Influenza virus, an RNA virus, synthesizes its messenger RNA in the nucleus of infected cells

Abstract

It had not been established whether influenza virus, an RNA virus of negative polarity, synthesizes its mRNA in the nucleus or cytoplasm of infected cells. To determine the site of synthesis, we fractionated infected chicken embryo fibroblasts and BHK-21 cells using both aqueous and nonaqueous procedures, and assayed the pulse-labeled viral RNA transcripts in the nucleus and cytoplasm by hybridization to virion RNA covalently linked to filter paper discs. With infected BHK-21 cells, most of the 3Huridine-pulse-labeled viral RNA transcripts, synthesized during both primary and amplified transcription, were found in the nuclear fraction after both aqueous and nonaqueous fractionation. The results with infected chicken embryo fibroblasts were not as clear-cut, but also pointed to the nucleus as the site of viral RNA transcription. Gel electrophoretic analysis of the pulse-labeled viral RNA transcripts in BHK-21 cells indicated that all eight viral mRNAs are synthesized in the nucleus. A pulse-chase experiment suggested that most of the viral RNA transcripts synthesized in the nucleus are transported to the cytoplasm. On the basis of these experiments as well as labeling experiments with 3H-methyl methionine (to label the caps of the viral mRNAs), it was established that: the nucleus is the site where cellular capped RNAs (that is, heterogeneous nuclear RNAs) prime viral RNA transcription and donate their 5′ caps and about 10 to 13 nucleotides to the viral mRNAs; and essentially only those capped cellular RNAs synthesized after infection, and not those synthesized before infection, are used as primers. Influenza virus is thus unique among nononcogenic RNA viruses in synthesizing its mRNA in the nucleus.