Substitutions at the Putative Receptor-Binding Site of an Encephalitic Flavivirus Alter Virulence and Host Cell Tropism and Reveal a Role for Glycosaminoglycans in Entry

Abstract

The flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (FG loop) Virus attachment to the host cell is the first stage of the virus replication cycle. It requires the molecular interaction between the virion surface and a host cell receptor and is often the basis for viral species and tissue tropisms as well as virulence properties. The cellular receptors for some viruses have been defined and reveal diverse strategies for virus attachment, ranging from binding to specific cell surface proteins to attachment via widely distributed carbohydrate moieties, such as sialic acid and heparan sulfate (for a review, see reference 48). For a large number of viruses, however, specific host cell receptors have not been identified. The use of multiple receptors on individual or different cells could be one reason for this lack of knowledge. This scenario has been proposed for the attachment of flaviviruses (35), a genus of approximately 70 mainly arthropod-borne, enveloped RNA viruses. Most flaviviruses replicate in vertebrate and arthropod cells and exhibit a wide species and tissue tropism. Numerous candidate receptor proteins with a molecular mass of 40 to 80 kDa have been found to associate with flaviviruses in binding assays (19,22,27,36,44). Furthermore, an important role of heparan sulfate has also been demonstrated for the attachment of dengue-2 virus to vertebrate cells (7). Interestingly, cell surface glycosaminoglycans (GAGs) are exploited as attachment molecules by other viruses in a process thought to concentrate virus particles at the cell surface for subsequent binding to high-affinity receptors (for a review, see reference 2). No experimental evidence on the use or nature of a high-affinity receptor for any of the flaviviruses exists at present.Flavivirus attachment and entry are mediated by the envelope (E) protein (ϳ50 kDa), the major glycoprotein on the flavivirus particle (for reviews, see references 6 and 33). The E protein forms an oligomer with the small membrane (M) protein (ϳ8 kDa) and constitutes most of the accessible virion surface; this is reflected in the dominance of the E protein as target antigen for virus-neutralizing and protective antibodies (33). The definition of the crystal structure of the ectodomain of the E protein of the flavivirus tick-borne encephalitis virus (TBE) (37), in combination with phenotypic analyses of E protein variants, has shed light on functional domains and mechanisms involved in flavivirus attachment and entry (for a review, see reference 33). An investigation by one of us on genotypic changes associated with host cell adaptation of the encephalitic flavivirus Murray Valley encephalitis virus (MVE) suggested an important role for residue 390 in the E protein in cell tropism and virulence (25). Asp 390 found in the prototype virus was altered to His, Gly, Ala, or Asn after passage of MVE in a human adenocarcinoma (SW13) cell line, resulting in improved growth in the human cell line as well as virulence attenuation in mice. Resi...

Complete genome characterization of Rocio virus (Flavivirus: Flaviviridae), a Brazilian flavivirus isolated from a fatal case of encephalitis during an epidemic in São Paulo state

“…Interestingly, in the E protein, the tripeptide (TGP) of ROCV within domain III was found to be different from those observed in all other JEV members (RGX, where X is D, E or T). As the RGD motif is speculated to be involved in the virus adsorption to host cells for JEV and MVEV, two important JEV group flaviruses (Bhardwaj et al, 2001;Lee & Lobigs, 2000) and as it was also found in ALFV and USUV, further experimental studies for ROCV are needed to investigate the role of the TGP tripeptide in the mechanisms that involve the viruscell interaction.…”

“…Interestingly, in the E protein, the tripeptide (TGP) of ROCV within domain III was found to be different from those observed in all other JEV members (RGX, where X is D, E or T). As the RGD motif is speculated to be involved in the virus adsorption to host cells for JEV and MVEV, two important JEV group flaviruses (Bhardwaj et al, 2001;Lee & Lobigs, 2000) and as it was also found in ALFV and USUV, further experimental studies for ROCV are needed to investigate the role of the TGP tripeptide in the mechanisms that involve the viruscell interaction.The functional roles of the 39-NCR of flaviviruses have been recently studied and the roles in the genomic cyclization between the 59-and 39-NCRs, viral replication and translation were elucidated. Moreover, the putative changes in the highly conserved regions related to important biological functions of the flaviviruses (survival and/or pathogenicity) were recognized (Bryant et al, 2005;Khromykh et al, 2001).…”

Encephalitic Arboviruses: Emergence, Clinical Presentation, and Neuropathogenesis

“…Indeed, mice lacking caspase-3 displayed tissue, placenta, and amniotic fluid of newborns with microcephaly and in the stillborn infants of women infected by Zika during pregnancy [28]. The envelope protein of encephalitic arboviruses is the major determinant of neurotropism and neurovirulence, owing to its role in receptor binding and virus entry [177][178][179]. However, sequence analysis of virulent and attenuated strains of these viruses revealed that other regions of the viral genome, including the 3´ untranslated region and nonstructural proteins, also contribute to neurovirulence and viral tropism [180].…”

Paradoxical effects of chondroitin sulfate-E on Japanese encephalitis viral infection

“…Flaviviruses, such as JEV, yellow fever virus, West Nile virus, Murray Valley encephalitis (MVE), and St. Louis encephalitis virus, have variable affinities for GAGs; this property contributes to viral attachment to host cells [8,12,[16][17][18][19][20][21].…”