Isolation and characterization of human cells resistant to retrovirus infection

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Identification of host cell proteins required for HIV-1 infection will add to our knowledge of the life cycle of HIV-1 and in the development of therapeutics to combat viral infection. We and other investigators have mutagenized rodent cells and isolated mutant cell lines resistant to retrovirus infection. Since there are differences in the efficiency of single round infection with VSVG pseudotyped HIV-1 on cells of different species, we conducted a genetic screen to isolate human cells resistant to HIV-1 infection. We chemically mutagenized human HeLa cells and validated our ability to isolate mutants at test diploid loci. We then executed a screen to isolate HeLa cell mutants resistant to infection by an HIV-1 vector coding for a toxic gene product. Results We isolated two mutant cell lines that exhibit up to 10-fold resistance to infection by HIV-1 vectors. We have verified that the cells are resistant to infection and not defective in gene expression. We have confirmed that the resistance phenotype is not due to an entry defect. Fusion experiments between mutant and wild-type cells have established that the mutations conferring resistance in the two clones are recessive. We have also determined the nature of the block in the two mutants. One clone exhibits a block at or before reverse transcription of viral RNA and the second clone has a retarded kinetic of viral DNA synthesis and a block at nuclear import of the preintegration complex. Conclusion Human cell mutants can be isolated that are resistant to infection by HIV-1. The mutants are genetically recessive and identify two points where host cell factors can be targeted to block HIV-1 infection.

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Published 01 January 2007
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Retrovirology
BioMedCentral
Open Access Research Isolation and characterization of human cells resistant to retrovirus infection 1 2 Patrycja Lech and Nikunj V Somia*
1 Address: Molecular, Cellular, Developmental Biology and Genetics Graduate Program, University of Minnesota, Minneapolis, Minnesota, USA 2 and Dept. of Genetics, Cell Biology and Development and the Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, USA Email: Patrycja Lech  lechx001@umn.edu; Nikunj V Somia*  somia001@umn.edu * Corresponding author
Published: 3 July 2007 Received: 18 December 2006 Accepted: 3 July 2007 Retrovirology2007,4:45 doi:10.1186/1742-4690-4-45 This article is available from: http://www.retrovirology.com/content/4/1/45 © 2007 Lech and Somia; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Background:Identification of host cell proteins required for HIV-1 infection will add to our knowledge of the life cycle of HIV-1 and in the development of therapeutics to combat viral infection. We and other investigators have mutagenized rodent cells and isolated mutant cell lines resistant to retrovirus infection. Since there are differences in the efficiency of single round infection with VSVG pseudotyped HIV-1 on cells of different species, we conducted a genetic screen to isolate human cells resistant to HIV-1 infection. We chemically mutagenized human HeLa cells and validated our ability to isolate mutants at test diploid loci. We then executed a screen to isolate HeLa cell mutants resistant to infection by an HIV-1 vector coding for a toxic gene product. Results:We isolated two mutant cell lines that exhibit up to 10-fold resistance to infection by HIV-1 vectors. We have verified that the cells are resistant to infection and not defective in gene expression. We have confirmed that the resistance phenotype is not due to an entry defect. Fusion experiments between mutant and wild-type cells have established that the mutations conferring resistance in the two clones are recessive. We have also determined the nature of the block in the two mutants. One clone exhibits a block at or before reverse transcription of viral RNA and the second clone has a retarded kinetic of viral DNA synthesis and a block at nuclear import of the preintegration complex. Conclusion:Human cell mutants can be isolated that are resistant to infection by HIV-1. The mutants are genetically recessive and identify two points where host cell factors can be targeted to block HIV-1 infection.
Background Intensive studies of the structure and function of HIV1 encoded genes has led to the development of a number of small molecule drugs to combat HIV1. However, the mutation rate of HIV1 is high (about one mutation in every 3 new genomes produced [1]) which leads to the evolution of viruses that are resistant to the drug blockade.
Indeed some antiviral drugs may accelerate the mutation rate of HIV1 [1]. This necessitates the development of new drugs and strategies to combat HIV1 infection. In this regard, a novel approach is to target cellular factors required by HIV1 to complete its lifecycle [2]. One method of identifying cellular factors essential for retrovi ral infection is through genetic screening of mutagenized
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