Abstract
Human sapovirus (SaV), an agent of human gastroenteritis, cannot be grown in cell culture, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the formation of virus-like particles (VLPs). In this study we compared the time-course expression of two different SaV rVP1 constructs. One construct had the native sequence (Wt construct), whereas the other had two nucleotide point mutations in which one mutation caused an amino acid substitution and one was silent (MEG-1076 construct). While both constructs formed VLPs morphologically similar to native SaV, Northern blot analysis indicated that the MEG-1076 rVP1 mRNA had increased steady-state levels. Furthermore, Western blot analysis and an antigen enzyme-linked immunosorbent assay showed that the MEG-1076 construct had increased expression levels of rVP1 and yields of VLPs. Interestingly, the position of the mutated residue was strictly conserved residue among other human SaV strains, suggesting an important role for rVP1 expression.
Highlights
The family Caliciviridae is made up of four genera, Sapovirus, Norovirus, Lagovirus, and Vesivirus, which contain sapovirus (SaV), norovirus (NoV), rabbit hemorrhagic disease virus, and feline calicivirus strains, respectively
During PCR amplification nucleotide point mutations occurred in our initial MQG-1076 construct, at nucleotide positions 4 and 1076 in VP1, which resulted in two amino acid substitutions at residues 2 and 358, respectively, and a silent nucleotide mutation at position 1895 in VP2 (Fig. 1)
In order to compare expression levels, we infected Wt and MEG-1076 recombinant baculoviruses each at a multiplicity of infection (MOI) of 14.5 in 2.7 × 106 confluent Tn5 cells in 1.5 ml of Ex-Cell 405 medium followed by incubation at 26°C
Summary
The family Caliciviridae is made up of four genera, Sapovirus, Norovirus, Lagovirus, and Vesivirus, which contain sapovirus (SaV), norovirus (NoV), rabbit hemorrhagic disease virus, and feline calicivirus strains, respectively. Human SaV and NoV strains are agents of gastroenteritis. The prototype strain of human SaV, the Sapporo virus, was originally discovered from an outbreak of gastroenteritis in an orphanage in Sapporo, Japan, in 1977 [1]. SaV strains were recently divided into five genogroups (GI to GV), of which GI, GII, GIV, and GV strains infect humans, while GIII strains infect porcine species [2]. The SaV GI, GIV, and GV genomes are each predicted to contain three main open reading frames (ORFs), whereas SaV GII and GIII have two ORFs. SaV ORF1 encodes for non-structural proteins and the major capsid protein (VP1). The NoV genome is organized in a slightly different way than the SaV, since ORF1 encodes all the nonstructural proteins, ORF2 encodes the capsid protein (VP1), and ORF3 encodes a small protein (VP2)
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