Erich R. Mackow

The VP8 fragment of VP4 is the rhesus rotavirus hemagglutinin.

The amino-terminal trypsin cleavage fragment of VP4, called VP8, was expressed from a recombinant baculovirus in Sf-9 cells. The baculovirus-expressed VP8 protein is antigenically conserved as demonstrated by its recognition by a library of neutralizing monoclonal antibodies. In Sf-9 cell sonicates, the expressed VP8 protein is capable of agglutinating human type O erythrocytes, indicating that the functionally intact rhesus rotavirus viral hemagglutinin is contained in the 247-amino acid VP8 trypsin cleavage fragment. Amino acid similarities between VP8 and the amino-terminal 282 amino acids of the reovirus sigma 1 protein suggests that the sigma 1 hemagglutination function resides within these amino-terminal amino acids as well. When the expressed VP8 protein was used to immunize mice, a broadly cross-reactive neutralizing antibody response was obtained. Antibodies elicited to the expressed VP8 protein neutralized viruses of serotypes 1-4 and 6 but not porcine strains OSU (st5) or Gottfried (st4). The neutralizing antibody response to VP8 appeared to be more cross-reactive than the immune response to expressed VP4 or to whole RRV virion. This suggests that subunit protein immunizations may broaden the neutralizing antibody immune responses to rotaviruses and enhance protective immunity to serotypically distinct strains.

Molecular determinant of rotavirus neutralization and protection

Publisher Summary Human group A rotaviruses were first identified by Bishop et al. in 1973. In the ensuing 15 years, a great deal was learned about these important pathogens. Rotaviruses are members of the Reoviridae family. They are icosahedral viruses with two concentric capsid layers and a diameter of approximately 75 nm. The outer capsid of the virus contains a t least two primary gene products, VP4 and VP7, the products of genes 4 and 8 or 9, respectively. The chapter takes into account the recently revised nomenclature for rotavirus proteins. Genetic studies have demonstrated that VP4 is the viral hemagglutinin. Rotaviruses are the single most important cause of severe, dehydrating diarrhea in children under the age of 2. These viruses are a major cause of infantile mortality in less developed countries and the most frequent cause of morbidity due to pediatric gastroenteritis in developed countries. Rotaviruses can be isolated from the young of many, if not most, mammalian species, and in many cases these viruses are the cause of infantile diarrhea in the mammalian species from which they are isolated.

Characterization of homotypic and heterotypic VP7 neutralization sites of rhesus rotavirus

The gene 9 nucleotide sequence was determined for rhesus rotavirus and each of 14 viral variants selected for their resistance to neutralizing monoclonal antibodies. Each variant contains a single gene 9, VP7, mutation which permits viral growth in the presence of the antibody. Variant mutations were identified in two distinct neutralization regions. Region A was identified by monoclonal antibodies that are involved in both serotype-specific and serotype cross-reactive neutralization. Region C was identified by serotype-specific neutralizing monoclonal antibodies. Heterotypic neutralizing monoclonal antibody 57-8 selected variants with a mutation at amino acid 94 in the A region, the same amino acid location selected by serotype-specific monoclonal antibodies. Monoclonal antibody 3 selected a VP7 mutation at amino acid 99 resulting in additional N-linked glycosylation of the VP7 protein. Despite the added VP7 glycosylation, variant v3 was not broadly resistant to additional VP7-specific neutralizing monoclonal antibodies.

Identification and partial characterization of a rhesus rotavirus binding glycoprotein on murine enterocytes

In order to assess the possibility that rotavirus binds to a specific cellular receptor on enterocytes, we have used a viral overlay protein blot assay to study viral binding to murine intestinal brush border membranes (BBM). Infectious double-shelled particles of rhesus rotavirus bound specifically to two approximately 300- and 330-kDa glycoproteins from BBM prepared from suckling mice. Significantly less rotavirus binding was observed when adult BBM were examined. Rats have never been shown to harbor natural group A rotavirus infection and correspondingly, rat BBM showed no rotavirus binding activity. In suckling mice, rotavirus was found to bind to villus tip membranes to a much greater extent than to crypt preparations. Rotavirus binding activity was abolished by treatment of membrane preparations with protease. Analysis by glycolytic digestion of BBM with N- and O-glyconases revealed evidence for both N- and O-linked glycosylation of the rotavirus binding protein. Also neuraminidase digestion showed that O-linked sialic acid residues were required for virus binding. Monoclonal antibodies which immunoprecipitate the 300-kDa viral binding glycoprotein react with the apical surface of suckling but not adult enterocytes by Western blot. Baculovirus-expressed vp4, the rotavirus outer capsid spike protein, bound to the 300- and 330-kDa proteins and competed with rotavirus particles for binding sites. The ability of rotavirus to bind via vp4 to large BBM glycoproteins correlates with in vivo rotavirus cell tropism and host range restriction. Specific host cell receptor expression may be important in rotavirus pathogenesis.

Identification of the gene encoding the group B rotavirus VP7 equivalent: Primary characterization of the ADRV segment 9 RNA

Gene segment 9 of the adult diarrheal rotavirus, ADRV, has been cloned, and the full-length gene 9 cDNA has been sequenced. Sequences at the 5 and 3 termini of the gene 9 double-stranded RNA were confirmed by direct RNA sequencing. The ninth RNA segment is 814 bases in length with a single open reading frame beginning at base 10 and terminating at base 757. The encoded polypeptide contains 249 amino acids with a calculated molecular weight of 28.5 kDa. The protein contains three potential N-linked glycosylation sites and a hydrophobic signal-like sequence at its amino terminus. A search of the NBRF protein data base with the encoded polypeptide revealed extensive similarities with VP7 proteins from a number of group A rotaviruses. Direct comparisons of the ADRV gene 9 polypeptide and the group A rotavirus VP7 demonstrate that the two proteins share 78% amino acid similarity and 28% identity as well as predicted secondary structure similarities. These findings suggest that the ADRV gene segment 9 encodes the VP7 polypeptide equivalent of group A rotaviruses.

Expression of the major inner capsid protein of the group B rotavirus ADRV: Primary characterization of genome segment 5 Primary characterization of genome segment 5☆

A complete cDNA copy of the fifth RNA segment of the human group B rotavirus, ADRV, has been cloned into plasmid AD512. Gene segment 5 contains 1269 bases and encodes one long open reading frame of 391 amino acids beginning at base 31 and terminating at base 1203. The gene 5 polypeptide, expressed in vitro in a rabbit reticulocyte lysate, comigrates with the 44-kDa major inner capsid protein present on EDTA treated ADRV virions. The gene 5 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent serum and by a group B-specific monoclonal antibody. In addition, this protein shares amino acid identity and similarity with the VP6 proteins from group C and group A rotavirus strains. The ADRV VP6 equivalent protein appears to be more closely related to the group C VP6 than the Group A VP6 polypeptide and a common ancestral rotavirus VP6 precursor protein is suggested. As a result, the fifth RNA segment of ADRV defines the major inner capsid protein, or VP6 equivalent, in the group B rotavirus. Expression of the ADRV VP6 equivalent protein is potentially useful for evaluating the prevalence of serum antibodies to group B rotavirus in human and animal populations as well as for generating antibodies for the direct detection of group B rotavirus antigen.

cDNA cloning of each genomic segment of the group B rotavirus ADRV: molecular characterization of the 11th RNA segment.

The group B noncultivatable rotavirus, ADRV, was purified from infected stool specimens. Double-stranded RNA was extracted, polyadenylated, reverse transcribed into cDNA, and cloned into plasmid vector pAT153. Each cDNA clone hybridized to a single ADRV RNA segment and cDNA clones of each genomic RNA segment were identified. Sequencing of genomic RNA and a full-length cDNA clone (AD63) determined that ADRV RNA segment 11 is 631 bases in length and contains a single open reading frame of 170 amino acids with a calculated molecular weight of 19.9 kDa and a pl of 6.2. The RNA 5- and 3-termini contain untranslated regions of 58 and 63 bases, respectively, and are complementary to each other. A comparison of encoded ADRV gene 11 amino acids with the NS26 protein of group A rotaviruses demonstrates a distant similarity between the two proteins. Further analysis and use of these ADRV clones should aid in determining the gene coding assignments for group B proteins as well as for diagnostic detection of group B or ADRV-specific nucleic acids in specimens.