Yunus, Mohammad and Shaila, Melkote S (2012) Establishment of an in vitro transcription system for Peste des petits ruminant virus. In: VIROLOGY JOURNAL, 9 .
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Abstract
Background: Peste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells. Results: RNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3' end to 5' end of the genome similar to that exhibited by the virus in infected cells. Conclusion: This report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.
| Item Type: | Journal Article |
|---|---|
| Publication: | VIROLOGY JOURNAL |
| Publisher: | BIOMED CENTRAL LTD |
| Additional Information: | Copyright for this article belongs to BIOMED CENTRAL LTD, ENGLAND |
| Keywords: | Peste-des-petits ruminants virus (PPRV);Transcription reconstitution;RNA dependent RNA polymerase;Morbillivirus |
| Department/Centre: | Division of Biological Sciences > Microbiology & Cell Biology |
| Date Deposited: | 02 Mar 2013 04:58 |
| Last Modified: | 02 Mar 2013 04:58 |
| URI: | http://eprints.iisc.ac.in/id/eprint/45964 |
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