Growth of rotaviruses in continuous human and monkey cell lines that vary in their expression of integrins

Abstract

Rotavirus replication occurs in vivo in intestinal epithelial cells. Cell lines fully permissive to rotavirus include kidney epithelial (MA104), colonic (Caco-2) and hepatic (HepG2) types. Previously, it has been shown that cellular integrins α2β1, α4β1 and αXβ2 are involved in rotavirus cell entry. As receptor usage is a major determinant of virus tropism, the levels of cell surface expression of these integrins have now been investigated by flow cytometry on cell lines of human (Caco-2, HepG2, RD, K562) and monkey (MA104, COS-7) origin in relation to cellular susceptibility to infection with monkey and human rotaviruses. Cells supporting any replication of human rotaviruses (RD, HepG2, Caco-2, COS-7 and MA104) expressed α2β1 and (when tested) αXβ2, whereas the non-permissive K562 cells did not express α2β1, α4β1 or αXβ2. Only RD cells expressed α4β1. Although SA11 grew to higher titres in RD, HepG2, Caco-2, COS-7 and MA104 cells, this virus still replicated at a low level in K562 cells. In all cell lines tested, SA11 replicated to higher titres than did human strains, consistent with the ability of SA11 to use sialic acids as alternative receptors. Levels of cell surface α2 integrin correlated with levels of rotavirus growth. The α2 integrin relative linear median fluorescence intensity on K562, RD, COS-7, MA104 and Caco-2 cells correlated linearly with the titre of SA11 produced in these cells at 20 h after infection at a multiplicity of 0n1, and the data best fitted a sigmoidal dose-response curve (r 2 l 1n00, P l 0n005). Thus, growth of rotaviruses in these cell lines correlates with their surface expression of α2β1 integrin and is consistent with their expression of αXβ2 and α4β1 integrins.

VLA-2 (α2β1) Integrin Promotes Rotavirus Entry into Cells but Is Not Necessary for Rotavirus Attachment

“…Although cell lines of renal and intestinal origin are the most susceptible to rotavirus infection, cell lines of human cervix (HeLa), liver (HepG2), myelogenous leukemic (K562), or embryonal rhabdomyosarcoma (RD) cells, mouse embryo (3T3) or fibroblast (L929) cells, and hamster ovary (CHO) cells have also been used for studies of rotavirus-cell interactions (5,23,33,36,42,44,56). For comparison, we retested some cell lines of nonrenal or nonintestinal origin, as well as nonrenal or nonintestinal cell lines never tested before, for susceptibility to rotavirus infection.…”

“…These heterodimeric glycoproteins act as bidirectional signaling receptors and can serve as cellular receptors or coreceptors for several viruses, including echoviruses 1 and 8 (8,9), coxsackievirus A9 (53), foot-and-mouth disease virus (39,40), adenoviruses (65), and papillomavirus (25). Recently, VLA-2 (␣2␤1), VLA-4 (␣4␤1), CR4 (␣x␤2), and ␣v␤3 integrins have been implicated in rotavirus cell attachment, entry, or postattachment events in infection (17,33,36,44,68). The rotavirus outer capsid proteins VP4 and VP7 contain sequence motifs that are potentially recognized by VLA-2, VLA-4, and CR4 but not ␣v␤3, and antibodies to these integrins or peptides containing these recognition motifs block approximately 30 to 50% of rotavirus infectivity (17,33,36,44).…”

“…Recently, VLA-2 (␣2␤1), VLA-4 (␣4␤1), CR4 (␣x␤2), and ␣v␤3 integrins have been implicated in rotavirus cell attachment, entry, or postattachment events in infection (17,33,36,44,68). The rotavirus outer capsid proteins VP4 and VP7 contain sequence motifs that are potentially recognized by VLA-2, VLA-4, and CR4 but not ␣v␤3, and antibodies to these integrins or peptides containing these recognition motifs block approximately 30 to 50% of rotavirus infectivity (17,33,36,44). These studies have shown that (i) VLA-2 and VLA-4 expressed in poorly susceptible human erythroleukemic K562 cells increase binding and infectivity of the SA-dependent SA11 strain (36), (ii) the susceptibility to rotavirus infection correlates with the amount of VLA-2 expressed on the surfaces of six human cell lines exhibiting various degrees of susceptibility to rotavirus infection (44), (iii) VLA-2 is the initial receptor for a neuraminidase-resistant (SA-independent) nar3 variant but not for the parental SA-dependent RRV strain (68), and (iv) ␣v␤3 promotes rotavirus cell entry at a postbinding step (33).…”

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

“…Flow cytometric data are expressed as the relative linear median fluorescence intensity (MFI), defined as the median fluorescence intensity with test MAb divided by the median fluorescence intensity with isotype-matched control MAb. An MFI of Ն1.20 was considered positive (34). The origins of the rotaviruses used in this study and their cultivation in MA104 cells have been described previously (10, 27, 31-33, 39, 40).…”

“…In the presence of MAb, cells were cooled on ice for 20 min. Virus infectivity was activated with 10 g of porcine trypsin (Sigma)/ml for 20 min at 37°C (34). Activated virus, which had been cooled to 4°C, at a multiplicity of infection (MOI) of 3.5 was allowed to attach to cells on ice for 1 h. This MOI was chosen because insufficient infectious virions bound to most cell lines for the infectivity counts to be reliable at lower MOI (Յ1).…”