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Isolation of suppressor genes that restore retrovirus susceptibility to a virus-resistant cell line
1Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA. gaogx@sun.im.ac.cn
Background:
Genetic selections in mammalian cell lines have recently been developed for the isolation of mutant cells that are refractory to infection by retroviruses. These selections have been used to recover lines that block early postentry stages of infection, either before reverse transcription or before nuclear entry. The mechanisms of action of these blocks remain unknown.
Results:
We have devised a method for the selection of genes from cDNA libraries that suppress the block to virus infection, and so restore virus susceptibility. The protocol involves the transformation of pools of resistant cells by cDNA expression libraries, followed by the selection for rare virus-sensitive cells, using multiple rounds of selection after infection by marked viral vector genomes. The suppressor genes were then recovered from these virus sensitive cells, and their ability to restore virus susceptibility was confirmed by reintroduction of these cDNAs into the resistant line.
Conclusions:
The identities of these genes provide insights into the mechanism of virus resistance and will help to define new pathways used during retrovirus infection. The methods for gene isolation developed here will also permit the identification of similar suppressors that modify or override other recently identified virus resistance genes.
Insights
Researchers developed a new method to identify genes that restore retrovirus susceptibility in resistant mammalian cells. This approach aids in understanding virus infection mechanisms and identifying novel resistance pathways.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Mammalian cell lines resistant to retroviruses are crucial for studying infection.
- Existing methods isolate resistant cells but don't reveal resistance mechanisms.
- The precise mechanisms of blocks in early postentry stages of retroviral infection are largely unknown.
Purpose of the Study:
- To develop a method for selecting genes that suppress blocks to retrovirus infection.
- To identify genes that restore virus susceptibility in resistant cells.
- To gain insights into retroviral infection pathways and resistance mechanisms.
Main Methods:
- Transformation of retrovirus-resistant cells with cDNA expression libraries.
- Selection for rare virus-sensitive cells after infection with marked viral vectors.
- Recovery and reintroduction of suppressor genes to confirm restored virus susceptibility.
Main Results:
- A novel method was devised to select for suppressor genes.
- The method successfully identified genes that restore retrovirus susceptibility.
- The identified genes offer insights into virus resistance mechanisms.
Conclusions:
- The identified genes provide crucial insights into retroviral infection pathways.
- This gene isolation method can be used to study other virus resistance genes.
- Understanding these pathways can lead to new antiviral strategies.
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