IF THIS HAS ALREADY BEEN POSTED, MODERATORS PLEASE DELETE Sadayuki Ohkura1, David C. Goldstone2, Melvyn W. Yap1, Kate Holden-Dye1, Ian A. Taylor2, Jonathan P. Stoye1* 1 Division of Virology, MRC National Institute for Medical Research, London, United Kingdom, 2 Division of Molecular Structure, MRC National Institute for Medical Research, London, United Kingdom Abstract Top After entry into target cells, retroviruses encounter the host restriction factors such as Fv1 and TRIM5?. While it is clear that these factors target retrovirus capsid proteins (CA), recognition remains poorly defined in the absence of structural information. To better understand the binding interaction between TRIM5? and CA, we selected a panel of novel N-tropic murine leukaemia virus (N-MLV) escape mutants by a serial passage of replication competent N-MLV in rhesus macaque TRIM5? (rhTRIM5?)-positive cells using a small percentage of unrestricted cells to allow multiple rounds of virus replication. The newly identified mutations, many of which involve changes in charge, are distributed over the outer top surface of N-MLV CA, including the N-terminal ?-hairpin, and map up to 29 Ao apart. Biological characterisation with a number of restriction factors revealed that only one of the new mutations affects restriction by human TRIM5?, indicating significant differences in the binding interaction between N-MLV and the two TRIM5?s, whereas three of the mutations result in dual sensitivity to Fv1n and Fv1b. Structural studies of two mutants show that no major changes in the overall CA conformation are associated with escape from restriction. We conclude that interactions involving much, if not all, of the surface of CA are vital for TRIM5? binding. Author Summary Top Host restriction factors such as TRIM5? are important for preventing cross species transmission of a variety of retroviruses. They act to block viral replication but their mode of virus recognition is poorly understood. To address this question we have developed a procedure for isolating viruses that replicate in the presence of restriction factors. Analysis of these viruses shows that individual mutations across the entire surface of the viral capsid molecule can relieve restriction. Escape from TRIM5? of one species does not necessarily lead to escape from another. It seems likely that restriction factor recognition involves extensive weak contacts between factor and virus. We suggest that this represents an important design feature in a system that recognizes multiple pathogens. Citation: Ohkura S, Goldstone DC, Yap MW, Holden-Dye K, Taylor IA, et al. (2011) Novel Escape Mutants Suggest an Extensive TRIM5? Binding Site Spanning the Entire Outer Surface of the Murine Leukemia Virus Capsid Protein. PLoS Pathog 7(3): e1002011. doi:10.1371/journal.ppat.1002011 Editor: Michael Emerman, Fred Hutchinson Cancer Research Center, United States of America Received: November 5, 2010; Accepted: January 28, 2011; Published: March 31, 2011 Copyright: 2011 Ohkura et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the UK Medical Research Council; file references U117512710 (JPS) and U117565647 (IAT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: email@example.com Huh? Does somebody want to take a crack at explaining this?