Giuseppe
Emeritus
[Source: US National Library of Medicine, (LINK). Edited.]
J Virol. 2010 May 12. [Epub ahead of print]
Genetic engineered, biarsenical labelled influenza virus allows visualization of viral NS1 protein in living cells.
Li Y, Lu X, Li J, B?rub? N, Giest KL, Liu Q, Anderson DH, Zhou Y. - Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 5E3; Cancer Research Unit, Health Research Division, Saskatchewan Cancer Agency, Saskatoon, Saskatchewan, Canada, S7N 4H4.
Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication competent fluorescence influenza A virus is that the segmented genome does not allow fusion of fluorescent protein gene to any viral gene. Here, we introduced the tetracysteine (TC) biarsenical labeling system into influenza virus in order to fluorescently label viral protein in virus life cycle. We generated infectious influenza A viruses bearing a small TC-tag (CCPGCC) in the loop/linker regions of the NS1 proteins. In the background of A/Puerto Rico/8/34 (H1N1) (PR8) virus, the TC-tag can be inserted into NS1 after AA52 (PR8-410), AA79 (PR8-412), AA102 (PR8-413), or replace AA79-84 (PR8-411), respectively. Although PR8-410, PR8-411 and PR8-412 attenuated to some extend in multiple cycle infection, they retain the similar growth potential during single cycle infection; and their NS1 subcellular localization, viral protein synthesis rate are quite similar to those of the wild type virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in the fluorescent NS1 in the context of virus infection. We could exploit this strategy on NS1 protein of A/Texas/36/91 (H1N1) (Tx91) by successfully rescuing a C-tagged virus Tx91-445, which carries the TC-tag replacement of AA79-84. Tx91-445 virus retained similar infectivity as WT Tx91 during multiple cycle as well as single cycle replication. The NS1 protein derived from Tx91-445 can be fluorescently labeled in living cells. Finally, with biarsenical labeling, the engineered replication-competent virus allowed us to real-time visualize NS1 protein nuclear import in virus infected cells.
PMID: 20463066 [PubMed - as supplied by publisher]
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J Virol. 2010 May 12. [Epub ahead of print]
Genetic engineered, biarsenical labelled influenza virus allows visualization of viral NS1 protein in living cells.
Li Y, Lu X, Li J, B?rub? N, Giest KL, Liu Q, Anderson DH, Zhou Y. - Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 5E3; Cancer Research Unit, Health Research Division, Saskatchewan Cancer Agency, Saskatoon, Saskatchewan, Canada, S7N 4H4.
Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication competent fluorescence influenza A virus is that the segmented genome does not allow fusion of fluorescent protein gene to any viral gene. Here, we introduced the tetracysteine (TC) biarsenical labeling system into influenza virus in order to fluorescently label viral protein in virus life cycle. We generated infectious influenza A viruses bearing a small TC-tag (CCPGCC) in the loop/linker regions of the NS1 proteins. In the background of A/Puerto Rico/8/34 (H1N1) (PR8) virus, the TC-tag can be inserted into NS1 after AA52 (PR8-410), AA79 (PR8-412), AA102 (PR8-413), or replace AA79-84 (PR8-411), respectively. Although PR8-410, PR8-411 and PR8-412 attenuated to some extend in multiple cycle infection, they retain the similar growth potential during single cycle infection; and their NS1 subcellular localization, viral protein synthesis rate are quite similar to those of the wild type virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in the fluorescent NS1 in the context of virus infection. We could exploit this strategy on NS1 protein of A/Texas/36/91 (H1N1) (Tx91) by successfully rescuing a C-tagged virus Tx91-445, which carries the TC-tag replacement of AA79-84. Tx91-445 virus retained similar infectivity as WT Tx91 during multiple cycle as well as single cycle replication. The NS1 protein derived from Tx91-445 can be fluorescently labeled in living cells. Finally, with biarsenical labeling, the engineered replication-competent virus allowed us to real-time visualize NS1 protein nuclear import in virus infected cells.
PMID: 20463066 [PubMed - as supplied by publisher]
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