Mikiko Yabe

Acquired Fusion Activity of a Murine Coronavirus MHV-2 Variant with Mutations in the Proteolytic Cleavage Site and the Signal Sequence of the S Protein

Abstract The spike (S) protein of a nonfusogenic murine coronavirus, MHV-2, was compared to the S protein of a variant with fusion activity, MHV-2f. Two amino acids differed between the S proteins of these viruses; one was located in the signal sequence and the other was in the putative cleavage site. The amino acid at position 12 in the signal sequence was S in MHV-2 and C in MHV-2f. The amino acid sequence of the cleavage site of MHV-2 was HRARS, while that of MHV-2f was HRARR, showing one amino acid replacement at position 757. In DBT cells infected with MHV-2, the S protein was not cleaved, while the S protein of MHV-2f was cleaved. The S protein of MHV-2f expressed in a transient vaccinia virus expression system was cleaved and was fusogenic in contrast to the nonfusogenic activity of uncleaved MHV-2 S protein. Because the signal sequence is assumed to be removed from the mature S protein soon after synthesis, and because the S protein of MHV-2 was expressed on the cell surface in the same way as the S protein of MHV-2f, the difference in the signal sequence seemed to have had little effect on the transportation and the fusion activity of the S protein. These results showed that MHV-2 does not fuse cells due to the lack of cleavage of its S protein. This conclusion differs from studies on the activity of syncytium formation by the S proteins of fusogenic MHV-JHM and -A59 strains. Possible reasons for these differences in fusion activity are discussed.

Unique N-linked glycosylation of murine coronavirus MHV-2 membrane protein at the conserved O-linked glycosylation site.

Abstract The membrane (M) proteins of murine coronavirus (MHV) strains have been reported to contain only O-linked oligosaccharides. The predicted O-glycosylation site consisting of four amino acid residues of Ser–Ser–Thr–Thr is located immediately adjacent to the initiator Met and is well conserved among MHV strains investigated so far. We analyzed the nucleotide sequence of a highly virulent strain MHV-2 M-coding region and demonstrated that MHV-2 had a unique amino acid, Asn, at position 2 at the conserved O-glycosylation site. We also demonstrated that this substitution added N-linked glycans to MHV-2 M protein resulting in increment of molecular mass of MHV-2 M protein compared with JHM strain having only O-linked glycans.

Phenotypic characterization of cynomolgus monkey natural killer cells

Natural killer (NK) activity of cynomolgus monkey peripheral blood lymphocytes (PBL) was determined using B95-8 cells as target cells. Examination for the reactivity of human NK-related monoclonal antibodies (mAbs), anti-Leu-7, anti-Leu-11b, anti-NKH1A, and NC-1, with cynomolgus PBL revealed that Leu-11b (CD16) was the only antigen expressed on cynomolgus PBL. The percentage of Leu-11b-positive (Leu-11b+) cells correlated well with the level of NK activity when PBL taken from 21 monkeys were tested. After depletion of Fc receptor-positive (FcR+) cells, NK activity was lost concomitantly with the disappearance of Leu-11b+ cells. These results show that cynomolgus NK cells are mainly FcR+ which can be detected by mAb directed to Leu-11b. Cynomolgus PBL were separated by Ficoll-Hypaque centrifugation after E rosette formation with 2-aminoethylisothiouronium bromide-treated sheep red blood cells, and NK activities of both E rosette-forming (E+) and nonforming (E-) fractions were determined. The high level of killing was observed in the E- fraction, suggesting that the majority of cynomolgus NK cells was contained in the E- fraction. The separation of PBL by Percoll discontinuous density gradient showed cynomolgus NK cells were enriched in the low density fractions.

Requirement of proteolytic cleavage of the murine coronavirus MHV-2 spike protein for fusion activity.

The spike (S) protein of a non-fusogenic murine coronavirus, MHV-2, was compared to that of a variant, MHV-2f, with fusion activity. Two amino acids differed between The S proteins of these viruses; one was located in the signal sequence (amino acid 12) and the other in the putative cleavage site (amino acid 757). To determine which one of these amino acid changes is important for the alteration of fusogenicity, chimeric S proteins between MHV-2 and -2f were constructed and expressed in DBT cells by a vaccinia virus expression system. The results revealed that one amino acid change (Ser to Arg) at position 757 is responsible for the acquisition of fusogenicity of the MHV-2f S protein. This change also altered the susceptibility to proteolytic cleavage of the MHV-2 S protein which was originally uncleavable. We concluded that the non-fusogenic activity of MHV-2 results from the lack of cleavage of its S protein.

Molecular cloning and expression of cynomolgus monkey interleukin-2 cDNA by the recombinant baculovirus system.

The cDNA encoding interleukin-2 (IL-2) of cynomolgus monkey was molecularly cloned from concanavalin-A-stimulated peripheral lymphocyte cDNA by the reverse transcription-polymerase chain reaction method. Cynomolgus monkey IL-2 cDNA displays a high degree of homology with the human equivalent both at nucleic acid (98%) and amino acid (96%) sequence levels, although having only one additional amino acid residue. Recombinant monkey IL-2 secreted by insect cells infected with the recombinant baculovirus migrated as two distinct bands with 15 and 18 kD corresponding to mature and precursor IL-2 molecules, respectively, which were revealed to cross-react with anti-human IL-2 polyclonal antibody in Western blot analysis. This monkey IL-2 exhibited high biological activity up to an order of 10(6) U/ml as assessed by an IL-2-dependent cell proliferation assay and was shown to be neutralized by anti-human IL-2 antibody.