Prion diseases have been observed to deregulate the transcription of erythroid genes, and prion protein knockout mice have demonstrated a diminished response to experimental anemia. To investigate the role of the cellular prion protein (PrP(C)) in erythropoiesis, we studied the protein's expression on mouse erythroid precursors in vivo and utilized an in vitro model of the erythroid differentiation of murine erythroleukemia cells (MEL) to evaluate the effect of silencing PrP(C) through RNA interference.The expression of PrP(C) and selected differentiation markers was analyzed by quantitative multicolor flow cytometry, western blot analysis and quantitative RT-PCR. The silencing of PrP(C) expression in MEL cells was achieved by expression of shRNAmir from an integrated retroviral vector genome. The initial upregulation of PrP(C) expression in differentiating erythroid precursors was detected both in vivo and in vitro, suggesting PrP(C)'s importance to the early stages of differentiation. The upregulation was highest on early erythroblasts (16200±3700 PrP(C) / cell) and was followed by the gradual decrease of PrP(C) level with the precursor's maturation reaching 470±230 PrP(C) / cell on most mature CD71(-)Ter119(+) small precursors. Interestingly, the downregulation of PrP(C) protein with maturation of MEL cells was not accompanied by the decrease of PrP mRNA. The stable expression of anti-Prnp shRNAmir in MEL cells led to the efficient (>80%) silencing of PrP(C) levels. Cell growth, viability, hemoglobin production and the transcription of selected differentiation markers were not affected by the downregulation of PrP(C).In conclusion, the regulation of PrP(C) expression in differentiating MEL cells mimics the pattern detected on mouse erythroid precursors in vivo. Decrease of PrP(C) protein expression during MEL cell maturation is not regulated on transcriptional level. The efficient silencing of PrP(C) levels, despite not affecting MEL cell differentiation, enables created MEL lines to be used for studies of PrP(C) cellular function.

• MeSH
• akutní erytroblastická leukemie genetika   patologie   MeSH
• buněčná diferenciace genetika   MeSH
• buňky kostní dřeně cytologie   MeSH
• down regulace MeSH
• erytroidní prekurzorové buňky cytologie   metabolismus   MeSH
• genetické vektory genetika   MeSH
• hemoglobiny biosyntéza   MeSH
• malá interferující RNA genetika   MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• priony genetika   MeSH
• regulace genové exprese u nádorů * MeSH
• Retroviridae genetika   MeSH
• RNA interference MeSH
• slezina cytologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hemoglobiny MeSH
• malá interferující RNA MeSH
• messenger RNA MeSH
• priony MeSH

The high-resolution structure of the N-terminal domain (NTD) of the retroviral capsid protein (CA) of Mason-Pfizer monkey virus (M-PMV), a member of the betaretrovirus family, has been determined by NMR. The M-PMV NTD CA structure is similar to the other retroviral capsid structures and is characterized by a six alpha-helix bundle and an N-terminal beta-hairpin, stabilized by an interaction of highly conserved residues, Pro1 and Asp57. Since the role of the beta-hairpin has been shown to be critical for formation of infectious viral core, we also investigated the functional role of M-PMV beta-hairpin in two mutants (i.e., DeltaP1NTDCA and D57ANTDCA) where the salt bridge stabilizing the wild-type structure was disrupted. NMR data obtained for these mutants were compared with those obtained for the wild type. The main structural changes were observed within the beta-hairpin structure; within helices 2, 3, and 5; and in the loop connecting helices 2 and 3. This observation is supported by biochemical data showing different cleavage patterns of the wild-type and the mutated capsid-nucleocapsid fusion protein (CANC) by M-PMV protease. Despite these structural changes, the mutants with disrupted salt bridge are still able to assemble into immature, spherical particles. This confirms that the mutual interaction and topology within the beta-hairpin and helix 3 might correlate with the changes in interaction between immature and mature lattices.

• MeSH
• Masonův-Pfizerův opičí virus chemie   MeSH
• molekulární modely MeSH
• mutantní proteiny chemie   MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• sekvenční delece MeSH
• substituce aminokyselin MeSH
• terciární struktura proteinů MeSH
• virové plášťové proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• srovnávací studie MeSH
• Názvy látek
• mutantní proteiny MeSH
• virové plášťové proteiny MeSH

Retroviral capsid protein (CA) mediates protein interactions driving the assembly of both immature viral particles and the core of the mature virions. Structurally conserved N-terminal domains of several retroviruses refold after proteolytic cleavage into a beta-hairpin, stabilized by a salt bridge between conserved N-terminal Pro and Asp residues. Based on comparison with other retroviral CA, we identified Asp50 and Asp57 as putative interacting partners for Pro1 in Mason-Pfizer monkey virus (M-PMV) CA. To investigate the importance of CA Pro1 and its interacting Asp in M-PMV core assembly and infectivity, P1A, P1Y, D50A, T54A and D57A mutations were introduced into M-PMV. The P1A and D57A mutations partially blocked Gag processing and the released viral particles exhibited aberrant cores and were non-infectious. These data indicate that the region spanning residues Asp50-Asp57 plays an important role in stabilization of the beta-hairpin and that Asp57 likely forms a salt-bridge with P1 in M-PMV CA.

• MeSH
• bodová mutace * MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   MeSH
• sestavení viru genetika   fyziologie   MeSH
• virion genetika   metabolismus   MeSH
• virové plášťové proteiny chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• virové plášťové proteiny MeSH