Rotavirus Attachment, Internalization, and Vesicular Traffic
Rotavirus Attachment, Internalization, and Vesicular Traffic
S. López Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México
Rotaviruses, members of the family Reoviridae, are nonenveloped cytopathic viruses that have a very exquisite tropism, infecting mainly mature enterocytes on the tips of the small intestinal villi and causing a severe gastroenteritis in children and in the young of many animal species. Over the past few years we have learned about the interactions of rotaviruses with cell surface molecules that allow them to recognize and enter their target cell through different endocytic pathways. The endocytic process leads viral particles onto an intracellular vesicular traffic during which they are uncoated in distinct endosomal compartments, depending on the virus strain, and end up as transcriptionally active viral particles in the cytosol. Here, we summarize our advances in this area.
attachment and postattachment interactions
receptors and coreceptors
1. Initial interactions of the virus with the host cell
Rotavirus particles are formed by a triple-layered capsid that surrounds the viral genome composed of eleven double-stranded RNA segments. The outermost layer of the viral particle is made of 780 copies of the glycoprotein VP7 arranged in trimers coordinated by calcium, which form a smooth surface layer from which spikes composed of trimers of VP4 protrude. To be infectious the virus depends on the specific cleavage by trypsin of VP4 into VP8 and VP5. Both cleavage domains of VP4, as well as VP7, have essential roles in the initial steps of the replication cycle of rotaviruses.
1.1. Virus Attachment
Rotavirus cell entry is a multistep process involving cellular glycans for cell binding and several cell surface molecules during postattachment steps (Lopez and Arias, 2006) (Fig. 2.2.1A). The VP8 domain of VP4 mediates the initial interaction of the virus with the cell surface, whereas the VP5 domain of VP4 and the surface glycoprotein VP7 interact with downstream postattachment molecules (Lopez and Arias, 2006). Different rotavirus strains were initially classified as neuraminidase (NA)-sensitive or NA-resistant, depending on their ability to infect cells previously treated with NA. Some animal rotavirus strains require sialic acid (SA) for attachment, whereas other animal and most human rotavirus strains are NA-resistant (Lopez and Arias, 2006). Recently, it was found that some rotavirus strains, which were originally classified as NA-resistant, bind to internal SAs, which are not cleaved by NA (Haselhorst et al., 2007), while others bind to human blood group antigens (HBGAs) (Table 2.2.1).
The SA binding domain of the virus is located on the tip of VP8 (Dormitzer et al., 2002; Isa et al., 1997). The crystal structure of several VP8 proteins has been resolved (Dormitzer et al., 2002; Blanchard et al., 2007; Monnier et al., 2006; Yu et al., 2011); this protein has a galectin-like fold with two β-sheets separated by a shallow cleft. The crystal structures of VP8 of NA-sensitive rotavirus strains complexed with SA showed that SA binds near the cleft region. Interestingly, when the VP8 proteins of NA-resistant strains were analyzed, this cleft was found to be wider (Haselhorst et al., 2009). Even though the structure of a human VP8 with a wider cleft in complex with a glycan has not been reported, NMR and modeling studies propose that a wider cleft would allow binding of gangliosides with internal SA (Venkataram Prasad et al., 2014).
Gangliosides and glycosphingolipids having one or more SA residues, are a large and heterogeneous family of lipids present on the extracellular leaflet of mammalian plasma membranes, and have been associated with rotavirus cell entry for some time. Using a thin-layer chromatography binding assay, NA-sensitive rotavirus strains were found to bind to gangliosides with terminal SA, while the NA-resistant strains tested interacted with gangliosides containing subterminal SA (Delorme et al., 2001). The role of gangliosides during the infection of MA104 cells by NA-sensitive and -resistant strains was also analyzed after silencing by RNA interference the expression of two key enzymes involved in ganglioside synthesis: UDP-glucose:ceramide glucosyltransferase and lactosyl ceramide-α-2,3-sialyl transferase 5 (Martinez et al., 2013). Knocking down the expression of these enzymes decreased ganglioside levels, resulting in a diminished infectivity of all rotavirus strains tested. Interestingly, the binding of virus to cells with low ganglioside levels was not affected, suggesting that the rotavirus-ganglioside interaction is not necessary for cell surface binding, but during a later step of the cell entry process.
Recently, it was demonstrated that many human rotavirus strains bind to human histo-blood group antigens (HBGAs). Some of them bind H-type glycans, while others have A-type glycan specificity (Bohm et al., 2015; Hu et al., 2012; Huang et al., 2012; Liu et al., 2012) (Table 2.2.1). In addition, the VP8 of a P neonatal strain, was shown to specifically bind a precursor of the H type II HBGA, which forms the core structure of type II glycans (Ramani et al., 2013). The crystal structure of a [P14] VP8 showed that it has a narrow cleft, like that present in NA-sensitive strains, and that the A-type HBGA binds in the same location in the cleft as SA in the animal VP8 structure (Venkataram Prasad et al., 2014). Of interest, glycan modification is thought to vary during neonatal development, which could explain the age-restricted infectivity of neonatal rotavirus strains. It is also of interest that the HBGA phenotype and the secretory status of children seem to correlate with the VP4 genotype of the infecting rotavirus strain (Nordgren et al., 2014; Van Trang et al., 2014).
1.2. Postattachment Interactions
After the initial attachment to glycans on the cell surface, rotaviruses interact with other surface molecules to gain access into the cell. Among these molecules there are several integrins (α2β1, α4β1, αXβ2, αVβ3) and the heat shock cognate protein 70 (hsc70). Whether all these molecules are used by all rotavirus strains, and whether these interactions are sequential or alternative, is not known. In the particular case of a rhesus rotavirus strain (RRV) it was shown that some of these interactions occur sequentially (Lopez and Arias, 2006). Interestingly, not all rotavirus strains interact with integrins, while all the strains tested require hsc70 for efficient cell infection (Gutierrez et al., 2010).
The interaction of rotavirus with integrin α2β1 is mediated by a DGE motif located towards the amino-terminal end of the VP5 domain of VP4, while the domain I of the integrin subunit α2 is involved in this interaction (Graham et al., 2003; Zarate et al., 2004). The amino acid residues on the I domain that interact with the virus spike protein were identified by expressing α2β1 integrin mutants in CHO cells (Fleming et al., 2011). On the other hand, integrin αVβ3 interacts with rotavirus through a linear sequence in VP7 (Graham et al., 2003; Zarate et al., 2004).
A cellular molecule used by all rotavirus strains is hsc70 (Gutierrez et al., 2010; Guerrero et al., 2002). The interaction between the viral particle and hsc70 is mediated by VP5, through a domain located between amino acids 642 and 659 (Zarate et al., 2003). Since a synthetic peptide corresponding to this region blocks virus infectivity but not cell binding, it has been suggested that the interaction between the virus and hsc70 is at a postattachment step. Apparently the ATPase domain of hsc70 is involved in promoting conformational changes in the viral particle that facilitate virus entry (Perez-Vargas et al., 2006).
Additionally, some of the molecules that participate in attachment and postattachment interactions (gangliosides, integrins α2β1, αVβ3, and hsc70) group together in detergent-resistant membrane domains (lipid rafts), where infectious viral particles are also present during cell infection (Isa et al., 2004). The integrity of these domains is fundamental for viral infection, since their destabilization severely decreases the infectivity of all rotavirus strains tested (Gutierrez et al., 2010; Guerrero et al., 2000) (Fig. 2.2.1A).
Rotaviruses are classical gastrointestinal viruses that infect the mature enterocytes located at the tip of the small intestinal villi. Given the basolateral localization of integrins in these cells, an important question is how rotaviruses reach these cell receptors to enter the cell. One explanation was offered when it was shown that a recombinant VP8 protein of RRV is able to decrease the transepithelial electrical resistance of polarized Madin–Darby canine kidney (MDCK) cells (Nava et al., 2004). Furthermore, this VP8 protein was shown to open the tight junctions, releasing basolateral proteins (integrins αVβ3, β1, and the Na+-K+-ATPase) to the apical side of the cells (Nava et al., 2004). In addition, it has recently been found that the tight-junction proteins JAM-A, occludin, and ZO-1 are important for the entry of some rotavirus strains (Torres-Flores et al., 2015).
It remains to be established whether all the described molecules work in concert, or represent alternative routes of entry. However, it is noteworthy that the assays used to block the interaction of rotaviruses with each of these proposed receptors and coreceptors using different approaches, such as proteases, antibodies, peptides, sugar analogues, or siRNAs, only decrease viral infectivity by less than 10-fold, suggesting that either a more relevant entry factor for rotavirus has yet to be found, the virus can use more than one route of entry, or the cellular factors that allow the entry of rotavirus are redundant.
2. Virus internalization
Most viruses hijack cellular endocytic pathways to reach the cell’s interior. The mechanisms more frequently used by these pathogens to enter the cell are either clathrin-dependent, or caveolae-mediated endocytosis, as well as macropinocytosis (Yamauchi and Helenius, 2013). Also, most endocytic pathways described to date depend on dynamin, a GTPase implicated in several membrane scission events and required during the endocytic process (Praefcke and McMahon, 2004).
It was originally proposed that rotaviruses enter cells via direct penetration at the plasma membrane. However, in recent years the role of the endocytic process in rotavirus cell entry has been carefully reevaluated using pharmacological inhibitors of endocytosis, overexpression of dominant-negative mutant proteins, and RNA interference to knockdown the expression of proteins implicated in different endocytic routes (Table 2.2.2). Using these tools it has been clearly established that rotaviruses enter cells by endocytosis, however, different strains use different endocytic pathways (Gutierrez et al., 2010) (Fig. 2.2.1B). Human rotavirus strains Wa, DS-1, WI69, and animal strains UK, YM, SA11-4S, and nar3 enter through clathrin-mediated endocytosis (Gutierrez et al., 2010; Diaz-Salinas et al., 2013), while RRV uses an atypical endocytic pathway which is clathrin- and caveolin-independent, but depends on dynamin 2, and on the presence of cholesterol (Sanchez-San Martin et al., 2004; Silva-Ayala et al., 2013). The requirement for cholesterol and dynamin is also shared by those rotaviruses that are internalized into MA104 cells by clathrin-dependent endocytosis (Gutierrez et al., 2010) (Fig. 2.2.1B, and Table 2.2.2). In contrast with these observations, it was found that the entry of RRV was not affected by dynasore, a chemical inhibitor of dynamin, in polarized MDCK, BSC-1, and MA104 cells (Abdelhakim et al., 2014; Wolf et al., 2011). Differences in the cells used, and in the methods employed to determine the role of dynamin on rotavirus infectivity might account for these discrepancies. That rotaviruses enter via endocytosis is also supported by the observation that actinin 4 and the small GTPases RhoA and Cdc42, as well as the activator of the latter, CDGAP, which are involved in different types of endocytic processes, have been implicated in the entry of all rotavirus tested so far (Silva-Ayala et al., 2013; Diaz-Salinas et al., 2014).