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Journal of Virology, September 2009, p. 8628-8637, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00873-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Low Endocytic pH and Capsid Protein Autocleavage Are Critical Components of Flock House Virus Cell Entry{triangledown}

Amy L. Odegard,1,{dagger} Maggie H. Kwan,1 Hanna E. Walukiewicz,2,{ddagger} Manidipa Banerjee,1 Anette Schneemann,1 and John E. Johnson1,2*

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037,1 Department of Chemistry and Biochemistry, University of California—San Diego, La Jolla, California 920932

Received 30 April 2009/ Accepted 12 June 2009

The process by which nonenveloped viruses cross cell membranes during host cell entry remains poorly defined; however, common themes are emerging. Here, we use correlated in vivo and in vitro studies to understand the mechanism of Flock House virus (FHV) entry and membrane penetration. We demonstrate that low endocytic pH is required for FHV infection, that exposure to acidic pH promotes FHV-mediated disruption of model membranes (liposomes), and particles exposed to low pH in vitro exhibit increased hydrophobicity. In addition, FHV particles perturbed by heating displayed a marked increase in liposome disruption, indicating that membrane-active regions of the capsid are exposed or released under these conditions. We also provide evidence that autoproteolytic cleavage, to generate the lipophilic {gamma} peptide (4.4 kDa), is required for membrane penetration. Mutant, cleavage-defective particles failed to mediate liposome lysis, regardless of pH or heat treatment, suggesting that these particles are not able to expose or release the requisite membrane-active regions of the capsid, namely, the {gamma} peptides. Based on these results, we propose an updated model for FHV entry in which (i) the virus enters the host cell by endocytosis, (ii) low pH within the endocytic pathway triggers the irreversible exposure or release of {gamma} peptides from the virus particle, and (iii) the exposed/released {gamma} peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.

* Corresponding author. Mailing address: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., MB-31, La Jolla, CA 92037. Phone: (858) 784-9705. Fax: (858) 784-8660. E-mail: jackj{at}scripps.edu

{triangledown} Published ahead of print on 24 June 2009.

{dagger} Present address: Department of Chemistry, University of Puget Sound, Tacoma, WA 98416.

{ddagger} Present address: Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801.

Journal of Virology, September 2009, p. 8628-8637, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00873-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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