Journal of Virological Methods 169 (2010) 244–247 Contents lists available at ScienceDirect Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet Short communication The effect of purification method on the completeness of the immature HIV-1 Gag shell Nitzan Kol a , Marianna Tsvitov a , Liron Hevroni a , Sharon G. Wolf b , Hong-Bo Pang c , Michael S. Kay c , Itay Rousso a,∗ a Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel b Electron Microscopy Center, Weizmann Institute of Science, Rehovot 76100, Israel c Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112-5650, USA Article history: Received 15 April 2010 Received in revised form 27 July 2010 Accepted 29 July 2010 Available online 4 August 2010 Keywords: HIV-1 Structure Immature Gag Purification Sucrose OptiPrep Iodixanol abstract Elucidating the structure of the immature HIV-1 Gag core is an important aspect of understanding the biology of this virus. In doing so, preservation of the fragile Gag lattice is essential. In this study, the effects of purification methods on the structural and mechanical integrity of immature HIV-1 are examined. The results show that the morphological and mechanical properties of the virion are preserved to a signifi- cantly higher degree by Iodixanol (OptiPrep) purification compared to the standard sucrose method. In conclusion, these results indicate that OptiPrep instead of sucrose purification should be employed when conducting structural studies on the HIV-1 virion. © 2010 Elsevier B.V. All rights reserved. Gag is the structural protein of HIV-1, which comprises the core of the virion and drives its assembly and budding at the plasma membrane. HIV-1 buds as a non-infectious immature par- ticle, composed of a dense doughnut-like shell consisting of Gag, located directly underneath the viral membrane. The Gag polypro- tein includes three main structural domains – MA, CA, and NC, which are cleaved after budding by the viral protease to form the mature infectious virion. The mature virus particle has a distinctly different morphology and contains a conical capsid. Elucidating the structure of the immature virion’s Gag shell has been the focus of several studies, as it may resolve important questions associated with the assembly, budding, and maturation processes of HIV-1. Initially Gag was assumed to generally form a complete lattice beneath the viral lipid bilayer membrane. Later it was shown that the lattice is hexagonal (Briggs et al., 2006, 2004; Mayo et al., 2003; Nermut et al., 1998), the closure of which requires the presence of defects. Defects can either be in form of pentamers at each of the 12 vertices yielding an icosohedral virion, like in the case of many viruses such as HSV, adenovirus, and bacteriophage, or gaps in the hexagonal lattice. In the last decade, several stud- ∗ Corresponding author. Tel.: +972 8 9343479; fax: +972 8 9344136. E-mail address: itay.rousso@weizmann.ac.il (I. Rousso). ies suggested that closure of the HIV shell is accomplished by the incorporation of defects in the form of gaps of various sizes (Briggs et al., 2009; Carlson et al., 2008; Fuller et al., 1997; Wilk et al., 2001; Wright et al., 2007). The most recent studies used electron cryoto- mography to estimate the size and nature of these gaps (Briggs et al., 2009; Carlson et al., 2008; Wright et al., 2007). The first study found the Gag shell to be ∼40% complete on average and to consist of several patches of Gag lattice (Wright et al., 2007). Two recent studies from the Krausslich group showed that the Gag shell has an average completeness of ∼70% and consists of one continuous lat- tice that contains small gaps throughout (Briggs et al., 2009; Carlson et al., 2008). All of these studies were carried out on viruses that were purified by pelleting through a sucrose cushion, a method that is used commonly in the HIV field. In the past, concerns were raised about the use of sucrose solutions to purify retroviruses due to possible damage by osmotic shock and the need for pelleting by ultracentrifugation. In fact, extensive damage to virus glycopro- teins was indeed demonstrated using this method (McGrath et al., 1978; Moennig et al., 1974). Therefore the structure of the Gag shell of the immature HIV-1 virion in its native state remains unknown. It has previously been reported that the Gag shell of the imma- ture HIV-1 particle is mechanically rigid (Kol et al., 2007). This finding is difficult to reconcile with the idea that the Gag shell is discontinuous and has large gaps. In this study, the impact on virus 0166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.07.035