Summarising the study, RSV is an important pathogen that causes oncogenic transformation in its host via the action of a protein kinase that it expresses. The RSV genome is reverse-transcribed into its complementary DNA, which then integrates into the host genome. This DNA thereafter serves as a template for transcription to manufacture viral proteins. The viral life cycle can, therefore, be inhibited if the functional elements of this DNA are altered. In this aspect, G4s may play an important role due to their involvement in hijacking the host machinery. Interestingly, the RSV-DNA contains multiple probable G4 forming elements, among which the sequences with the highest G4 forming propensity are located within the GAG and POL genes. Additionally, a sequence within the SRC oncogene also has G4 forming potential. In this study, we verified the G4 formation in these sequences via various biophysical assays. Further, the structural topology of these G4s has also been studied using computational and biophysical methods. We have established that GG4 forms a parallel G4 structure while PG4 and SG4 form highly dynamic G4s, switching between various structural forms. Such molecular switching behaviour may also aid in the functional properties of these G4s in vivo. However, further studies are required to elucidate the functional properties of these elements. We have also analysed the binding of these G4s to specific small-molecule ligands and the structural changes induced by the binding of Braco-19 on the G4s. Finally, we have observed that the G4 forming sequences in the RSV-DNA are recognised and bound by human nucleolin, which is highly similar in structure to the chicken nucleolin. This suggests that the G4s in the RSV-DNA may be implicated in various biological functions. These studies conclude that G4s are formed in the RSV-DNA at multiple locations, and these G4s show molecular switching properties under physiological conditions. Further, these G4s are also bound by small-molecule ligands and proteins, which induce structural changes. Thus, these G4s may be targetable sites for the control of RSV infection.
- MeSH
- antivirové látky * farmakologie chemie MeSH
- DNA virů * chemie genetika metabolismus MeSH
- G-kvadruplexy * MeSH
- genom virový * MeSH
- lidé MeSH
- virus Rousova sarkomu * genetika účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antivirové látky * MeSH
- DNA virů * MeSH
The extent of virus transmission among individuals and species is generally determined by the presence of specific membrane-embedded virus receptors required for virus entry. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor is the first and crucial step in determining host specificity. Using a well-established retroviral model-avian Rous sarcoma virus (RSV)-we analyzed changes in an RSV variant that had repeatedly been able to infect rodents. By envelope gene (env) sequencing, we identified eight mutations that do not match the already described mutations influencing the host range. Two of these mutations-one at the beginning (D32G) of the surface Env subunit (SU) and the other at the end of the fusion peptide region (L378S)-were found to be of critical importance, ensuring transmission to rodent, human, and chicken cells lacking the appropriate receptor. Furthermore, we carried out assays to examine the virus entry mechanism and concluded that these two mutations cause conformational changes in the Env variant and that these changes lead to an activated, or primed, state of Env (normally induced after Env interaction with the receptor). In summary, our results indicate that retroviral host range extension is caused by spontaneous Env activation, which circumvents the need for original cell receptor. This activation is, in turn, caused by mutations in various env regions.
- Klíčová slova
- Rous sarcoma virus, envelope glycoprotein, receptor-independent entry, retrovirus, virus entry,
- MeSH
- genetické vektory * genetika metabolismus MeSH
- genové produkty env * genetika metabolismus MeSH
- krysa rodu Rattus MeSH
- kur domácí MeSH
- lidé MeSH
- missense mutace * MeSH
- nádorové buněčné linie MeSH
- substituce aminokyselin MeSH
- transdukce genetická * MeSH
- virus Rousova sarkomu * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- genové produkty env * MeSH
Systems of antigen delivery into antigen-presenting cells represent an important novel strategy in chicken vaccine development. In this study, we verified the ability of Rous sarcoma virus (RSV) antigens fused with streptavidin to be targeted by specific biotinylated monoclonal antibody (anti-CD205) into dendritic cells and induce virus-specific protective immunity. The method was tested in four congenic lines of chickens that are either resistant or susceptible to the progressive growth of RSV-induced tumors. Our analyses confirmed that the biot-anti-CD205-SA-FITC complex was internalized by chicken splenocytes. In the cytokine expression profile, several significant differences were evident between RSV-challenged progressor and regressor chicken lines. A significant up-regulation of IL-2, IL-12, IL-15, and IL-18 expression was detected in immunized chickens of both regressor and progressor groups. Of these cytokines, IL-2 and IL-12 were most up-regulated 14 days post-challenge (dpc), while IL-15 and IL-18 were most up-regulated at 28 dpc. On the contrary, IL-10 expression was significantly down-regulated in all immunized groups of progressor chickens at 14 dpc. We detected significant up-regulation of IL-17 in the group of immunized progressors. LITAF down-regulation with iNOS up-regulation was especially observed in the progressor group of immunized chickens that developed large tumors. Based on the increased expression of cytokines specific for activated dendritic cells, we conclude that our system is able to induce partial stimulation of specific cell types involved in cell-mediated immunity.
- MeSH
- antigeny virové imunologie MeSH
- buněčná imunita imunologie MeSH
- CD antigeny imunologie MeSH
- cytokiny fyziologie MeSH
- dendritické buňky imunologie virologie MeSH
- kur domácí imunologie virologie MeSH
- lektiny typu C imunologie MeSH
- protilátky bispecifické imunologie MeSH
- ptačí sarkom imunologie prevence a kontrola MeSH
- receptory buněčného povrchu imunologie MeSH
- vedlejší histokompatibilní antigeny imunologie MeSH
- virové vakcíny imunologie MeSH
- virus Rousova sarkomu imunologie MeSH
- zvířata kongenní imunologie virologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antigeny virové MeSH
- CD antigeny MeSH
- cytokiny MeSH
- DEC-205 receptor MeSH Prohlížeč
- lektiny typu C MeSH
- protilátky bispecifické MeSH
- receptory buněčného povrchu MeSH
- vedlejší histokompatibilní antigeny MeSH
- virové vakcíny MeSH
This article summarizes the essential steps in understanding the chicken Rous sarcoma virus (RSV) genome association with a nonpermissive rodent host cell genome. This insight was made possible by in-depth study of RSV-transformed rat XC cells, which were called virogenic because they indefinitely carry virus genetic information in the absence of any infectious virus production. However, the virus was rescued by association of XC cells with chicken fibroblasts, allowing cell fusion between both partners. This and additional studies led to the interpretation that the RSV genome gets integrated into the host cell genome as a provirus. Study of additional rodent virogenic cell lines provided evidence that the transcript of oncogene v-src can be transmitted to other retroviruses and produce cell transformation by itself. As discussed in the text, two main questions related to nonpermissiveness to retrovirus infection remain to be solved. The first is changes in the retrovirus envelope gene allowing virus entry into a nonpermissive cell. The second is the nature of the permissive cell functions required by the nonpermissive cell to ensure infectious virus production. Both lines of investigation are being pursued.
- Klíčová slova
- cell transformation, nonpermissiveness to virus infection, virus integration, virus rescue,
- MeSH
- buněčné linie MeSH
- fúze buněk * MeSH
- genom virový genetika MeSH
- genové produkty env genetika MeSH
- krysa rodu Rattus MeSH
- kur domácí virologie MeSH
- onkogenní protein pp60(v-src) genetika MeSH
- proviry genetika růst a vývoj MeSH
- virová transformace buněk MeSH
- virus Rousova sarkomu genetika růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- genové produkty env MeSH
- onkogenní protein pp60(v-src) MeSH
In my article I tried to present the results of early experiments suggesting a significant role for cell association in Rous sarcoma virus transformation of non-permissive cells and revealing that infectious virus can be efficiently rescued from such cells by their fusion with permissive chicken fibroblasts.
- MeSH
- krysa rodu Rattus MeSH
- kur domácí virologie MeSH
- proviry patogenita fyziologie MeSH
- ptačí sarkom virologie MeSH
- replikace viru MeSH
- virová transformace buněk MeSH
- virus Rousova sarkomu patogenita fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
UNLABELLED: Transformation of rodent cells with avian Rous sarcoma virus (RSV) opened new ways to studying virus integration and expression in nonpermissive cells. We were interested in (i) the molecular changes accompanying fusion of RSV-transformed mammalian cells with avian cells leading to virus rescue and (ii) enhancement of this process by retroviral gene products. The RSV-transformed hamster RSCh cell line was characterized as producing only a marginal amount of env mRNA, no envelope glycoprotein, and a small amount of unprocessed Gag protein. Egress of viral unspliced genomic RNA from the nucleus was hampered, and its stability decreased. Cell fusion of the chicken DF-1 cell line with RSCh cells led to production of env mRNA, envelope glycoprotein, and processed Gag and virus-like particle formation. Proteosynthesis inhibition in DF-1 cells suppressed steps leading to virus rescue. Furthermore, new aberrantly spliced env mRNA species were found in the RSCh cells. Finally, we demonstrated that virus rescue efficiency can be significantly increased by complementation with the env gene and the highly expressed gag gene and can be increased the most by a helper virus infection. In summary, Env and Gag synthesis is increased after RSV-transformed hamster cell fusion with chicken fibroblasts, and both proteins provided in trans enhance RSV rescue. We conclude that the chicken fibroblast yields some factor(s) needed for RSV replication, particularly Env and Gag synthesis, in nonpermissive rodent cells. IMPORTANCE: One of the important issues in retrovirus heterotransmission is related to cellular factors that prevent virus replication. Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, is able to infect and transform mammalian cells; however, such transformed cells do not produce infectious virus particles. Using the well-defined model of RSV-transformed rodent cells, we established that the lack of virus replication is due to the absence of chicken factor(s), which can be supplemented by cell fusion. Cell fusion with permissive chicken cells led to an increase in RNA splicing and nuclear export of specific viral mRNAs, as well as synthesis of respective viral proteins and production of virus-like particles. RSV rescue by cell fusion can be potentiated by in trans expression of viral genes in chicken cells. We conclude that rodent cells lack some chicken factor(s) required for proper viral RNA processing and viral protein synthesis.
- MeSH
- fúze buněk MeSH
- genové produkty env genetika metabolismus MeSH
- genové produkty gag genetika metabolismus MeSH
- křečci praví MeSH
- kur domácí MeSH
- nemoci drůbeže virologie MeSH
- ptačí sarkom virologie MeSH
- testy genetické komplementace MeSH
- transformované buněčné linie MeSH
- virová transformace buněk MeSH
- virus Rousova sarkomu genetika fyziologie MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- genové produkty env MeSH
- genové produkty gag MeSH
- MeSH
- dějiny 20. století MeSH
- dějiny 21. století MeSH
- geny src * MeSH
- heterografty MeSH
- integrace viru MeSH
- krysa rodu Rattus MeSH
- kur domácí virologie MeSH
- nádorové buněčné linie MeSH
- nemoci drůbeže dějiny virologie MeSH
- periodika jako téma dějiny MeSH
- proviry genetika fyziologie MeSH
- ptačí sarkom dějiny virologie MeSH
- transplantace nádorů MeSH
- virová transformace buněk MeSH
- virus Rousova sarkomu genetika izolace a purifikace fyziologie MeSH
- zvířata MeSH
- Check Tag
- dějiny 20. století MeSH
- dějiny 21. století MeSH
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- autobiografie MeSH
- biografie MeSH
- historické články MeSH
- úvodníky MeSH
- Geografické názvy
- Československo MeSH
- Maryland MeSH
- O autorovi
- Svoboda, Jan
- Rous, Francis Peyton
Most retroviruses employ a frameshift mechanism during polyprotein synthesis to balance appropriate ratios of structural proteins and enzymes. To investigate the requirements for individual precursors in retrovirus assembly, we modified the polyprotein repertoire of Mason-Pfizer monkey virus (M-PMV) by mutating the frameshift sites to imitate the polyprotein organization of Rous sarcoma virus (Gag-Pro and Gag-Pro-Pol) or Human immunodeficiency virus (Gag and Gag-Pro-Pol). For the "Rous-like" virus, assembly was impaired with no incorporation of Gag-Pro-Pol into particles and for the "HIV-like" virus an altered morphogenesis was observed. A mutant expressing Gag and Gag-Pro polyproteins and lacking Gag-Pro-Pol assembled intracellular particles at a level similar to the wild-type. Gag-Pro-Pol polyprotein alone neither formed immature particles nor processed the precursor. All the mutants were non-infectious except the "HIV-like", which retained fractional infectivity.
- MeSH
- AIDS opičí virologie MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- genové produkty gag genetika MeSH
- genové produkty pol genetika MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus genetika patogenita MeSH
- messenger RNA genetika MeSH
- posunová mutace MeSH
- proteosyntéza MeSH
- RNA virová genetika MeSH
- transfekce MeSH
- virion genetika patogenita MeSH
- virové proteiny genetika MeSH
- virus Rousova sarkomu genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- genové produkty gag MeSH
- genové produkty pol MeSH
- messenger RNA MeSH
- RNA virová MeSH
- virové proteiny MeSH
This chapter provides a personal insight into the scientific and social atmosphere in former Czechoslovakia. It covers the period of the rise of Hasek's immunologic school and application of immunologic tolerance to Rous sarcoma virus (RSV) heterotransmission. These approaches permitted establishment of a new model of mammalian cells transformed by RSV (virogenic XC cells), where the noninfectious viral genome was kept indefinitely as new genetic information (provirus). RSV was rescued from nonpermissive mammalian cells by fusion (complementation) with permissive chicken fibroblasts; this opened the way to understanding virus nonpermissiveness. Mammalian cells transformed by the reverse transcript of v-src mRNA were characterized, and the resulting provirus was shown to be highly oncogenic for chickens and to carry tumor-specific transplantation antigen. Other areas covering epigenetic reversion of RSV-transformed cells and long-term persistence of chicken leucosis viruses in foreign avian species are discussed.
- MeSH
- dějiny 20. století MeSH
- geny src MeSH
- integrace viru MeSH
- lékařská onkologie dějiny MeSH
- onkogenní viry fyziologie MeSH
- virová transformace buněk MeSH
- virus Rousova sarkomu fyziologie MeSH
- výzkum * MeSH
- zvířata MeSH
- Check Tag
- dějiny 20. století MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- historické články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Československo MeSH
Unmethylated CpG islands are known to keep adjacent promoters transcriptionally active. In the CpG island adjacent to the adenosine phosphoribosyltransferase gene, the protection against transcriptional silencing can be attributed to the short CpG-rich core element containing Sp1 binding sites. We report here the insertion of this CpG island core element, IE, into the long terminal repeat of a retroviral vector derived from Rous sarcoma virus, which normally suffers from progressive transcriptional silencing in mammalian cells. IE insertion into a specific position between enhancer and promoter sequences led to efficient protection of the integrated vector from silencing and gradual CpG methylation in rodent and human cells. Individual cell clones with IE-modified reporter vectors display high levels of reporter expression for a sustained period and without substantial variegation in the cell culture. The presence of Sp1 binding sites is important for the protective effect of IE, but at least some part of the entire antisilencing capacity is maintained in IE with mutated Sp1 sites. We suggest that this strategy of antisilencing protection by the CpG island core element may prove generally useful in retroviral vectors.
- MeSH
- biologické modely MeSH
- CpG ostrůvky * MeSH
- genetická transkripce * MeSH
- koncové repetice MeSH
- lidé MeSH
- mutace MeSH
- průtoková cytometrie MeSH
- ptačí sarkom genetika virologie MeSH
- ptáci MeSH
- reportérové geny MeSH
- transkripční faktor Sp1 metabolismus MeSH
- umlčování genů * MeSH
- vazebná místa MeSH
- virus ptačí leukózy metabolismus MeSH
- virus Rousova sarkomu metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- transkripční faktor Sp1 MeSH
