Insights into the structural properties of M-PMV reverse transcriptase

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60461373%3A22330%2F25%3A43932640" target="_blank" >RIV/60461373:22330/25:43932640 - isvavai.cz</a>

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Insights into the structural properties of M-PMV reverse transcriptase

Popis výsledku v původním jazyce

Background: Reverse transcriptase (RT) is a key enzyme in retroviral replication, catalyzing the synthesis of DNA from an RNA template. While the RT of HIV-1, a lentivirus, has been well-characterized as a heterodimer, the RT of Mason-Pfizer monkey virus (M-PMV), a betaretrovirus, remains poorly understood. Unlike other betaretroviral RTs, which typically exist as monomers, evidence suggests that M-PMV RT may adopt a heterodimeric structure similar to HIV-1 RT. No structural data has yet been reported for RT M-PMV.Objectives: This study aims to characterize the RT of M-PMV, determine its structural properties, and provide insights into its enzymatic activity.Methods: We expressed and purified M-PMV RT from Sf9 insect cells. To identify N-terminus of M-PMV RT truncated form, proteolytic cleavage performed using M-PMV protease, followed by N-terminal sequence analysis and electrospray ionization mass spectrometry (ESI-MS) was used. The enzymatic activity of cleaved and uncleaved RT was evaluated using quantitative PCR (qPCR). Additionally, crosslinking experiments were conducted to examine the oligomerization of RT.Results: Proteolytic cleavage revealed that approximately 50% of RT was processed at the RT/RNase H site. ESI-MS analysis identified the precise cleavage site, and both cleavage products were found to have identical N-terminal sequences. Cleaved RT exhibited higher activity compared to the uncleaved enzyme. Crosslinking experiments detected a 115 kDa band on the western blot, corresponding to a heterodimeric RT complex.Conclusions: Our results suggest that M-PMV RT predominantly adopts a heterodimeric form, based on indirect evidence.This work was supported by The project National Institute of virology and bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) - Funded by the European Union - Next Generation EU.

Název v anglickém jazyce

Insights into the structural properties of M-PMV reverse transcriptase

Popis výsledku anglicky

Background: Reverse transcriptase (RT) is a key enzyme in retroviral replication, catalyzing the synthesis of DNA from an RNA template. While the RT of HIV-1, a lentivirus, has been well-characterized as a heterodimer, the RT of Mason-Pfizer monkey virus (M-PMV), a betaretrovirus, remains poorly understood. Unlike other betaretroviral RTs, which typically exist as monomers, evidence suggests that M-PMV RT may adopt a heterodimeric structure similar to HIV-1 RT. No structural data has yet been reported for RT M-PMV.Objectives: This study aims to characterize the RT of M-PMV, determine its structural properties, and provide insights into its enzymatic activity.Methods: We expressed and purified M-PMV RT from Sf9 insect cells. To identify N-terminus of M-PMV RT truncated form, proteolytic cleavage performed using M-PMV protease, followed by N-terminal sequence analysis and electrospray ionization mass spectrometry (ESI-MS) was used. The enzymatic activity of cleaved and uncleaved RT was evaluated using quantitative PCR (qPCR). Additionally, crosslinking experiments were conducted to examine the oligomerization of RT.Results: Proteolytic cleavage revealed that approximately 50% of RT was processed at the RT/RNase H site. ESI-MS analysis identified the precise cleavage site, and both cleavage products were found to have identical N-terminal sequences. Cleaved RT exhibited higher activity compared to the uncleaved enzyme. Crosslinking experiments detected a 115 kDa band on the western blot, corresponding to a heterodimeric RT complex.Conclusions: Our results suggest that M-PMV RT predominantly adopts a heterodimeric form, based on indirect evidence.This work was supported by The project National Institute of virology and bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) - Funded by the European Union - Next Generation EU.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10607 - Virology

Projekt

<a href="/cs/project/LX22NPO5103" target="_blank" >LX22NPO5103: Národní institut virologie a bakteriologie</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2025

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů