Expression and Functional Characterization of Bluetongue Virus VP2 Protein: Role in Cell Entry

Abstract

Segment 2 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP2, was tagged with the S-peptide fragment of RNase A and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity by virtue of the S tag, and the oligomeric nature of the purified protein was determined. The data obtained indicated that the majority of the protein forms a dimer and, to a lesser extent, some trimer. The recombinant protein was used to determine various biological functions of VP2. The purified VP2 was shown to have virus hemagglutinin activity and was antigenically indistinguishable from the VP2 of the virion. Whether VP2 is responsible for BTV entry into permissive cells was subsequently assessed by cell surface attachment and internalization studies with an immunofluorescence assay system. The results demonstrated that VP2 alone is responsible for virus entry into mammalian cells. By competition assay, it appeared that both VP2 and the BTV virion attached to the same cell surface molecule(s). The purified VP2 also had a strong affinity for binding to glycophorin A, a sialoglycoprotein component of erythrocytes, indicating that VP2 may be responsible for BTV transmission by the Culicoides vector to vertebrate hosts during blood feeding. Further, by various enzymatic treatments of BTV-permissive L929 cells, preliminary data have been obtained which indicated that the BTV receptor molecule(s) is likely to be a glycoprotein and that either the protein moiety of the glycoprotein or a second protein molecule could also serve as a coreceptor for BTV infection.

Bluetongue virus coat protein VP2 contains sialic acid-binding domains, and VP5 resembles enveloped virus fusion proteins

“…The density map shows the protruding nature of the tip domain and its possession of a cavity lined with β-sheets. The outermost location of this surface rich in β-sheets suggests that the tip domain is well positioned for the initial encounter of the virus with the host cell plasma membrane (13).…”

“…Even though there is no sequence homology between the rotavirus VP8 and the BTV VP2, our above structural findings suggest that VP2 binds SA, and this assignment was further investigated using biochemical approaches. BTV particles agglutinate erythrocytes of ruminants (15), and VP2 alone is responsible for this activity (13). To determine whether SA is available on the plasma membrane of permissive cells for BTV infection, we stained HeLa cells with fluorescently labeled wheat germ agglutinin (WGA), a lectin that binds SA and N-acetylglucosamine sugar residues (16) Previous biochemical studies have shown that VP5 causes membrane leakiness and that its expression at the cell surface induces cell-cell fusion to produce syncytia upon exposure to low pH, similar to that found in the endosomal milieu (11).…”

African horse sickness in naturally infected, immunised horses

“…It is likely that the RT-PCR assay detected erythrocyte-associated AHSV RNA, which was not necessarily infectious. Bluetongue virus, an orbivirus closely related to AHSV, has been shown to be erythrocyte bound [5], and the lifespan of an erythrocyte in the circulation can be in excess of 145 days [6], which may also explain the extended detection of AHSV nucleic acid in the blood of infected horses by RT-PCR. The period during which horses can be infectious to midges is not known but, given the potential importance to virus transmission in the field, this aspect of the pathogenesis of AHSV infection of horses warrants further investigation.…”

A Clathrin Independent Macropinocytosis-Like Entry Mechanism Used by Bluetongue Virus-1 during Infection of BHK Cells

“…The outer capsid is formed from two viral proteins, VP2 and VP5, while the inner core-particle consists of a 'core-surface' layer composed of VP7, which surrounds a sub-core shell composed of VP3. VP2 interacts with host cellsurface receptors serving as a virus-attachment protein while VP5 is involved in host-cell membrane penetration [41,42,43]. The outer capsid proteins are removed during cell entry, and transcriptionally active core particles are released into the cytosol where virus replication occurs [44,45].…”