ABSTRACT
The non-structural protein NS3 was investigated in Ibaraki virus (IBAV), an epizootic hemorrhagic disease virus, serotype 2. Degree of NS3 glycosylation, cytopathic effect, and virus release efficiency were compared between mammalian and insect cells. The molecular weight of synthesized NS3 was compared in Western blot analysis following the removal of the glycochain by PNGase F treatment and revealed that glycosylation of NS3 occurred only in mammalian cells. Also, it was revealed that the amount of infectious IBAV in the extracellular fraction continued to increase for insect cells even after 60h post infection without disrupting cells. These results suggested that glycosylation of NS3 controls pathogenicity of IBAV in host cells to protect vector insects by altering the release pathway of assembled progeny viruses.
Subject(s)
Hemorrhagic Disease Virus, Epizootic/metabolism , Hemorrhagic Disease Virus, Epizootic/pathogenicity , Host-Pathogen Interactions , Viral Nonstructural Proteins/metabolism , Animals , Cell Line , Cricetinae , Cytopathogenic Effect, Viral , Gene Expression , Glycosylation , Hemorrhagic Disease Virus, Epizootic/genetics , Humans , Molecular Weight , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Virulence , Virus ReplicationABSTRACT
The complete nucleotide sequence of the gene encoding the major outer capsid protein VP2 from the Alberta isolate of epizootic hemorrhagic disease virus serotype 2 (EHDV-2) was determined. Complementary DNA (cDNA) corresponding to segment 2 was 3002 nucleotides in length with a single open reading frame that encoded a VP2 of 982 amino acids. Although the VP2 from EHDV-2 was only 34% homologous to the cognate protein from EHDV-1, their predicted hydropathic profiles were similar, suggesting that conservation of structure is important biologically to these capsid proteins. Sequence analysis of six North American EHDV-2 field isolates showed a high degree of comparative genetic identity (> 97%). Phylogenetic profiles constructed suggest that regionalization of the viruses within the North American continent has contributed to the genetic diversity.
Subject(s)
Capsid/genetics , Genetic Variation , Hemorrhagic Disease Virus, Epizootic/genetics , Amino Acid Sequence , Animals , Capsid Proteins , Hemorrhagic Disease Virus, Epizootic/classification , Hemorrhagic Disease Virus, Epizootic/metabolism , Molecular Sequence Data , Phylogeny , RNA, Viral , Sequence Analysis, RNA , Sequence Homology, Amino Acid , SerotypingABSTRACT
The structure and single-stranded (ss) RNA-binding by the nonstructural protein NS2 of three different orbiviruses were studied and compared. African horsesickness virus (AHSV), bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV) were analyzed in recombinant baculovirus-infected cells and in cells infected with BTV and AHSV. Sedimentation analysis and nonreducing SDS-PAGE revealed that NS2 of all three orbiviruses is a 7S multimer with both inter- and intramolecular disulfide bonds, probably consisting of six or more NS2 molecules. The 7S NS2 multimer of all three viruses binds ssRNA but there is a marked disparity in the ssRNA-binding ability between the three proteins. At physiological salt concentration, BTV NS2 binds ssRNA very efficiently, whereas AHSV NS2 shows only a low efficiency for binding ssRNA. EHDV NS2 binds with intermediate efficiency. The result was the same irrespective of whether poly(U)-Sepharose or viral mRNA was used, indicating that ssRNA-binding by NS2 is nonspecific. The difference in RNA-binding ability may be related to the alpha-helix content of the respective proteins. NS2 of BTV has the highest predicted alpha-helix content followed by EHDV and AHSV. The ability of the NS2 proteins to form virus inclusion body-like structures in baculovirus-infected cells is not affected by the ssRNA-binding disparity.
