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Journal of Virology, October 2005, p. 12969-12978, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12969-12978.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Isolation of Cell Lines That Show Novel, Murine Leukemia Virus-Specific Blocks to Early Steps of Retroviral Replication

James W. Bruce,1 Kenneth A. Bradley,2 Paul Ahlquist,1,3 and John A. T. Young4*

Institute for Molecular Virology, University of Wisconsin, Madison, Wisconsin 53706-1596,1 Department of Microbiology, Immunology, and Molecular Genetics, University of California at Los Angeles, Los Angeles, California 90095,2 Howard Hughes Medical Institute University of Wisconsin, Madison, Wisconsin 53706-1596,3 Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California 920374

Received 9 May 2005/ Accepted 26 July 2005

In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.

* Corresponding author. Mailing address: Infectious Disease Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 453-4100. Fax: (858) 554-0341. E-mail: jyoung{at}salk.edu.

Journal of Virology, October 2005, p. 12969-12978, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12969-12978.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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