Interactions of viruses with lipid rafts

Viruses are dependent on the cellular machinery for many steps in their replication cycle. Sev-eral cellular mechanisms used by viruses either for entry, such as endocytosis or signal trans-duction pathways, or for morphogenesis, such as protein transport can involve lipid rafts. Not surprisingly it has been shown for several viruses that lipid rafts are crucial for their replication cycle (reviewed in Chazal and Gerlier (2003); Manes et al. (2003); Nayak and Barman (2002);

Ono and Freed (2005)).

1.2.3.1 Virus entry

Viruses have evolved different strategies to enter their host cells. All animal viruses that have extracellular phases initiate their replication by binding to one or more cellular receptor(s). In all cases viruses have to cross the cellular membrane to gain access to the cytoplasm. Non-enveloped viruses are usually internalised by endocytosis. Intracellular (endosomal, lysosomal or caveosomal) membranes may be overcome either by membrane lysis or by pore formation.

Enveloped viruses overcome the membrane barrier by fusion of their own lipid envelope with a cellular membrane. Viruses, which are internalised by endocytosis fuse with an internal mem-brane; others fuse at the plasma membrane (Flint et al., 2004).

For non-enveloped viruses a role for lipid rafts is restricted to the cellular membrane, like for coxsackieviruses (Triantafilou and Triantafilou, 2003, 2004). In the case of enveloped viruses, two membranes, cell membrane and viral envelope are involved in the fusion process. Lipid rafts may be required in either membrane, in both or entry may be lipid raft independent.

Murine leukemia virusis sensitive to cholesterol depletion from the cellular, but not from the viral membrane (Lu et al., 2002), whereas the reverse has been demonstrated for influenza virus (Sieczkarski and Whittaker, 2002; Sun and Whittaker, 2003; Takeda et al., 2003). Human im-munodeficiency virus (HIV) andHerpes simplex virus require cholesterol in both membranes (Liao et al., 2003, 2001; Manes et al., 2000; Graham et al., 2003; Lee et al., 2003; Bender et al., 2003). In contrastVesicular stomatitis virus(VSV) appears to be cholesterol independent (Thorp and Gallagher, 2004; Scheiffele et al., 1999; Brown and Lyles, 2003; Pessin and Glaser, 1980).

1.2. Lipid rafts 39

There are several different ways in which lipid rafts are exploited by viruses for their entry.

Perhaps the most direct way is the internalisation by a lipid raft dependent machinery such as via caveolae or by other, yet not well described, lipid raft mediated pathways (Pelkmans, 2005). The non-enveloped virusSimian virus 40(SV40) is probably the best known virus using caveolae as entry portals (Anderson et al., 1996; Stang et al., 1997; Chen and Norkin, 1999).

Some enveloped viruses, likeHuman coronavirus 229Ealso interact with caveolae for efficent internalisation (Nomura et al., 2004). A non-enveloped virus, Coxsackievirus B4, appears to exploit a lipid raft-dependent endocytic route other than caveolae to gain access to the cell (Triantafilou and Triantafilou, 2004).

Lipid rafts can also be used in more indirect ways to support virus entry. They can act as platform to concentrate proteins. Cholesterol, highly concentrated in lipid rafts, can support a special protein conformation. A prominent example is HIV which exploits lipid rafts in many ways. It has been proposed that cell membrane rafts are the regions where the HIV receptor CD4 and its co-receptors CXCR4 or CCR5 cluster upon binding of the HIV envelope protein, thereby meeting the requirement for three to six HIV envelope trimers to bind to several CD4 and four to six CCR5 molecules (Viard et al., 2002; Kuhmann et al., 2000; Manes et al., 2003).

Furthermore it has been shown that cholesterol is necessary for a correct conformation of the HIV co-creceptors CCR5 and CXCR4 (Nguyen and Taub, 2002b,a). For influenza virus which concentrates its envelope protein HA during assembly within lipid rafts it has similarly been discussed that virus envelope lipid rafts concentrate multiple HA trimers thereby facilitating successful fusion during entry (Takeda et al., 2003). The fusion process can also be supported by lipid rafts in a different way, as has been shown forSemliki forest virus, whose fusion peptide directly interacts with lipid rafts (Ahn et al., 2002).

No reports are available describing a role of cellular or envelope cholesterol for the entry process of morbilliviruses. For paramyxoviruses, there is only one recent publication reporting that although NDV assembles at lipid rafts and incorporates lipid rafts into the envelope, it does not require envelope cholesterol for cell entry (Laliberte et al., 2006). The requirement of cellular cholesterol for entry has not been investigated yet for any paramyxovirus.

40 1. Introduction

1.2.3.2 Assembly and budding

Assembly of viruses includes the association of all viral proteins and the genome in an ordered fashion. This process comprises transport and correct targeting of the proteins and concentra-tion of the proteins at the site of assembly. Enveloped viruses have to obtain their envelope membrane either by budding from an internal or from the plasma membrane. Interestingly, in many cases the composition of the virus membrane does not reflect the composition of the cellular membrane from which it is derived. This led to the proposal that virus budding often occurs at microdomains of the cell membrane (Pessin and Glaser, 1980).

Interactions of viral components with membranes during assembly are obvious for enveloped viruses, but not for non-enveloped viruses. Therefore, membrane interactions of virus proteins during assembly have been intensively investigated for enveloped viruses only (Ono and Freed, 2005). Most results concerning enveloped viruses have been obtained by investigating the in-teraction of viral wild type or mutant proteins with DRMs. Only more recent investigations include techniques such as FRET.

Lipid rafts can exert many functions during virus assembly. The interactions of viral proteins with lipid rafts have been best investigated for influenza virus and this virus exploits lipid rafts for a variety of purposes. Influenza viruses have three transmembrane proteins, the haemagglu-tinin (HA) protein, the neuraminidase (NA) protein and an ion-channel forming protein (M2), and one peripherally associated protein, the matrix (M1) protein. It has been shown that HA and NA partition, in a cholesterol dependent manner, into DRMs. Interactions of the HA and NA proteins with DRMs are necessary for a correct polar transport of these proteins to the apical side of epithelial cells (the role for lipid rafts in apical protein delivery is discussed in 1.3.2).

Finally, the high amount of cholesterol within the influenza virus envelope is dependent on the presence of intact HA and NA proteins as tailless mutants incorporate less cholesterol into the virion. The peripheral membrane protein M1 was found to be DRM associated in a timede-pendent manner and this association was also detimede-pendent on the expression of HA and NA with intact cytoplasmic tails. For M2, no DRM association was identified, but incorporation of this protein into virions is very low (reviewd in Ono and Freed (2005); Nayak and Barman (2002)).

These data suggest the following mechanism for influenza virus assembly: lipid rafts transport and target the HA and NA proteins to the apical side of epithelial cells. Additionally lipid rafts concentrate these proteins, thereby creating an assembly site. The M1 protein is dragged to

1.2. Lipid rafts 41

this site by interaction with the cytoplasmic tails of the glycoproteins and also directs the ri-bonucleocapsid via interactions to this site. The M2 protein, which is targeted to the apical side in a lipid raft independent manner, is probably mainly excluded from the lipid raft assembly site, making incorporation of this protein rare (reviewed in Ono and Freed (2005); Nayak and Barman (2002)).

Except for the functions exploited by influenza virus, lipid rafts can also be beneficial during the assembly process by facilitating multimerisation of viral proteins. In addition, specific inclusion or exclusion of cellular proteins can be advantageous. For example, HIV specifically includes the cellular decay accelerating factor (DAF or CD59), which protects cells from complement lysis, while CD45 is excluded from the viral particle in spite of its great abundance on the cell surface (Nguyen and Hildreth, 2000).

Several reports give strong evidence for an involvement of lipid rafts inparamyxovirus assem-bly. For MV, it has been shown that the H, F, M and N proteins partition into DRMs (Manie et al., 2000). The F protein has an intrinsic abilitiy to segregate into DRMs, while the H proteins becomes only detergent resistant if co-expressed with the F protein (Vincent et al., 2000). In contrast to influenza virus, the M (and N) proteins do no require the co-expression of the viral glycoproteins for partitioning into DRMs (Manie et al., 2000), but detergent resistance of the N protein requires the presence of the genome as well as of the M protein (Vincent et al., 2000).

This body of data suggests that lipid rafts are the assembly sites for MV. A model by Vincent et al. (2000) proposes that the F protein partitions into lipid rafts and additionally drags the H protein into these domains. The N protein, as RNP complex, is dragged to the assembly site by an interaction with the raft associated M protein. Association of the M protein with lipid rafts as well as interaction of the M protein with the cytoplasmic tail of the F protein, concentrates all viral components to a common assembly site within lipid rafts (Vincent et al., 2000). Indeed it has been shown that MV-components isolated from DRMs are infectious (Manie et al., 2000).

Sendai virusproteins also partition into DRMs, but in contrast to MV,Sendai virusF and HN both have intrinsic signals for a DRM association. However, mutational analyses as well as kinetic studies propose that the M protein has to interact with the cytoplasmic tail of HN or F, residing in DRMs, to also gain detergent resistance (Ali and Nayak, 2000).

Several reports also describe a role for lipid rafts in the assembly ofRespiratory syncytial virus.

Similar toSendai virusthe F proteins has intrinsic signals for association with DRMs, while the association of the M protein with DRMs depends on interaction with the F protein (Henderson et al., 2002; Fleming et al., 2006; Oomens et al., 2006; Marty et al., 2004).

42 1. Introduction

The F protein ofNewcastle disease virus(NDV) is also DRM resident (Dolganiuc et al., 2003) and very recently it has been published that also NDV HN and NP protein partition into DRMs and that virus assembly takes place at lipid rafts (Laliberte et al., 2006).