Infectious measles virus from cloned cDNA.

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.

Altered Interaction of the Matrix Protein with the Cytoplasmic Tail of Hemagglutinin Modulates Measles Virus Growth by Affecting Virus Assembly and Cell-Cell Fusion

“…We found that when the M protein had the R175G substitution, the inhibitory effect on cell-cell fusion due to the P64S and E89K substitutions was partially cancelled (data not shown). The Edmonston tag strain is a recombinant virus generated from cloned cDNAs, and MV strains used to construct the Edmonston tag cDNAs were passaged in CD46 ϩ cell lines (3,36). Furthermore, the H protein of the Edmonston tag strain can use CD46 efficiently as a receptor (44).…”

Multiple Amino Acid Substitutions in Hemagglutinin Are Necessary for Wild-Type MeaslesVirus To Acquire the Ability To Use Receptor CD46 Efficiently

“…The Edmonston tag strain is an infectious clone generated from cDNAs derived from the Edmonston B strain (34). However, a short segment at the junction of the N and P genes of the full-length genomic cDNA was derived from a different source, the subacute sclerosing panencephalitis-derived IP-3-Ca cell line (2). When the infectious cDNA was constructed, this short segment tag remained (34).…”

Rescue of a foreign gene by Sendai virus.

“…Recombinant infectious virus systems have also been developed for double-stranded RNA viruses such as bacteriophage 46 (10) and reovirus (11). Finally, for paramyxoviruses, infectious measles virus has been generated from cloned measles virus cDNA by microinjecting "committed transcription complexes" into helper cells (12). Thus these studies provide examples of alternative and rewarding approaches to manipulate and understand negative-sense RNA virus genomes and their gene products.…”