Severe acute respiratory syndrome (SARS) which emerged in the winter of 2002 caused a mortality rate as high as 9.6%. SARS-coronavirus (SARS-CoV) is a causative agent of SARS. It is an enveloped, positive single-stranded RNA virus with an RNA genome about 30,000 nucleotides in length, which encodes 14 open reading frames. The virus particle consists of four major structural proteins: spike (S), membrane (M), envelope (E) and nucleocapsid (N). The S protein initiates virus entry by binding to cell surface receptors followed by conformational changes that lead to membrane fusion. The specific aim of this study is to identify surface molecules of host cells involved in the process of SARS-CoV infection. Virus-like particles (VLPs) consist of three major envelope proteins E, M and S that mimic the organization and conformation of native virus particles. VLPs can undergo infection, following cell attachment and membrane fusion. In order to generate SARS-VLPs, Sf9 cells were co-infected with recombinant baculoviruses that express E, M, and S proteins. Four days post-infection, cells were lysed and SARS-VLPs were isolated from culture medium and purified by sucrose gradient centrifugation. Electron microscopic and immunogold labeling analysis of negatively stained SARS-VLPs exhibited SARS-CoV-like structures. In this study, spike protein was found to form complexes with ACE2 when SARS-VLPs were co-incubated with Vero E6 cells for 10 min. Immunoprecipitation followed by LC-MS/MS analysis identified the association of actin, vimentin and annexin A2 with ACE2. The specific interaction between ACE2 and vimentin was further confirmed by immunoprecipitation and Western blot analysis. In addition, colocalization of the spike protein of SARS-CoV and vimentin on the cell membrane was demonstrated by confocal microscopic analysis. Taken together, these results indicate that vimentin may form complexes with spike and ACE2 during virus infection. The role of vimentin involved in virus entry needs to be further studied.