The specific Cell Entry Pathway of the Human Endogenous Retrovirus-K (HML-2) and the Role of Heparan Sulfate Proteoglycans

Abstract

A lot of viruses have been shown to use a wide variety of attachment molecules on cell surfaces and exploit them as receptors and establish an infection (Rostand and Esko, 1997). Viral entry, spread and pathogenesis can be better understood by identification of the cell surface receptor(s) of particular virus targets (Shukla and Spear, 2001). Virus entry is a two-step process, with the first step being attachment of the virus to a primary receptor, followed by the interaction with a co-receptor that is usually a cell-specific transmembrane protein (Shukla et al., 1999; Summerford and Samulski, 1998). Understanding the interaction between proteins on the surface of virus particles and the cell surface receptors which are used by the virus particles to enter cells is essential to understand viral tropism, which further helps in creating effective antiviral therapies. Several published studies have suggested that a number of viruses use the heparan sulfate (HS) component of cell surface heparan sulfate proteoglycans (HSPGs), as an main receptor to attach to cells (Liu and Thorp, 2002; Rostand and Esko, 1997; Zhu et al., 2011). HS is a glycosaminoglycan (GAG) composed of repeating disaccharides of glucosamine and hexuronic acid, joined in alternating sequences by 1,4-glycosidic linkages, which gives these carbohydrate chains the flexibility to bind to many different proteins (Li and Vlodavsky, 2009; Lindahl, 1990). There are a lot of HS-binding viruses from different families and include herpes simplex virus (HSV) (WuDunn and Spear, 1989), human papillomavirus (HPV) (Giroglou et al., 2001), hepatitis B virus (HBV) (Cooper et al., 2005), respiratory syncytial virus (RSV) (Hallak et al., 2000), foot-and-mouth disease virus (FMDV) (Jackson et al., 1996) and the human immunodeficiency virus type 1 (HIV-1) (Roderiquez et al., 1995). For this reason, it was important to analyze which proteins are responsible for the entry of HERV-K (HML-2). Therefore, the receptors ASCT1/2 (for syncytin-1) and MFSD2A (for syncytin-2) were transfected into HEK293T cells. Infection with various pseudotyped HIV showed that viral entry into the cells could not be increased. For this reason, it could be assumed that syncytin receptors are not primarily responsible for entry. This was the reason that new receptor candidates had to be used for further experiments. Previos work by N. Bnnert and A. Richter compared different gene profiles of successfully infected cells with HERV-K and thus identified potential receptors that might be specifically involved in the onset of the virus. These receptors were the G protein-coupled receptor 56 and 161, the cell surface receptor CD63, the neural cell adhesion molecule L1CAM, the golgi sorting receptor sotilin-1 and the cell surface protein neuroligin-1. Overexpression of these receptors revealed that the g protein-coupled receptor 56 must have a crucial role in the onset of HERV-K (HML-2), as it could increase the infection rate by 3-fold. Furthermore, this study investigated the influence of heparan sulfates on the entry of HERV-K into the cells. For this, free heparan sulfate was incubated before infection with the cells and pseudotyped HIV. It turned out that an increase of the heparan sulfate concentration up to 400μg/ml led to a reduction of the infection rate up to 40%. A change in the cell surface profile was achieved by enzymatic digestion with heparinase I and III and a chemical reaction with sodium chlorate. While heparinase is responsible for the digestion of heparan sulfates, sodium chlorate prevents the sulfation of glycosaminoglycan’s. Both experiments showed that with increasing concentration, the infection rate of HERV-K decreased. The viral entry after enzymatic digestion was reduced up to 50% and after chemical inactivation of sulfation by up to 30%. Another approach to verify HS functionality was the overexpression of a specific heparan sulfate. Syndecan-1 is a transmembrane (type I) heparan sulfate proteoglycan and is a member of the syndecan family. This protein was amplified in HEK293T and HCT116 cells and infected with pseudotyped HIV. However, analysis of the resulting luciferase revealed that this had no effect on the rate of infection. In further experiments, syndecan-1 was reduced by means of special shRNAs on the cell surface. A short hairpin RNA is an artificial RNA molecule with a tight hairpin turn. The expression of shRNA in the cell lines was done by the transfection of specific plasmids and blocks the specific mRNA of the examined gen. The reduction of the syndecan-1 level on the cell surface led to a reduction in the infection rate of HERV-K down to 30% in HEK293T cells. The entry of the viruses into the cells should be measured by means of the formed firefly luciferase. In the luciferase assay, reporter constructs carrying a firefly luciferase gene behind a CMV promotor and after this, the sequence which is important to produce virus particles. Based on these facts, it can be said that various heparan sulfates and GPR56 might play a crucial role in the entry of HERV-K (HML-2). This should be one of the main theses for future work on this topic.