09:00 〜 09:30
▲ [14a-3A-1] Sensing molecular events of virus in live cells
キーワード：live cell,,molecular imaging,fluorescent protein
Viruses are pathogens of many infectious diseases. Monitoring viral behaviors and their molecular events in live cells help deep understanding mechanisms of viral infections. We have constructed a series of molecular biosensors for visualization of viral behaviors in their host cells.
Molecular beacons were designed for imaging behaviors of Poliovirus and Influenza A viruses in live cells. With the biosensors, we characterized viral RNA distribution and movement patterns in the cellular region, meanwhile found that virus may induced microtubule-associated membrane rearrangement [1], and that the transport of influenza A virus mRNA, in both nucleus and cytoplasm, was energy dependent[2]. We also built an aptamer beacon to visualize endogenous proteins in living cells. Transiently expressed HIV-1 reverse transcriptase (RT) was specifically imaged[3].
Red, far-red fluorescent proteins and infrared emission proteins were selected to construct bimolecular complementary (BiFC) and trimolecular complementation (TriFC) systems. The systems feature brilliant redness, shorter maturation time, and the longer excitation and emission wavelengths. By combined use of the mCherry-based red BiFC with a Venus-based yellow BiFC system, simultaneous detection of multi protein-protein interactions and mRNA-protein interaction were realized in Vero cells[4, 5]. The results obtained helps to understand the nuclear export mechanism of influenza A viral mRNAs[5]. The mNeptune based far-red TriFC system, when applied to study HIV activation from latency, were verified as useful tools for imaging protein-protein and mRNA-protein interactions, respectively, in live cells and mice[6]. A bacterial phytochrome-based, near-infrared fluorescent protein (iRFP) was also used to construct the BiFC for visualizing interaction between HIV-1 integrase and celluar cofactor protein LEDGF/p75) in live cells and live mice[7].
The methods developed were verified as powerful tools for imaging virus-host interactions under physiological conditions in live cells and in live mice.
Molecular beacons were designed for imaging behaviors of Poliovirus and Influenza A viruses in live cells. With the biosensors, we characterized viral RNA distribution and movement patterns in the cellular region, meanwhile found that virus may induced microtubule-associated membrane rearrangement [1], and that the transport of influenza A virus mRNA, in both nucleus and cytoplasm, was energy dependent[2]. We also built an aptamer beacon to visualize endogenous proteins in living cells. Transiently expressed HIV-1 reverse transcriptase (RT) was specifically imaged[3].
Red, far-red fluorescent proteins and infrared emission proteins were selected to construct bimolecular complementary (BiFC) and trimolecular complementation (TriFC) systems. The systems feature brilliant redness, shorter maturation time, and the longer excitation and emission wavelengths. By combined use of the mCherry-based red BiFC with a Venus-based yellow BiFC system, simultaneous detection of multi protein-protein interactions and mRNA-protein interaction were realized in Vero cells[4, 5]. The results obtained helps to understand the nuclear export mechanism of influenza A viral mRNAs[5]. The mNeptune based far-red TriFC system, when applied to study HIV activation from latency, were verified as useful tools for imaging protein-protein and mRNA-protein interactions, respectively, in live cells and mice[6]. A bacterial phytochrome-based, near-infrared fluorescent protein (iRFP) was also used to construct the BiFC for visualizing interaction between HIV-1 integrase and celluar cofactor protein LEDGF/p75) in live cells and live mice[7].
The methods developed were verified as powerful tools for imaging virus-host interactions under physiological conditions in live cells and in live mice.