Control of Influenza Virus Gene Expression: Quantitative Analysis of Each Viral RNA Species in Infected Cells

Abstract

We established a quantitative hybridization system by which three types of influenza virus RNAs (vRNA, mRNA, and cRNA) for the 8 genome segments were measured individually. As the hybridization probes, 32P-labeled RNAs of both plus and minus polarity were produced employing an SP-6 transcription system and used in a large molar excess, sufficient to overcome complementary RNAs present in the viral RNA samples. Employing the system, we studied the control of the synthesis of each viral RNA species in MDCK cells infected with A/Udorn/72 (H3N2). Our new observations were as follows. 1) Segment-specific transcription was observed at the primary transcription. 2) Replication of the virus genome began simultaneously for all segments. No delay was observed in the replication of the segments carrying late genes. 3) In addition to control at the transcriptional levels, the expression of viral late genes was regulated at some post-transcriptional step(s). These results are not compatible with the concepts reported previously, and lead us to propose unique regulations operating on the expression of the viral late genes.

Influenza infection modulates vesicular trafficking and induces Golgi complex disruption

“…The dependence of IFV virus export on the CRM1 pathway is well established and the TAP pathway has also been shown to be involved in IFV export [31]. In addition, several studies indicate specific inhibition of late viral gene expression after UV irradiation, LB, actinomycin D or DRB treatment [1,12,21,22,27]. These studies reported that the drugs DRB and LB, which were added at the beginning of the infection, did not inhibit viral mRNA synthesis completely and that viral transcripts were retained in the nucleus, consistent with our microscopy results.…”

Involvement of the influenza A virus PB2 protein in the regulation of viral gene expression

“…The resulting RNA was extracted again with phenol-chloroform. A quantitative hybridization system was employed to measure the amount of the three types of virus-specific RNA (mRNA, cRNA and vRNA) for each of the eight genome segments as reported elsewhere (Hatada et al, 1989), in which RNA probes intensely labelled with 32p were used in a molar excess sufficient to overpower complementary RNAs present in the viral RNA samples. The probes for mRNA and cRNA were obtained by transcribing SP 6 recombinant DNAs, which gave the intact 5' end sequences of vRNAs.…”

“…Several studies have been carried out to reveal the relationship between the synthesis of viral RNAs and proteins (Lamb & Choppin, 1976;Hay et al, 1977b;Inglis & Mahy, 1979;Barrett et al, 1979;Smith & Hay, 1982;Enami et al, 1985 ;Shapiro et al, 1987;Hatada et al, 1989). In MDCK cells infected with A/Udorn/72 (H3N2) virus, the synthesis of virus-specific RNAs proceeds as follows (Hatada et al, 1989).…”

“…In MDCK cells infected with A/Udorn/72 (H3N2) virus, the synthesis of virus-specific RNAs proceeds as follows (Hatada et al, 1989). During primary transcription at early times of infection, considerable amounts of mRNA are produced from the non-structural protein (NS), nucleoprotein (NP) and the three polymerase genes (early proteins), but very low amounts from the haemagglutinin (HA), neuraminidase (NA) and matrix protein (M) genes (late proteins).…”

Genome packaging in influenza A virus

“…Consequently, it was concluded that there must be some form of segment specificity in genome packaging (Laver & Downie, 1976;Nakajima & Sugiura, 1977). Further support was lent to the segment-specific packaging model by the observation that segments were present at equimolar levels in virions (Hatada et al, 1989;McGeoch et al, 1976) even when their ratios in infected cells differed (Bergmann & Muster, 1995;Smith & Hay, 1982), suggesting that some form of selection process was operating.…”