Focal Adhesion Proteins Talin-1 and Vinculin Negatively Affect Paxillin Phosphorylation and Limit Retroviral Infection Craig Brown 1,2 , Scott G. Morham 3 , Derek Walsh 2 and Mojgan H. Naghavi 1,4 ⁎ 1 Centre for Research in Infectious Diseases, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland 2 National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland 3 Myrexis, Inc., Salt Lake City, UT 84108, USA 4 Department of Biochemistry and Molecular Biophysics, Department of Microbiology and Immunology, Columbia University, New York, NY 10032, USA Received 31 January 2011; received in revised form 30 March 2011; accepted 31 March 2011 Edited by M. F. Summers Keywords: retrovirus; HIV-1; cytoskeleton; focal adhesions; early block Many of the early events in retroviral infection are not well understood, but it is known that the host cytoskeleton and signaling pathways play integral roles in various entry and post-entry processes. Focal adhesion complexes act as sites of integration for both cytoskeletal organization and integrin signaling at the cell surface. Here, we show that talin-1 and vinculin, two interacting proteins that localize in focal adhesions to mediate integrin linkage to the actin cytoskeleton, function during retroviral infection. Transient overexpression of either talin-1 or vinculin reduced the susceptibility of human cells to infection with pseudotyped human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus. In contrast, transient short interfering RNA-mediated knockdown of talin-1 or vinculin increased infection by pseudotyped HIV- 1 and simian immunodeficiency virus, demonstrating that the endogenous forms of these proteins also impaired retroviral infection. Talin-1 or vinculin overexpression inhibited infection by retroviruses that entered the cell by either fusion or endocytosis, while analysis of HIV-1 DNA synthesis demonstrated that the block occurred early in infection and prior to the initiation of reverse transcription. Both factors retained antiviral activity in the presence of actin or microtubule depolymerizing agents. Finally, talin-1 and vinculin expression was found to negatively influence tyrosine phosphorylation of paxillin, a major focal adhesion scaffolding protein whose transient knockdown decreased pseudotyped HIV-1 infection. Together, these findings demonstrate that talin-1 and vinculin negatively affect tyrosine phosphorylation of paxillin, a novel positive regulator of *Corresponding author. Department of Biochemistry and Molecular Biophysics, Department of Microbiology and Immunology, Columbia University, HHSC 1313, 701 West 168th Street, New York, NY 10032, USA. E-mail address: mn2034@columbia.edu. Abbreviations used: HIV-1, human immunodeficiency virus type 1; M-MuLV, Moloney murine leukemia virus; SIV, simian immunodeficiency virus; VSV-G, vesicular stomatitis virus; MT, microtubule; FAK, focal adhesion kinase; ECM, extracellular matrix; ERM, ezrin/radixin/moesin; ERK, extracellular signal-regulated kinase; siRNA, short interfering RNA; GFP, green fluorescent protein; MSS, minus-strand strong stop; DMSO, dimethyl sulfoxide; FERM, 4.1/ezrin/ radixin/moesin; KS, Kaposi's sarcoma; qPCR, quantitative real-time PCR; TBS-T, Tris-buffered saline and 1% Tween. doi:10.1016/j.jmb.2011.03.076 J. Mol. Biol. (2011) 410, 761–777 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter. Published by Elsevier Ltd.