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Published January 1982 | Published
Journal Article Open

Sequence analysis of cDNA's derived from the RNA of Sindbis virions and of defective interfering particles


Sindbis virus generates defective interfering (DI) particles during serial high-multiplicity passage in cultured cells. These DI particles inhibit the replication of infectious virus and can be an important factor in the establishment and maintenance of persistent infection in BHK cells. In an effort to understand how these DI particles are generated and how they interfere with the replication of standard virus, we performed a partial sequence analysis of the RNA obtained from two independently isolated populations of DI particles and from two Sindbis virus variants and compared these with the RNA of the parental wild-type virus. The 3'-terminal regions of the RNAs were sequenced by the dideoxy chain terminating method. Internal regions of the RNA were examined by restriction endonuclease digestion of cDNA's made to the various RNAs and by direct chemical sequencing of 5' end-labeled restriction fragments from cDNA made to the DI RNAs. One of the variant viruses examined was originally derived from cells persistently infected with Sindbis virus for 16 months and is resistant to interference by the DI strains used. In the 3'-terminal region of the RNA from this variant, only two base changes were found; one of these occurs in the 20-nucleotide 3'-terminal sequence which is highly conserved among alphaviruses. The DI RNA sequences were found to have been produced not by a single deletional event, but by multiple deletion steps combined with sequence rearrangements; all sequences examined are derived from the plus strand of Sindbis virion RNA. Both DI RNAs had at least 50 nucleotides of wild-type sequence conserved at the 3' terminus; in addition, they both contained conserved and perhaps amplified sequences derived from the non-26S region of the genome which may be of importance in their replication and interference ability.

Additional Information

© 1982 American Society for Microbiology. Received 15 June 1981; Accepted 28 August 1981. We thank T. Hunkapiller for the computer program which made it possible to determine the sequence homologies reported here and Barbara Weiss for encouragement, critical comments, and help in preparing the RNA. This research was supported by Public Health Service grants Al 11377 (to S.S.) and AI 10793 (to J.H.S.) from the National Institute of Allergy and Infectious Diseases, GM 06965 (to J.H.S.) from the National Institute of General Medical Science, and P30CA16217 (to S.S.) from the National Cancer Institute, and by grant PCM-8022830 (to J.H.S.) from the National Science Foundation.

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