The interaction of rotavirus with its host cell is a multistep process

Several lines of evidence suggest that rotaviruses need to interact with more than one cell surface molecule to enter the cell, using during this process different

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FIG. 1. Distinctive structural features of the outer shell protein VP4. The trypsin cleavage region is indicated by an arrow, which defines the boundary between VP8 and VP5. In VP8, the haemagglutination domain (HA) (aa 93 to 208) is shadowed; the asterisks below this domain indicate aa 155 and 188—190, which are important in the SA binding activity of this protein. The disulfide bridges between Cys203 and Cys216, and between Cys318 and Cys380, are indicated by S=S. In VP5, the position of the DGE and IDA tripeptide sequence binding motifs which might putatively be recognized by integrins a2^1 and a4;81, respectively, are shown. The hydrophobic region (HR), which has been proposed to be a putative fusion domain, and a predicted heptad repeat (coiled-coil) which might form part of a coiled-coil structure are also depicted.

FIG. 1. Distinctive structural features of the outer shell protein VP4. The trypsin cleavage region is indicated by an arrow, which defines the boundary between VP8 and VP5. In VP8, the haemagglutination domain (HA) (aa 93 to 208) is shadowed; the asterisks below this domain indicate aa 155 and 188—190, which are important in the SA binding activity of this protein. The disulfide bridges between Cys203 and Cys216, and between Cys318 and Cys380, are indicated by S=S. In VP5, the position of the DGE and IDA tripeptide sequence binding motifs which might putatively be recognized by integrins a2^1 and a4;81, respectively, are shown. The hydrophobic region (HR), which has been proposed to be a putative fusion domain, and a predicted heptad repeat (coiled-coil) which might form part of a coiled-coil structure are also depicted.

domains of the virus surface protein VP4 (Fig. 1). The following studies, which support these multiple interactions, were carried out in the rhesus monkey kidney epithelial cell line MA104, which is highly susceptible for rotavirus infection.

(a) In an infection assay designed to detect competition for cell surface molecules at both attachment and post-attachment steps (Mendez et al 1999), it was found that HRV Wa efficiently competed with the infectivity of the SA-dependent porcine rotavirus strain YM, and that of the variant nar3 both in untreated, as well as in NA-treated cells. This competition was non-reciprocal since YM and nar3 did not compete with the infectivity of Wa. In contrast, a two-direction competition between the variant nar3 and a SA-dependent strain was found. The fact that the competition between the two NA-resistant strains nar3 and Wa was not reciprocal indicates that they bind to different molecules. In addition, the SA-dependence phenotype clearly differentiates strains, like RRV or YM, from nar3 and Wa. Altogether, these findings suggest the existence of at least three cellular structures involved in rotavirus cell infection, with at least one being shared by human, SA-dependent, and animal, NA-resistant, variant strains.

(b) The comparison of the binding characteristics of wild-type RRV (wtRRV) and nar3 to MA104 cells showed that both the SA-dependent and SA-independent interactions of these viruses with the cell are mediated through two different domains of VP4 (Mendez et al 1993). It was shown that RRV

TABLE 1 Inhibition of binding and infectivity of RRV and nar3 viruses by MAbs to VP4 and by VP8 and VP5 recombinant proteins

% Binding and infectivity in the presence of the indicated MA bs or recombinantproteins

TABLE 1 Inhibition of binding and infectivity of RRV and nar3 viruses by MAbs to VP4 and by VP8 and VP5 recombinant proteins

% Binding and infectivity in the presence of the indicated MA bs or recombinantproteins

no

aVP8

a.VP5

GST-

GST-

Virus

MAb

(7A12)

(2G4)

GST

VP8

VPS

Bindinga

RRV

100

9

84

102

25

97

nar3

100

72

9

99

100

24

Infectivityb

RRV

100

8

9

87

44

102

nar3

100

95

16

110

104

50

aExpressed as the percentage of virus binding in the absence of antibodies or recombinant proteins. ^Expressed as percentage of the virus infectivity obtained in the absence of antibodies or recombinant proteins. The arithmetic means from two independent experiments performed in duplicate are shown.

aExpressed as the percentage of virus binding in the absence of antibodies or recombinant proteins. ^Expressed as percentage of the virus infectivity obtained in the absence of antibodies or recombinant proteins. The arithmetic means from two independent experiments performed in duplicate are shown.

attaches to the cell through VP8, while nar3 does so through the VP5 domain of VP4 (Zarate et al 2000a). This observation is supported by the fact that neutralizing antibodies to VP8 block the attachment to cells of RRV, but not of its variant nar3, while a monoclonal antibody (MAb) to VP5 (2G4) inhibits the binding of nar3, but not that of RRV. In addition, recombinant VP8 and VP5 proteins produced in bacteria as fusion products with glutathione S-transferase (GST), are capable of inhibiting the binding and infection of wild-type and variant viruses, respectively, when pre-incubated with the cell (Table 1, Zarate et al 2000b). While nar3 only needs to interact (through VP5) with the NA-resistant receptor, wtRRV seems to engage in the two interactions described in a sequential manner, since MAb 2G4, despite selectively blocking the binding of nar3, efficiently neutralizes the infectivity of both viruses (see also below). (c) The sequential interaction of RRV with two molecules on the surface of MA104 cells is further supported by the observation that MAb 2D9, which is directed to a cell surface antigen, specifically blocks the infectivity of both wtRRV and nar3, but competes only with the attachment of the variant, indicating that wtRRV is blocked at a post-binding step (Lopez et al 2000). Since MAb 2D9 also blocks the infectivity of nar3 in NA-treated cells, and prevents the cell attachment of the recombinant protein GST—VP5, but does not affect the binding of GST—VP8 (Fig. 3), it would seem to be directed to the NA-resistant receptor used by nar3 to attach to the cell, or to a molecule closely associated with it.

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