Letter: Evidence for non-repetitive subunits in the genome of Rous sarcoma virus

The complexity of Rous sarcoma virus RNA has been determined using molecular hybridization. Relative to poliovirus RNA, the complexity of Rous sarcoma virus is 9·3 × 10⁶ daltons, a value close to its physically-determined molecular weight of about 10⁷. Our interpretation is that the 35 S RNA subunits of the 70 S virus genome are non-repetitive, that is, each possesses a unique nucleotide sequence, although a limited amount of redundancy cannot be excluded.     

A method for infection of cultured myogenic cells with rous sarcoma virus using polybrene

Summary A method for efficiently infecting primary myogenic cultures with a temperature-sensitive variant of the Prague strain of Rous sarcoma virus (RSV, tsLA24) to obtain a high yield of transformed myogenic cells is reported. It incorporates the use of an amorphous polymer of polycations, Polybrene, to enhance the absorption of the virus by the muscle cells. In addition, other steps which were shown to be important were a) to allow cell attachment before infection, b) to infect at 35° C in low protein medium, c) to use a density of 1 to 1.5×10⁶ cells/60-mm dish, d) gentle agitation during infection, and e) to minimize the number of passages after infection. The use of the temperature-sensitive virus provided a means of confirming the presence of myogenic cells in transformed cultures. When infected cells were maintained at 35° C (the permissive temperature for virus activity) they exhibited the characteristics of transformed cells. These characteristics included altered cell morphology, the absence of contact-inhibited growth, growth in semisolid medium, and expression of thesrc oncogene. In contrast, not expresssrc and showed normal myogenic development and ultimately formed myotubes. This work was supported by grants from the Australian Research Grants Committee and the Cancer Foundation of Western Australia.     

Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis

To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 degrees C (nonpermissive) to 32 degrees C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of approximately 40kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.     

Defective and nondefective adenovirus vectors for expressing foreign genes in vitro and in vivo.

We have constructed recombinant adenoviruses (Ad), with functional or defective E1a genes, which harbor either the hepatitis B (HB) virus s gene encoding the HB surface antigen, as well as the pre-S2 epitopes, or the bacterial gene encoding chloramphenicol acetyltransferase (CAT) under control of the Ad major late promoter (MLP). The recombinant viruses defective for E1a (Ad.MLP.S2 and Ad.CAT), which can be efficiently propagated only on 293 cells that complement this defect, and the nondefective (Ad.MLP.S2.E1A) recombinant were used to infect a wide spectrum of cells of different origin. The yields of HBs and CAT proteins obtained with these different recombinant viruses demonstrate no real advantage to using nondefective vectors, whatever the cell type infected. The injection into chimpanzees of Ad.MLP.S2 does not elicit the production of antibodies, but can immunologically prime the animals, resulting in a partial protection against HBV challenge.     

Robust and ubiquitous GFP expression in a single generation of chicken embryos using the avian retroviral vector,RCASBP

Functional genomics in avian models has lagged behind that of mammals, and the production of transgenic birds has proven to be challenging and time-consuming. All current methods rely upon breeding chimeric birds through at least one generation. Here, we report a rapid method for the ubiquitous expression of GFP in chicken embryos in a single generation (G-0), using the avian retroviral vector, Replication-Competent Avian sarcoma-leukosis virus, with a Splice acceptor, Bryan RSV Pol (RCASBP). High-titre RCASBP retrovirus carrying eGFP was injected into unincubated (stage X) blastoderms in ovo. This resulted in stable and widespread expression of eGFP throughout development in a very high proportion of embryos. Transgenic tissues were identified by fluorescence and immunohistochemistry. These results indicate that chicken blastodermal cells are permissive for infection by the RCASBP virus. This system represents a rapid and efficient method of producing global gene expression in the chicken embryo. The method can be used to generate avian cells with a stable genetic marker, or to induce global expression of a gene of choice. Interestingly, in day 8.5 embryos, somatic cells the embryonic gonads were predominantly GFP positive but primordial germ cells were GFP negative, indicating viral silencing in the embryonic germline. This dichotomy in the gonads allows the isolation or enrichment of the germ cells through negative selection during embryonic stages. This transgenic chicken model is of value in developmental studies, and for the isolation and study of avian primordial germ cells.     

A molecular switch required for retrovirus assembly participates in the hexagonal immature lattice

In the Rous sarcoma virus (RSV) Gag protein, the 25 amino-acid residues of the p10 domain immediately upstream of the CA domain are essential for immature particle formation. We performed systematic mutagenesis on this region and found excellent correlation between the amino-acid side chains required for in vitro assembly and those that participate in the p10-CA dimer interface in a previously described crystal structure. We introduced exogenous cysteine residues that were predicted to form disulphide bonds across the dimer interface. Upon oxidation of immature particles, a disulphide-linked Gag hexamer was formed, implying that p10 participates in and stabilizes the immature Gag hexamer. This is the first example of a critical interaction between two different Gag domains. Molecular modeling of the RSV immature hexamer indicates that the N-terminal domains of CA must expand relative to the murine leukaemia virus mature hexamer to accommodate the p10 contact; this expansion is strikingly similar to recent cryotomography results for immature human immunodeficiency virus particles.     

Analysis of the cellular heterogeneity in the basal layer of mouse ear epidermis: an approach from partial decomposition in vitro and retroviral cell marking in vivo

In the thin epidermis, the existence of epidermal proliferation units was hypothesized. Each unit is supposed to be partitioned into each column of polygonal-shaped cornified plates, estimated to contain a central stem cell in its basal layer. We attempted to verify this hypothesis in vitro by analyzing the partially decomposed fragment of mouse ear epidermis and in vivo using retroviral cell marking. Partially decomposed fragments of the mouse ear epidermis, mostly composed of cytokeratin 14-expressing basal keratinocytes, formed multicellular colonies in vitro. They were composed of heterogeneously shaped cells, morphologically resembling the cells in each single cell-derived colony, including potential stem cells with great proliferative potency in vitro. The estimated frequency of the candidates of stem cells in the fragments was much lower than the prediction from the representative hypothesis. Retroviral cell marking with nuclear localizing LacZ protein in vivo suggested the existence of a large clonal cellular unit for epidermal renewal. From these in vitro and in vivo observations, we propose a new model for the epidermal proliferation unit.     

Adaptation of chimeric retroviruses in vitro and in vivo: isolation of avian retroviral vectors with extended host range

We have designed and characterized two new replication-competent avian sarcoma/leukosis virus-based retroviral vectors with amphotropic and ecotropic host ranges. The amphotropic vector RCASBP-M2C(797-8), was obtained by passaging the chimeric retroviral vector RCASBP-M2C(4070A) (6) in chicken embryos. The ecotropic vector, RCASBP(Eco), was created by replacing the env-coding region in the retroviral vector RCASBP(A) with the env region from an ecotropic murine leukemia virus. It replicates efficiently in avian DFJ8 cells that express murine ecotropic receptor. For both vectors, permanent cell lines that produce viral stocks with titers of about 5 x 10(6) CFU/ml on mammalian cells can be easily established by passaging transfected avian cells. Some chimeric viruses, for example, RCASBP(Eco), replicate efficiently without modifications. For those chimeric viruses that do require modification, adaptation by passage in vitro or in vivo is a general strategy. This strategy has been used to prepare vectors with altered host range and could potentially be used to develop vectors that would be useful for targeted gene delivery.     

A transient three-plasmid expression system for the production of hepatocytes targeting retroviral vectors

Targeting of retroviral vectors to specific cells was attempted through modifying the surface protein of the murine leukemia viruses （MLVs）, but in many cases the protein function was affected, and it is difficult to achieve the targeted delivery. In this study, we have tried to engineer ecotropic Moloney murine leukemia viruses （MoMLV）-based retroviral vectors to transduce hepatocytes. A chimeric envelope （Env） expression plasmid was constructed containing the hepatitis B virus PreS2 peptide fused to aa ＋1 at the Nterminus of Env. Following simultaneous transfection of pgag-pol, pLEGFP and chimeric env plasmids into 293T cells, helper-free retrovirus stocks with the titer of approximately 104 infectious units/ml were achieved at 48 h post-transfection. These pseudotype vectors showed the normal host range of retrovirus, infecting host NIH 3T3 cells, although the efficiency was reduced compared with that of virions carrying wild-type ecotropic MoMLV envelope. In addition, the resultant pseudotype viruses could transduce human hepatoma cells mediated by polymerized human serum albumin with relatively high titers in comparison with those transductions without polymerized human serum albumin. This approach can be used to target hepatocytes selectively.     

Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer

Retroviral vectors have become an important tool for gene transfer in vitro and in vivo. Classical Moloney murine leukemia virus (MLV) based retroviral vectors have been used for over 20 years to transfer genes into dividing cells. Cell lines for production of retroviral vectors have become commonly available and modifications in retroviral vector design and use of envelope proteins have made the production of high titer, helper-free, infectious virus stocks relatively easy. More recently, lentiviral vectors, another class of retroviruses, have been modified for in vitro and in vivo gene transfer. The ability of lentiviral vectors to transduce non-dividing cells has made them especially attractive for in vivo gene transfer into differentiated, non-dividing tissues. Several improvements in helper plasmids and vectors have made lentivirus a safe vector system for ex vivo and in vivo gene transfer. This review will briefly summarize the background of these vector systems and provide some common protocols available for the preparation of MLV based retroviral vectors and HIV-1 based lentiviral vectors.     

Appropriate in vivo expression of a muscle-specific promoter by using avian retroviral vectors for gene transfer [corrected]

The promoter regions of the chicken skeletal muscle alpha-actin (alpha sk-actin) and the cytoplasmic beta-actin genes were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Replication-competent retroviral vectors were used to introduce these two actin/CAT cassettes into the chicken genome. Chickens infected with retroviruses containing the alpha sk-actin promoter expressed high levels of CAT activity in striated muscle (skeletal muscle and heart); much lower levels of CAT activity were produced in the other nonmuscle tissues. In contrast, chickens infected with retroviruses containing the beta-actin promoter linked to the CAT gene expressed low levels of CAT activity in many different tissue types and with no discernible tissue specificity. Data are presented to demonstrate that the high levels of CAT activity that were detected in the skeletal muscle of chickens infected with the retrovirus containing the alpha sk-actin promoter/CAT cassette were not due to preferential infectivity, integration, or replication of the retrovirus vector in the striated muscles of these animals.     

Close linkage of genes encoding receptors for subgroups A and C of avian sarcoma/leucosis virus on chicken chromosome 28

Avian sarcoma and leucosis viruses (ASLV) are classified into six major subgroups (A to E and J) according to the properties of the viral envelope proteins and the usage of cellular receptors for virus entry. Subgroup A and B receptors are identified molecularly and their genomic positions TVA and TVB are mapped. The subgroup C receptor is unknown, its genomic locus TVC is reported to be genetically linked to TVA, which resides on chicken chromosome 28. In this study, we used two chicken inbred lines that carry different alleles coding for resistance (TVC(R) and sensitivity (TVC(S)) to infection by subgroup C viruses. A backross population of these lines was tested for susceptibility to subgroup C infection and genotyped for markers from chicken chromosome 28. We confirmed the close linkage between TVA and TVC loci. Further, we have described the position of TVC on chromosome 28 relative to markers from the consensus map of the chicken genome.     

In vivo diversification and migrations of chick embryo heart muscle cells: a morphometric analysis with ALV- and SNV-based non-replicative vectors

 By means of a reporter gene we previously demonstrated that non-replicative Avian Leukemia Virus- and Spleen Necrosis Virus-based retroviral vectors were preferentially expressed in the heart of avian embryos from different species. Using a computer-assisted approach, we now compare clones tagged by the two types of vectors, for volume, anatomical and subanatomical localisation, number of Hoechst-stained cell nuclei and mean cell division time during the period of heart morphogenesis, i.e. from stages 17–19 to 34 of Hamburger and Hamilton (1951). This analysis demonstrates that clones labelled by the two types of viruses display similar features and bring about new insights on the relationships between mitotic and migratory properties of the myocardial cells and histogenesis of the heart. Since only exteriormost cells were tagged with our inoculation procedure, our analysis shows that: (1) at stages 17–19, the myocardium is composed of cells with diverse potentials; some cells still retain the capacity to divide extensively and participate to different heart muscle layers, whilst most are restricted in their multiplication potential and contribute to single muscle layers; (2) about half of the clones are located deep in the heart wall, revealing extensive cell migrations from the heart surface to the ventricular trabeculae, the first migrating cells tagged being detected 20 h after viral inoculation. The presence of these cells is consistent with the finding of a large number of compact trabecular clones 5 days later suggesting that these cells divide mainly after completing migration. Our approach provides new insights as well as quantitative data on the different processes involved in heart morphogenesis, namely multiplication, migration and localisation of heart muscle cells. Received: 10 March 1996 / Accepted in revised form: 7 July 1996     

Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus

Summary Prompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions, from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain.