Identification of Nucleocapsid Binding Sites within Coronavirus-Defective Genomes

Virology
Volume 277, Issue 2, 25 November 2000, Pages 235-249

Raymond Cologna, Jeannie F. Spagnolo, and Brenda G. Hogue

Abstract

The coronavirus nucleocapsid (N) protein is a major structural component of virions that associates with the genomic RNA to form a helical nucleocapsid. N appears to be a multifunctional protein since data also suggest that the protein may be involved in viral RNA replication and translation. All of these functions presumably involve interactions between N and viral RNAs. As a step toward understanding how N interacts with viral RNAs, we mapped high-efficiency N-binding sites within BCV- and MHV-defective genomes. Both in vivo and in vitro assays were used to study binding of BCV and MHV N proteins to viral and nonviral RNAs. N–viral RNA complexes were detected in bovine coronavirus (BCV)-infected cells and in cells transiently expressing the N protein. Filter binding was used to map N-binding sites within Drep, a BCV-defective genome that is replicated and packaged in the presence of helper virus. One high-efficiency N-binding site was identified between nucleotides 1441 and 1875 at the 3′ end of the N ORF within Drep. For comparative purposes N-binding sites were also mapped for the mouse hepatitis coronavirus (MHV)-defective interfering (DI) RNA MIDI-C. Binding efficiencies similar to those for Drep were measured for RNA transcripts of a region encompassing the MHV packaging signal (nts 3949–4524), as well as a region at the 3′ end of the MHV N ORF (nts 4837–5197) within MIDI-C. Binding to the full-length MIDI-C transcript (∼5500 nts) and to an ∼1-kb transcript from the gene 1a region (nts 935–1986) of MIDI-C that excluded the packaging signal were both significantly higher than that measured for the smaller transcripts. This is the first identification of N-binding sequences for BCV. It is also the first report to demonstrate that N interacts in vitro with sequences other than the packaging signal and leader within the MHV genome. The data clearly demonstrate that N binds coronavirus RNAs more efficiently than nonviral RNAs. The results have implications with regard to the multifunctional role of N.

Keywords

nonviral RNAs, coronavirus nucleocapsid (N) protein, in vivo, in vitro, hepatitis

Lactogenic immunity following vaccination of cattle with bovine coronavirus

Vaccine
Volume 19, Issues 2–3, 15 September 2000, Pages 189-196

C.F Crouch, S Oliver, D.C Hearle, A Buckley, A.J Chapman, M.J Francis

Abstract

In order to investigate the ability of an oil adjuvanted vaccine containing bovine coronavirus antigen to enhance lactogenic immunity in the calf, pregnant cows and heifers were vaccinated and specific virus neutralising antibody levels determined in serum, colostrum and milk. Pre-existing antibody titres (as a result of natural infection) in the serum of these animals were found to be significantly increased as a result of a single shot vaccination carried out between 2 and 12 weeks before calving. This was reflected in a similar increase in the titre and duration of specific antibody in milk and colostrum that was passed on to the calves. The overall response observed was highly dependent on an adequate antigen payload being incorporated within the single dose vaccine. No abnormal local or systemic reactions were observed as a result of vaccination. It is hoped that this approach will lead to the production of a superior commercial vaccine for the protection of neonatal calves against enteric coronavirus infection.

Keywords

Bovine coronavirus, Cattle, Lactogenic immunity

Downstream Ribosomal Entry for Translation of Coronavirus TGEV Gene 3b

Virology
Volume 269, Issue 1, 30 March 2000, Pages 172-182

Jennifer Black O'Connor, David A.Brian

Abstract

Gene 3b (ORF 3b) in porcine transmissible gastroenteritis coronavirus (TGEV) encodes a putative nonstructural polypeptide of 27.7 kDa with unknown function that during translation in vitro is capable of becoming a glycosylated integral membrane protein of 31 kDa. In the virulent Miller strain of TGEV, ORF 3b is 5′-terminal on mRNA 3–1 and is presumably translated following 5′ cap-dependent ribosomal entry. For three other strains of TGEV, the virulent British FS772/70 and Taiwanese TFI and avirulent Purdue-116, mRNA species 3–1 is not made and ORF 3b is present as a non-overlapping second ORF on mRNA 3. ORF 3b begins at base 432 on mRNA 3 in Purdue strain. In vitro expression of ORF 3b from Purdue mRNA 3-like transcripts did not fully conform to a predicted leaky scanning pattern, suggesting ribosomes might also be entering internally. With mRNA 3-like transcripts modified to carry large ORFs upstream of ORF 3a, it was demonstrated that ribosomes can reach ORF 3b by entering at a distant downstream site in a manner resembling ribosomal shunting. Deletion analysis failed to identify a postulated internal ribosomal entry structure (IRES) within ORF 3a. The results indicate that an internal entry mechanism, possibly in conjunction with leaky scanning, is used for the expression of ORF 3b from TGEV mRNA 3. One possible consequence of this feature is that ORF 3b might also be expressed from mRNAs 1 and 2.

Keywords

porcine transmissible gastroenteritis coronavirus, gene 3b, ribosomal scanning, ribosomal shunting

Capture ELISA systems for the detection of bovine coronavirus-specific IgA and IgM antibodies in milk and serum

Veterinary Microbiology
Volume 72, Issues 3–4, 15 March 2000, Pages 183-206

K. Naslund, M. Traven, B. Larsson, A. Silvan, N. Linde

Abstract

Isotype-capture ELISAs for BCV-specific IgA and IgM were developed and tested on milk and serum samples from Swedish cattle. The capture ELISAs showed higher sensitivity than indirect ELISAs for detection of BCV-specific IgA and IgM. In the capture ELISAs the agreement between detection in milk and serum samples was 94% for IgA and 86% for IgM. The correlation between log10 titres in milk and serum was r=0.82 (P<0.001) for IgA and 0.84 (P<0.001) for IgM. Milk seemed a better target than serum for diagnosing specific IgA at low levels. There was no variation in the isotype-specific BCV antibody titres between healthy quarters of the same udder, but subclinical mastitis was associated with higher levels of IgA antibodies and weak false IgM positive reactions in undiluted milk. Bovine IgA and IgM antibodies in milk and serum showed high stability towards freezing and thawing and storage at room temperature.

The antibody responses to BCV were followed in milk and serum from six dairy cows and in serum from four calves for a period of 1 year after an outbreak of winter dysentery (WD). In this outbreak some animals became reinfected with BCV. The IgA and IgM capture ELISAs differentiated between primarily BCV infected and reinfected animals. In the primarily infected cattle, IgM antibodies were first detected in milk and serum four to nine days after the first WD symptoms observed, and were subsequently detected for at least 2–3 weeks. IgM was also detected in the reinfected cows, but mostly at lower levels and for a shorter period of time than in the primarily infected animals. In milk, however, the IgM response of the reinfected cows was detected for a longer period of time than in serum. Six months after the outbreak, IgA was still detected in both serum and milk of all six cows and also in serum of one calf. The reinfected cows showed higher and more long-lasting peak levels of IgA in milk and serum than the primarily infected cows, indicating boosting of the IgA response.

Keywords

Cattle, IgA, IgM, Isotype-capture, Bovine coronavirus, Milk, Reinfection

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

Virus Research
Volume 66, Issue 2, February 2000, Pages 149-154

Yasuko K. Yamada, Mikiko Yabe, Takahiro Ohtsuki, Fumihiro Taguchi

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.

Keywords

Murine coronavirus, MHV, M protein, N-glycosylation, O-glycosylation