Minimum protein requirements for transcription and RNA replication of a minigenome of human parainfluenza virus type 3 and evaluation of the rule of six

Anna P. Durbin, Jeffrey W. Siew, Brian R. Murphy, Peter L. Collins

Research output: Contribution to journalArticlepeer-review

Abstract

A reconstituted transcription and RNA replication system for human parainfluenza virus type 3 (HPIV3) was developed using components expressed intracellularly from transfected plasmids driven by T7 RNA polymerase supplied by a vaccinia virus recombinant. The system is based on a negative-sense analog of HPIV3 genomic RNA in which the viral genes were deleted and replaced with that encoding bacterial chloramphenicol acetyl transferase (CAT). The N, P, and L proteins expressed from cotransfected plasmids were necessary and sufficient to direct efficient transcription and RNA replication. Transcription yielded subgenomic polyadenylated mRNA, which was isolated by oligo(dT) chromatography. RNA replication yielded a mini-antigenome and progeny minigenome, which were shown to be encapsidated based on resistance to digestion with micrococcal nuclease. A panel of cDNAs was constructed to encode minigenomes which differed in length by single-nucleotide increments. Transcription and RNA replication in the reconstituted system were most efficient for the minigenome whose length was an even multiple of six. Both RNA replication and transcription appeared to be governed by the rule. However, minigenomes whose lengths were one nucleotide greater than or less than an even multiple of six also were very active, especially in RNA replication, indicating that the rule was not absolute.

Original languageEnglish (US)
Pages (from-to)74-83
Number of pages10
JournalVirology
Volume234
Issue number1
DOIs
StatePublished - Jul 21 1997
Externally publishedYes

ASJC Scopus subject areas

  • Virology

Fingerprint Dive into the research topics of 'Minimum protein requirements for transcription and RNA replication of a minigenome of human parainfluenza virus type 3 and evaluation of the rule of six'. Together they form a unique fingerprint.

Cite this