R7 gag-tmos ORF, we demonstrated that R7 does synthesize a Gag-tMos fusion protein. Using R7 gag deletion mutants with and without the FLAG sequence, we further demonstrated that (i) deletion of the entire gag sequence abolished R7's transforming activity; (ii) the ability of the virus to transform cultured NIH/3T3 cells was significantly reduced only when most of gag was deleted; (iii) the ability of the virus to induce brain lesions was inversely proportional to the extent of its gag deletions; and (iv) the insertion of FLAG at the Mos C-terminus did not reduce the in vitro transforming activity of the FLAG-tagged viruses but did reduce their ability to induce brain lesions. Thus, we have demonstrated that altering the N- or C-terminus of the R7 Gag-tMos fusion protein can affect disease manifestation.

Studies on the pathology, especially brain hemorrhage and angioendotheliomas, induced by two new mos-containing viruses.

Recombinant virus 7 (R7), a spontaneous deletion mutant of SV7, which is itself a molecular clone of Moloney murine sarcoma virus 124 (MoMuSV 124), induces brain lesions and tumors of the subcutaneous tissue and spleen in all infected mice. In contrast, SV7 only induces tumors of the spleen and subcutaneous tissues. One of the genetic differences between R7 and SV7 is that R7 encodes a Gag-Mos protein whereas SV7 encodes an Env-Mos protein. To investigate whether the novel R7 gag-mos oncogene is required for brain lesion induction, two viruses (SV7d1 and SVM1) were constructed in which the R7 gag-mos sequences and the adjacent 53 bp of the 5' noncoding sequence were replaced by either the SV7 or myeloproliferative sarcoma virus (MPSV) env-mos oncogenes, respectively. Like R7, SV7d1 and SVM1 induced brain lesions and tumors in the spleen and subcutaneous tissues. A prominent component of R7-, SV7d1-, and SVM1-induced tumors of the brain, subcutaneous tissues, and spleen was the presence of abnormally enlarged cells with eccentric nuclei lining vessels, scattered singly or in small clusters. Their size, localization to the luminal surface of distended vessels, and binding to Bandeiraea simplicifolia (BS-1) lectin, an endothelial cell (EC) marker, suggest that they are most likely transformed ECs. Our findings therefore indicate that the induction of brain lesions is not limited to the expression of the R7 Gag-Mos protein. However, our findings also indicate that expression of the different forms of the Mos protein results in differences in the relative abundance of ECs in brain angioendotheliomas and subcutaneous and spleen tumors induced by these viruses.

In vivo gene transfer of a suicide gene under the transcriptional control of the carcinoembryonic antigen promoter results in bone marrow transduction but can avoid bone marrow suppression.

We constructed the CEA419/CD retrovirus vector carrying the cytosine deaminase (CD) gene directed by the carcinoembryonic antigen (CEA) promoter. pCD2 retrovirus vector carrying the CD gene directed by the retrovirus long terminal repeat promoter was also used. When mice bearing intraperitoneally disseminated colorectal carcinomas (CRCs) were infused intraperitoneally with pCD2 or CEA419/CD retrovirus-producing cells, a CD fragment was detected in CRCs and bone marrow cells. It was shown that the CD gene was expressed both in CRCs and in the bone marrow of animals infused with pCD2 retrovirus-producing cells, while the CD gene was expressed solely in CRCs of animals infused with CEA419/CD retrovirus-producing cells. These results indicate that the use of a tumor-selective promoter may warrant the safety of in vivo gene therapy using suicide genes.

Anti-tumor immunity generated by tumor cells engineered to express B7-1 via retroviral or adenoviral gene transfer.

We engineered B7-1 retroviral and adenoviral gene transfer systems and studied them in four immunogenic tumor models. M-MSV tumor cells, but not K-Balb, 38.2 and 205 tumor cells, when expressing B7-1 by retroviral transduction were rejected and conferred protection against a tumor challenge. Transient expression of B7-1 after transduction with adenoviruses was less efficient. We observed enhanced cytotoxic T-lymphocyte activity accompanied by increased secretion of IL-6, IFNgamma and GM-CSF. GM-CSF secretion correlated with tumor rejection. Enhanced IFNgamma but unchanged IL-4 secretion suggested a T-helper 1-mediated anti-tumor immune response.

Studies on the pathology, especially brain lesions, induced by R7, a spontaneous mutant of Moloney murine sarcoma virus 124.

We have recently isolated R7, a spontaneous Moloney murine sarcoma virus (MoMuSV) 124 variant. Molecular cloning and sequence analysis showed that, relative to MoMuSV 124, R7 has an extra repeat in each enhancer and a truncated mos gene in frame with the truncated gag coding sequence. This report presents a detailed study on the pathology induced by R7. R7 induced not only sarcomas with well developed angiomatous components but also brain lesions. Brain lesions were observed in all less-than-48-hour-old BALB/c mice inoculated with greater than 2 x 10(5) R7 focus-forming units (FFUs). R7 was detected in all brains examined by day 9 after inoculation, and brain lesions were observed in two of four mice examined by day 14 after inoculation. Light microscopy of brains revealed that approximately 15% of the lesions were unenclosed blood pools of varying sizes containing red blood cells and inflammatory cells spreading into surrounding brain tissues. The remainder of the brain lesions had tumor cells. These lesions ranged from a few enlarged vascular endothelial cells intermixed with blood cells to large circumscribed lesions consisting of well developed tangled masses of vessels surrounded by blood pools. Activated astrocytes surrounded and infiltrated the tumors. In addition, the thymus of R7-infected mice regressed significantly and precipitously due to apoptosis (especially of cortical thymocytes) at the end stage of the disease.

R7, a spontaneous mutant of Moloney murine sarcoma virus 124 with three direct repeats and an in-frame truncated gag-mos gene, induces brain lesions.

We have isolated Recombinant 7 (R7), a spontaneous mutant of SV7, a molecular clone of MoMuSV124. Like SV7, R7 induces subcutaneous fibrosarcomas, spleen tumors, and mesentery tumors infiltrated by proliferating vessels lined by transformed endothelial cells. However, it also induces brain lesions. We have molecularly cloned and sequenced the R7 proviral DNA and shown that the R7 genome consists of 3401 bp. It has three direct repeats in each enhancer. Its coding sequence consists of only 176 bp of p15, 263 bp of p30, a 7-bp insertion, and 853 bp of an N-terminally truncated mos gene. From the sequence of R7 we have deduced that the truncated mos sequence is in-frame with all of the gag sequence and the 7-bp insertion. The incorporation of the 3' end of the p15 sequence further suggests that the R7 Gag-Mos is myristylated. We have also shown that the molecularly cloned R7 virus transformed NIH/3T3 fibroblasts about sevenfold better than the parental SV7. We have also confirmed that molecularly cloned R7 induces the same disease phenotype as that induced by the nonmolecularly cloned R7.

Utilization of nonhomologous minus-strand DNA transfer to generate recombinant retroviruses.

During reverse transcription, minus-strand DNA transfer connects the sequences located at the two ends of the viral RNA to generate a long terminal repeat. It is thought that the homology in the repeat (R) regions located at the two ends of the viral RNA sequences facilitate minus-strand DNA transfer. In this report, the effects of diminished R-region homology on DNA synthesis and virus titer were examined. A retrovirus vector, PY31, was constructed to contain the 5' and 3' cis-acting elements from Moloney murine sarcoma virus and spleen necrosis virus. These two viruses are genetically distinct, and the two R regions contain little homology. In one round of replication, the PY31 titer was approximately 3,000-fold lower than that of a control vector with highly homologous R regions. The molecular characteristics of the junctions of minus-strand DNA transfer were analyzed in both unintegrated DNA and integrated proviruses. Short stretches of homology were found at the transfer junctions and were likely to be used to facilitate minus-strand DNA transfer. Both minus-strand strong-stop DNA and weak-stop DNA were observed to mediate strand transfer. The ability of PY31 to complete reverse transcription indicates that minus-strand DNA transfer can be used to join sequences from two different viruses to form recombinant viruses. These results suggest the provocative possibility that genetically distinct viruses can interact through this mechanism.

Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability.

Constitutive activation of mitogen-activated protein kinase (MAPK) is a property common to many oncoproteins, including Mos, Ras, and Raf, and is essential for their transforming activities. We have shown that high levels of expression of the Mos/MAPK pathway in Swiss 3T3 fibroblast cause cells in S phase to undergo apoptosis, while cells in G1 irreversibly growth arrest. Interestingly, cells in G2 and M phases also arrest at a G1-like checkpoint after proceeding through mitosis. These cells fail to undergo cytokinesis and are binucleated. Thus, constitutive overexpression of Mos and MAPK cannot be tolerated, and fibroblasts transformed by Mos express only low levels of the mos oncogene product. Here, we show that p53 plays a key role in preventing oncogene-mediated activation of MAPK. In the absence of p53 (p53-/-), the growth arrest normally observed in wild-type p53 (p53+/+) mouse embryo fibroblasts (MEFs) is markedly reduced. The mos transformation efficiency in p53-/- MEFs is two to three orders of magnitude higher than that in p53+/+ cells, and p53-/- cells tolerate > 10-fold higher levels of both Mos and activated MAPK. Moreover, we show that, like Mos, both v-ras and v-raf oncogene products induce apoptosis in p53+/+ MEFs. These oncogenes also display a high transforming activity in p53-/- MEFs, as does a gain-of-function MAPK kinase mutant (MEK*). Thus, the p53-dependent checkpoint pathway is responsive to oncogene-mediated MAPK activation in inducing irreversible G1 growth arrest and apoptosis. Moreover, we show that the chromosome instability induced by the loss of p53 is greatly enhanced by the constitutive activation of the Mos/MAPK pathway.

The Moloney murine sarcoma virus ts110 5' splice site signal contributes to the regulation of splicing efficiency and thermosensitivity.

The 5' splice site signal (5'ss) in Moloney murine sarcoma virus ts110 (MuSVts110) RNA was found to participate in the regulation of its splicing phenotype. This 5'ss (CAG/GUAGGA) departs from the mammalian consensus (CAG/GURAGU) at positions +4 and +6, both of which base pair with U1 and U6 small nuclear RNAs during splicing. A doubling in splicing efficiency and near elimination of the splicing thermosensitivity characteristic of MuSVts110 were observed in 5'ss mutants containing a U at position +6 (termed 5' A6U), even in those in which U1-5'ss complementarity had been reduced. At the permissive temperature (28 degrees C), the 5' A6U mutation increased the efficiency of the second splicing reaction, while at the nonpermissive temperature (39 degrees C), both splicing reactions were positively affected.

Viral interference during simultaneous transduction with two independent helper-free retroviral vectors.

The ability to stably transduce a single cell with two independent retroviral vectors would have distinct advantages for gene therapy. We determined that cells can be transduced with two distinct retroviral vectors and have quantitated transduction efficiencies in cells infected sequentially and simultaneously. Two amphotropic, helper virus-free, retroviral vectors, a murine Moloney sarcoma virus-based vector containing the nuclear beta-galactosidase and neomycin resistance genes (MMSVn beta-gal/neoR) and a Harvey virus-derived vector containing the human multidrug resistance gene (HaMDR) were introduced into NIH-3T3 cells, pig keratinocytes, and primary pig fibroblasts simultaneously and sequentially. Analytical flow cytometry was utilized to determine retroviral transduction efficiency by assessing the percentage of cells transduced by either one or both retroviruses, in the absence of selection. Simultaneous retroviral transductions were infrequent events. In addition, transduction of previously infected cells (sequential transductions) occurred at lower than expected frequencies. Our data suggest that there is quantifiable viral interference in sequential retroviral transductions. This interference occurs by a mechanism that appears to be independent of the amphotropic retroviral receptor. Thus, such dual transductions will likely require in vitro selection or the use of a single retrovirus which contains both desired genes on the same genome.

Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.