Studies on the RNA and protein species synthesised in cells infected with ts mutants of VSV New Jersey
Başlık:
Studies on the RNA and protein species synthesised in cells infected with ts mutants of VSV New Jersey
Yazar:
Paterson, Reay Gilmour, author.
ISBN:
9780438052833
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (279 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Advisors: J. F. Szilagyi.
Özet:
The work described here concerns the study of macromolecular synthesis in cells infected with the wild type and the temperature sensitive mutants belonging to the six complementation groups (A to F) of vesicular stomatitis virus New Jersey serotype. This was done in order to analyse the various RNA and protein species synthesised at the permissive (31°C) and non-permissive (39°C) temperatures and to determine how the temperature sensitive mutation in each complementation group affects these syntheses. The [3-H] uridine labelled RNA was analysed by acid-urea agarose gel electrophoresis and the proteins, labelled with either [35-S] methionine or [32-P] orthophosphate, were analysed by polyacrylamide gel electrophoresis. The results obtained in each case were compared to the results of in vitro transcriptase assays and to the known gene assignments for each complementation group. At 31°C in infected cells the wild type virus and the temperature sensitive mutants synthesised the mRNAs for the L, G, N, n and NS polypeptides as well as genome-sized RNA, In addition to these RNAs four minor RNA species (a, b, c, d) were also recognised in infected cells. Two of these, c and d, were also detected amongst the products of in vitro transcription. Although the nature and function of the minor RNA species has not been determined the reproducibility with which they are detected relative to the major RNA species indicates that they are produced by a specific mechanism and are not the products of a fortuitous event. At 39°C in vivo wild type RNA synthesis is indistinguishable from that at 31°C, These results are confirmed by the observation that wild type New Jersey synthesises similar amounts of viral proteins at both the permissive and non-permissive temperatures, Both group A mutants studied, ts A1 and ts A4, synthesised RNA in vitro at 39°C similarly to wild type virus. In vivo ts A1 was able to carry out primary transcription at the non-permissive temperature while in the case of A4 even primary transcription was severely restricted. The amounts of the mRNAs synthesised by ts A4 at 39°C in vivo were insufficient to result in detectable levels of protein synthesis which emphasised the extent to which ts A4 RNA synthesis was restricted. Mutants ts A1 and A4 both possess altered N polypeptides. In the case of ts A4 this is readily detected as the ts A4 N polypeptide differs in electrophoretic mobility from that of the wild type virus. This difference in mobility was also observed in the case of intracellular N polypeptides and after in vitro translation of mRNA synthesised both in vivo and in vitro. Examination of the ribonuclease sensitivity of the ribonucleoprotein cores showed that the A4 genomic RNA was more sensitive to ribonuclease digestion than that of wild type virus. However, as ts A4 genomic RNA exhibited this increased ribonuclease sensitivity both at 31°C and 39°C this cannot be the reason for the small amounts of RNA detected at the non-permissive temperature. Nevertheless, these results suggest that a mutation in the N polypeptide can have an effect on transcription and replication, Hutant B1 is unable to transcribe RNA in vitro at the non-permissive temperature and was therefore classified as transcriptase negative. However, in vivo transcription, and most probably even replication, did take place at the non-permissive temperature in ts B1 infected BHK cells although not in chick embryo cells. These results uiere confirmed by the detection of ts B1 viral polypeptides in infected BHK cells but not in chick embryo cells incubated at 39°C, Mutant ts B1 has been shown to possess a mutated L polypeptide and it is suggested that there is a host factor present in BHK cells but not in chick embryo cells which helps to overcome the temperature sensitive mutation of this mutant. In this respect ts B1 shows some similarities to the temperature-dependent host range mutant tdCE3, where a similar host factor involvement was proposed. Although ts B1 was able to synthesise RNA and protein at 39°C in BHK cells the virus development remained restricted possibly due to a host-cell factor- L polypeptide interaction. Attempts to demonstrate the activity of such a host-cell factor in vitro uiere unsuccessful. The three group E mutants differ in their ability to synthesise RNA at 39°C in vitro; ts E1 synthesising negligible amounts of RNA, ts E2 synthesising intermediate amounts of RNA and ts E3 synthesising RNA similarly to wild type virus. These mutants were also observed to differ in vivo since ts E1 failed to synthesise RNA at the non-permissive temperature while ts E3 was able to carry out primary transcription and ts E2 was apparently able to carry out both transcription and replication at 39°C, although the amounts of RNA produced were reduced in comparison to wild type virus. These results support the suggestion that the N5 polypeptide, which is affected by the temperature sensitive mutation in this complementation group, is multifunctional having a role in transcription, replication and a late event in virus development. In vitro at 39°C ts F1 was unable to synthesise significant amounts of RNA while ts F2 transcription at this temperature was similar to that of wild type virus, Mutant ts F1 also failed to synthesise detectable amounts of RNA at the non-permissive temperature in vivo and F2 was observed to carry out primary transcription only. These results were confirmed by those obtained on examination of the proteins synthesised at 39°C in cells infected by the two group F mutants. Thus the polypeptide affected by the mutation in complementation group F appears to be involved both in the transcription and replication of the VSV genome. The representative mutant from complementation group C, ts C1, was observed to synthesise RNA similarly to wild type virus at 39°C both in vivo and in vitro. This mutant was also found to synthesise virtually identical amounts of viral proteins at both temperatures in infected BHK cells. Thus, C1 is a late mutant since its macromolecular synthesis is unaffected by the temperature sensitive mutation and therefore the block in virus development is most likely to occur at the stage of virus maturation. The single mutant belonging to complementation group D, ts D1, synthesised RNA in vitro at 39°C as well as, or even better than, wild type virus. In addition, ts D1 was able to transcribe and almost certainly replicate its RNA at the non-permissive temperature in infected cells, although the levels of RNA synthesis were somewhat reduced in comparison to those detected at 31°C, These results therefore suggest that in the case of ts D1, the production of progeny virions at the non-permissive temperature is blocked at a later stage in virus development than replication of genomic RNA.
Notlar:
School code: 0547
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684429.1) | 684429-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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