Killen, Helen (1983) Biochemical and serological characterisation of a calf rotavirus. PhD thesis, University of Warwick.

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Abstract

mRNA was transcribed m vitro by the endogenous RNA transcriptase of calf rotavirus. This mRNA had a mean sedimentation coefficient of 12S, and hybridised to genomic double stranded RNA. Rotavirus mRNA appeared not to be polyadenylated as less than 1% of it was retained by oligo dT cellulose. Methyl groups from S-adenosyl methionine were
incorporated in this system into RNA which co-sedimented with 32
P-labelled mRNA on sucrose gradients, and the methyl groups were shown to be present in the form of an a 1ka1i-resistant cap which co-migrated with the reovirus 5' cap on DEAE cellulose with a net negative charge of -5 to -6.

In vitro translation of the in vitro transcription products gave 12 polypeptides, 11 of which had the same mobility on PAGE as virus-specific polypeptides seen in infected cells treated with tunlcamycin (l.e. in which glycosylation was inhibited).
These were VP1, VP2, VP3, VP4, VP5, VP6, VP8, VP9, vpr7, VP 11 and VP12. The VP2, VP3, VP5, VP6, VP8, VP9 and VP12 from Jn vitro translation and from infected cells were compared by peptide mapping and were shown to be the same proteins. Two of the primary gene products (VP 12 and vpr7) become glycosylated in infected cells to VP 10 and VP7 respectively, and these are cleaved to VPIOc, VP7.1 and VP7.2. Virus structural proteins which are also seen in infected cells are VP1, VP2, VP6, VP7.1, VP7.2 and VPIOc. Virus particles also have polypeptides VP3*, VP1**, VP1*.2, VP5* and VP1*. 3 which are not found in infected cells, and are all produced during trypsin activation of virus pa rt i c 1 es. VP1*. 2 and VP1*.3 are produced by cleavage of VP3 by trypsin, and VP3*, VP1** and VP5* are produced by cleavage of some of VP2. Thus, only three primary gene products (VP1, VP2 and VP6) are seen in virus particles while the other eight structural proteins (VP3*, VP1**, VP1*.2, VP5*, VP7.1, VP7.2, VPIOc and VP1*.3) are the result of post-translational modification of primary gene products.

Immune precipitation of polypeptides from infected cells revealed that VP2, VP3*,1**, VP6, VP7/7.1 and VP8 were efficiently precipitated, VP10/10c were inefficiently precipitated, and VP1 and VP12 were not precipitated by anti-calf rotavirus serum.

Immune precipitation using heterologous antisera led us to conclude that VP2, VP6 and VP8 are group-specific antigens, and VP3, VP1*.2 and VP 7/7.1 behave as type-spec if ic antigens.

Monospecific polyclonal antisera were raised in guinea- pigs against the purified calf rotavirus polypeptides VP1, VP2, VP3*,1**, VP1*.2, VP6, VP7.1, VP7.2 and VP10.

All of the antisera gave a similar pattern of cytoplasmic immunofluorescence in rotavirus-infected cells, and spots of fluorescence of varying intensity with different antisera were also seen over the nucleus.

Immune precipitation showed that VP2 was precipitated by antiserum to VP2 (a-VP2) and aVP3*, <**. and VP6 by aVP6, aVP7.1 and aVP7.2 both precipitated the same range of proteins from infected cells (VP7, VP7.1 and VP7.2) or from virions (VP7.1 and VP7.2) VP10, either from virions or infected cells, was not precipitated by aVPIO. The only antiserum which efficiently neutralized infectivity was aVP7.2. There were low levels of neutralization with aVPIO (but the results varied from experiment to experiment), and trace levels with aVP6. Neutralization by aVP7.2 and aVPIO was enhanced by complement. aVPIO and aVP6 both appeared to block neutralization by aVP7.2. aVP7. and the other antisera did not neutralize even though aVP7.1 agglutinated double shelled particles as seen in immune electron microscopy to a greater extent than aVP7.2. Both VP7.1 and VP7.2 were shown to be glycoproteins by tunicamycin treatment of infected cells.

Core particles only were agglutinated by aVPIO. All the evidence leads to conclude that there are major neutralizing antigenic determinants present on VP7.2, a minor component of the outer shell of the virion.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology Q Science > QR Microbiology > QR355 Virology
Library of Congress Subject Headings (LCSH):	Rotaviruses, Immunospecificity, Genetic transcription, RNA
Official Date:	1983
Dates:	Date Event 1983 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Dimmock, N. J.
Sponsors:	Medical Research Council (Great Britain)
Format of File:	pdf
Extent:	xvi, 396 leaves
Language:	eng

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