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Since its discovery in 1989, efforts to grow clinical isolates of the hepatitis C virus (HCV) in cell culture have met with limited success. Only the JFH-1 isolate has the capacity to replicate efficiently in cultured hepatoma cells without cell culture-adaptive mutations (1-3). We hypothesized that cultured cells lack one or more factors required for the replication of clinical isolates. To identify the missing factors, we transduced Huh-7.5 human hepatoma cells with a pooled lentivirus-based human complementary DNA (cDNA) library, transfected the cells with HCV subgenomic replicons lacking adaptive mutations, and selected for stable replicon colonies. This led to the identification of a single cDNA, SEC14L2, that enabled RNA replication of diverse HCV genotypes in several hepatoma cell lines. This effect was dose-dependent, and required the continuous presence of SEC14L2. Full-length HCV genomes also replicated and produced low levels of infectious virus. Remarkably, SEC14L2-expressing Huh-7.5 cells also supported HCV replication following inoculation with patient sera. Mechanistic studies suggest that SEC14L2 promotes HCV infection by enhancing vitamin E-mediated protection against lipid peroxidation. This provides a foundation for development of in vitro replication systems for all HCV isolates, creating a useful platform to dissect the mechanisms by which cell culture-adaptive mutations act., Hepatitis C virus (HCV) is a leading cause of liver disease worldwide, with an estimated global burden of 185 million chronic infections (4). Studies of this important human pathogen have [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.01.035 Byline: Robert D. Fisher (1), Hyo-Young Chung (1), Qianting Zhai (1), Howard Robinson (2), Wesley I. Sundquist (1), Christopher P. Hill (1) Abstract: ALIX/AIP1 functions in enveloped virus budding, endosomal protein sorting, and many other cellular processes. Retroviruses, including HIV-1, SIV, and EIAV, bind and recruit ALIX through YPX.sub.nL late-domain motifs (X = any residue; n = 1-3). Crystal structures reveal that human ALIX is composed of an N-terminal Bro1 domain and a central domain that is composed of two extended three-helix bundles that form elongated arms that fold back into a 'V.' The structures also reveal conformational flexibility in the arms that suggests that the V domain may act as a flexible hinge in response to ligand binding. YPX.sub.nL late domains bind in a conserved hydrophobic pocket on the second arm near the apex of the V, whereas CHMP4/ESCRT-III proteins bind a conserved hydrophobic patch on the Bro1 domain, and both interactions are required for virus budding. ALIX therefore serves as a flexible, extended scaffold that connects retroviral Gag proteins to ESCRT-III and other cellular-budding machinery. Author Affiliation: (1) Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112, USA (2) Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA Article History: Received 13 November 2006; Revised 22 December 2006; Accepted 5 January 2007 Article Note: (miscellaneous) Published: March 8, 2007

Research demonstrates that 22 class E proteins along with a protein network consisting of 43 other protein-protein complexes mediate human multivesicular bodies biogenesis as well as participate in the release of HIV and other viruses by budding. Data indicate that AIP1 plays a key role in this viral budding process.

Retrovirus budding requires short peptide motifs (late domains) located within the viral Gag protein that function by recruiting cellular factors. The YPX(n)L late domains of HIV and other lentiviruses recruit the protein ALIX (also known as AIP1), which also functions in vesicle formation at the multivesicular body and in the abscission stage of cytokinesis. Here, we report the crystal structures of ALIX in complex with the YPX(n)L late domains from HIV-1 and EIAV. The two distinct late domains bind at the same site on the ALIX V domain but adopt different conformations that allow them to make equivalent contacts. Binding studies and functional assays verified the importance of key interface residues and revealed that binding affinities are tuned by context-dependent effects. These results reveal how YPX(n)L late domains recruit ALIX to facilitate virus budding and how ALIX can bind YPX(n)L sequences with both n = 1 and n = 3.