Virus Research 152 (2010) 180–183 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Short communication Cholesterol depletion affects infectivity and stability of pseudorabies virus Ann S. Desplanques, Maria Pontes, Natasha De Corte, Nele Verheyen, Hans J. Nauwynck, Dries Vercauteren, Herman W. Favoreel ∗ Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium article info Article history: Received 13 April 2010 Received in revised form 11 June 2010 Accepted 11 June 2010 Available online 22 June 2010 Keywords: Cholesterol Pseudorabies virus Alphaherpesviruses Stability Infectivity Lovastatin Methyl-beta-cyclodextrin abstract Statins, such as lovastatin, inhibit the cellular cholesterol biosynthesis. Addition of lovastatin to SK cells and subsequent infection with the alphaherpesvirus pseudorabies virus (PRV) did not affect the intracel- lular production of viral structural proteins but reduced virus titers. Addition of methyl-beta-cyclodextrin to deplete cholesterol from the viral envelope also resulted in reduced virus titers. Addition of exoge- nous cholesterol restored virus titers in both experimental assays. Further analysis showed that reducing cholesterol levels reduced both the infectivity of newly produced infectious virus and their stability, as assessed by determining virus titers immediately after treatment or upon storage of the virus at room temperature or frozen. This is the first report to demonstrate that cholesterol is involved in the stability of infectious alphaherpesvirus, and that treatment of host cells with statins reduces alphaherpesvirus titers. Hence, cholesterol is important for pseudorabies virus infectivity and stability. © 2010 Elsevier B.V. All rights reserved. Herpesviruses are large, enveloped DNA viruses and alphaher- pesviruses represent the largest subfamily of the herpesviruses. The alphaherpesvirus subfamily contains different important, and relatively closely related pathogens of man and animal, including herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseu- dorabies virus (PRV). The swine alphaherpesvirus PRV is often used as a model to study general aspects of alphaherpesvirus biology (Pomeranz et al., 2005). Cholesterol is known to provide rigidity to eukaryotic mem- branes and membrane cholesterol has been described to be important for succesful cryopreservation of oocytes, sperm cells, and embryos (Medeiros et al., 2002; Seidel, 2006). Statins such as lovastatin are the most widely prescribed drugs in the world. They inhibit intracellular cholesterol biosynthesis by inhibiting HMG CoA reductase (Futterman and Lemberg, 2005). They have been widely used in research to assess the importance of cholesterol in a plethora of different processes (Tobert, 2003). The aim of the current study was to determine a possible role of cellular cholesterol in infectivity of progeny pseudorabies virus particles. Others and we have shown before that plasma membrane cholesterol is involved in efficient alphaherpesvirus entry in host cells (Bender et al., 2003; Desplanques et al., 2008; Hambleton et al., 2007). Cholesterol depletion of host cells does not pre- ∗ Corresponding author. Tel.: +32 9 264 73 74; fax: +32 9 264 74 95. E-mail address: Herman.Favoreel@UGent.be (H.W. Favoreel). vent alphaherpesvirus entry, but reduces the efficiency, possibly by reducing fusion efficiency of the viral envelope with the cel- lular plasma membrane (Desplanques et al., 2008; Hannah et al., 2009; Zhu et al., 2010). Our aim was to determine a potential effect of lovastatin treatment on later stages of infection: progeny virus production and infectivity. Therefore, it was important to ensure that any effects observed from the depletion of choles- terol using lovastatin were not due to effects on virus entry. To this end, SK cells were pretreated with 4 M lovastatin (Sigma) for 48 h, a cholesterol-reducing treatment that has been described before (Keller and Simons, 1998). Subsequently, PRV Becker was added at an m.o.i. of 10. This general treatment ensured equal efficiency of PRV entry in the different treatment groups. 2 h post inoculation (hpi), remaining extracellular virus was inactivated by treatment with citrate buffer as described before (Favoreel et al., 2005), cells were kept in medium without fetal bovine serum (to avoid an unwanted/uncontrolled increase in cholesterol levels) and were split in two populations. One population continued to receive lovastatin and thereby retained low cholesterol levels (LC), whereas cholesterol levels in the other population were restored by the addition of 15 g/ml exogenous cholesterol (Sigma), resulting in a population with high cholesterol (HC). As an additional control, SK cells were not treated with lovastatin and inoculated with PRV as described above, referred to as cells with normal cholesterol (NC). Analysis of the cholesterol content of the different populations of cells using the AmplexRed assay (Invitrogen) as described before (Desplanques et al., 2008), and comparison with the NC population showed that lovastatin efficiently reduced the cellular cholesterol 0168-1702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2010.06.008