Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

Abstract

Integrins ␣2␤1, ␣X␤2, and ␣V␤3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the ␣2␤1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the ␣X␤2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of ␣2␤1, ␣X␤2, and ␣V␤3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound ␣2␤1, and VP7 interacted with ␣X␤2 and ␣V␤3 at a postbinding stage. DGEA inhibited rotavirus binding to ␣2␤1 and infectivity, whereas GPRP binding to ␣X␤2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed ␣2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the ␣2 I domain. In a novel process, integrin-using viruses bind the ␣2 I domain of ␣2␤1 via DGE in VP4 and interact with ␣X␤2 (via GPR) and ␣V␤3 by using VP7 to facilitate cell entry and infection.Rotaviruses are leading causes of acute gastroenteritis in human infants and young animals. Their restricted tropism suggests that very specific virus-host cell interactions are necessary to establish infection. The viral spike protein VP4, the major cell attachment protein, is cleaved by trypsin for enhanced infectivity into two subunits, VP5* (60 kDa) and VP8* (28 kDa). VP4 and the major outer capsid protein VP7 independently define serotype specificities. The inner capsid protein VP6 contains group-specific antigenic determinants. Reassortant rotaviruses containing combinations of the 11 double-stranded RNA genes from two parental viruses can be generated (27) which occasionally show unexpected phenotypes due to VP4-VP7 interactions (43).Integrins ␣2␤1, ␣X␤2, and ␣4␤1 were implicated in group A rotavirus cell attachment and entry (11, 23), and ␣V␤3 was proposed to mediate rotavirus cell entry (20). Integrin usage by rotaviruses was discovered when VP5* was shown to contain the type I collagen-derived, ␣2␤1 ligand sequence DGE at amino acids 308 to 310, and the fibrinogen-derived ␣X␤2 integrin ligand sequence GPR was identified in VP7 at amino acids 253 to 255 (11). Peptides containing these viral integrin ligand sequences (GPRP and RDGEE), monoclonal antibodies (MAbs) to ␣2␤1 and MAbs to ␤2, inhibited the infection of monolayers of MA104 and Caco-2 cells by simian rotavirus SA11 and/or human rotavirus RV-5 by 30 to 90% in additive fashion (9, 11). Infectivity blockade in MA104 cell monolayers...

Cholangiocyte expression of α2β1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

“…The peptide sequence arginine-glycine-aspartic acid (RGD) is the laminin-binding domain to the ␣ 2␤1-integrin (28). The peptide sequence glycine-proline-arginine (GPR) is the fibrinogen-binding domain to the ␣ v␤3-integrin (13). The peptide sequence GHRP served as a negative control.…”

“…Consistent with the in vivo findings, RRV was better able to replicate in cholangiocytes than hepatocytes. Because the ability of rotavirus to infect cells appears to be regulated by cell-surface expression of the integrins ␣ 2 ␤ 1 , ␣ 4 ␤ 1 , ␣ v ␤ 3 , and ␣ x ␤ 2 , which serve as viral receptors (8,13,14,16,21), the cholangiocyte was surveyed for integrin expression and found to uniquely express the ␣ 2 ␤ 1 -integrin when compared with hepatocytes. Based on this information, we hypothesized that expression of the ␣ 2 ␤ 1 -integrin serves as a determinant governing cholangiocyte susceptibility to RRV infection contributing to the mechanism by which rotavirus initiates the experimental model of biliary atresia.…”

Integrin-dependent phagocytosis – spreading from microadhesion to new concepts

“…Similarly, the uptake of adenovirus type 3 (Ad3) was recently shown to be dependent on the αV integrin and on Rac1-and Pak-1-dependent actin remodelling (Amstutz et al, 2008). Finally, α2β1, αXβ2, α4β1 and α4β7 integrins promote the entry of rotaviruses (Coulson et al, 1997;Graham et al, 2005;Graham et al, 2003;Graham et al, 2006;Guerrero et al, 2000;Hewish et al, 2000), although it remains to be seen whether this follows a phagocyticlike process.Overall, several viral and bacterial pathogens use integrin signalling to enter host cells in an actin-dependent process. Deciphering the molecular pathways that are used for local, transient, integrin-triggered actin and membrane remodelling should aid in the design of drugs that will help fight infection in vitro and in vivo.…”

Rotavirus specific maternal antibodies and immune response to RV3-BB neonatal rotavirus vaccine in New Zealand

“…The RV3-BB vaccine is derived from a neonatal rotavirus strain which binds to specific receptors in the newborn gut and is integrin independent and, may inherently offer protection from maternal antibodies. 34 In this study, high levels of IgA in colostrum or breast milk did not appear to inhibit the serum IgA response or stool excretion following 3 doses of RV3-BB rotavirus vaccine in either a neonatal schedule with the first dose at age 0-5 d or an infant schedule with the first dose at 8 weeks of age. These findings are consistent with studies from North India 25 and South Africa 26 which found little difference in anti-rotavirus IgA seroconversion irrespective of whether breastfeeding was withheld for either 30 minutes or one hour before and after RV1 vaccine administration.…”

“…However, a potential advantage of a vaccine based on a human neonatal rotavirus strain delivered at birth is the proposed intrinsic structural characteristics that enable it to avoid neutralisation by maternal and breast milk antibodies. 34 Data from the placebo group provided a negative control for the rate of natural rotavirus infection in this study population. Interestingly one of the participants in the placebo group was excreting RV3-BB rotavirus vaccine in the stool at both 20 weeks and 28 weeks, which suggested presence of this strain in the community.…”