To cleave or not to cleave: effects of cleavage site mutations on M-PMV reverse transcriptase activity and viral infectivity

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60461373%3A22810%2F25%3A43932654" target="_blank" >RIV/60461373:22810/25:43932654 - isvavai.cz</a>

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RIV/60461373:22330/25:43932654

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

To cleave or not to cleave: effects of cleavage site mutations on M-PMV reverse transcriptase activity and viral infectivity

Popis výsledku v původním jazyce

Reverse transcriptase (RT) plays a central role in retroviral replication by converting viral RNA into DNA. While RTs from most betaretroviruses are monomeric, the RT of Mason‑Pfizer monkey virus (M‑PMV) appears to form a heterodimer, resembling the lentiviral RT of HIV‑1. In M‑PMV, the RT is initially translated as a part of a Gag‑Pro‑Pol polyprotein and during maturation it undergoes proteolytic cleavage not only at its N‑ and C‑terminus, as well as between the polymerase and RNase H domains. This internal cleavage occurs in only one subunit, yielding a functional heterodimer. To investigate the role of precise proteolytic processing between the polymerase and RNase H domains, we introduced point mutations into the reverse transcriptase cleavage site. HEK293 cells were transfected with these mutant constructs, and viral particles were harvested. RT activity was quantified using a qPCR‑based assay, protein processing was analyzed by western blotting, and virion infectivity was assessed by measuring GFP fluorescence in target cells using flow cytometry.Although none of the mutations completely blocked the internal cleavage, two appeared to shift the cleavage site, as indicated by changes in the molecular weights of the polymerase and RNase H fragments. One mutation resulted in the complete loss of enzymatic activity, despite RT expression, while other mutations caused a partial reduction in RT activity and infectivity. Altogether these data suggest that the accuracy of the internal cleavage is critical for RT function.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

To cleave or not to cleave: effects of cleavage site mutations on M-PMV reverse transcriptase activity and viral infectivity

Popis výsledku anglicky

Reverse transcriptase (RT) plays a central role in retroviral replication by converting viral RNA into DNA. While RTs from most betaretroviruses are monomeric, the RT of Mason‑Pfizer monkey virus (M‑PMV) appears to form a heterodimer, resembling the lentiviral RT of HIV‑1. In M‑PMV, the RT is initially translated as a part of a Gag‑Pro‑Pol polyprotein and during maturation it undergoes proteolytic cleavage not only at its N‑ and C‑terminus, as well as between the polymerase and RNase H domains. This internal cleavage occurs in only one subunit, yielding a functional heterodimer. To investigate the role of precise proteolytic processing between the polymerase and RNase H domains, we introduced point mutations into the reverse transcriptase cleavage site. HEK293 cells were transfected with these mutant constructs, and viral particles were harvested. RT activity was quantified using a qPCR‑based assay, protein processing was analyzed by western blotting, and virion infectivity was assessed by measuring GFP fluorescence in target cells using flow cytometry.Although none of the mutations completely blocked the internal cleavage, two appeared to shift the cleavage site, as indicated by changes in the molecular weights of the polymerase and RNase H fragments. One mutation resulted in the complete loss of enzymatic activity, despite RT expression, while other mutations caused a partial reduction in RT activity and infectivity. Altogether these data suggest that the accuracy of the internal cleavage is critical for RT function.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