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Infectious measles virus from cloned cDNA
I Ballart1, D Eschle, R Cattaneo
1Institut für Molekularbiologie I, Universität Zürich, Switzerland.
Abstract:
The study of measles virus (MV) and of negative strand RNA viruses in general has been hampered by the lack of an experimental system for genetic manipulation. Here we describe a procedure for generating infectious MV from cloned MV cDNA. First we assembled a genetically marked DNA copy of the MV genome in plasmids, under the control of phage T3 or T7 promoters, allowing production of transcripts almost identical to the MV genome or antigenome. Incubation of these linearized plasmid DNAs with the appropriate phage polymerase and only two ribonucleoside triphosphates yielded committed transcription complexes. Microinjection of these complexes into the cytoplasm of helper cells which provide the proteins necessary for MV genome encapsidation and transcription/replication, reproducibly give rise to lytic MVs. The transcripts of one of these viruses were analysed by sequencing after reverse transcription followed by DNA amplification, and found to contain the genetic tags. The described procedure permits the analysis of a negative strand RNA virus with the same genetic tools previously applicable only to positive strand RNA viruses and retroviruses.
Insights
Researchers created infectious measles virus (MV) from cloned DNA, enabling genetic manipulation. This breakthrough allows studying negative-strand RNA viruses like MV with advanced genetic tools.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Studying measles virus (MV) and negative-strand RNA viruses has been limited by the absence of genetic manipulation systems.
- Existing methods do not allow for comprehensive genetic analysis of these important viral families.
Purpose of the Study:
- To develop a novel experimental system for generating infectious measles virus (MV) from cloned cDNA.
- To enable genetic manipulation of MV, facilitating deeper understanding of negative-strand RNA viruses.
Main Methods:
- Assembled a genetically marked DNA copy of the MV genome in plasmids.
- Produced transcripts using phage T3 or T7 promoters and specific polymerases.
- Microinjected transcription complexes into helper cells for virus rescue and replication.
Main Results:
- Successfully generated infectious, lytic measles viruses (MVs) from cloned cDNA.
- Confirmed the presence of genetic tags in the rescued MV transcripts via sequencing.
- Demonstrated a reproducible method for MV recovery and genetic analysis.
Conclusions:
- The developed procedure provides the first experimental system for genetic manipulation of measles virus (MV).
- This breakthrough extends the application of genetic tools, previously limited to positive-strand RNA viruses and retroviruses, to negative-strand RNA viruses.
- Facilitates in-depth analysis of MV and other negative-strand RNA viruses, advancing virological research.