Polyuridylated mRNA synthesized by a recombinant influenza virus is defective in nuclear export

Abstract

The poly(A) tail of influenza virus mRNA is synthesized by reiterative copying of a U track near the 5' end of the virion RNA (vRNA) template by the viral RNA polymerase. We have engineered a novel influenza A/WSN/33 virus which contains a neuraminidase (NA) vRNA with its U track mutated into an A track. Instead of synthesizing poly(A)-tailed NA mRNA, this novel virus synthesizes poly(U)-tailed NA mRNA. In infected cells, most poly(U)-tailed NA mRNA was retained in the nucleus, while most control polyadenylated NA mRNA was transported to the cytoplasm. These results suggest that the poly(A) tail is important for efficient nuclear export of NA mRNA. The mutant virus produced a reduced amount of NA and showed an attenuated phenotype, suggesting that poly(A) signal mutants of this type might be useful as potential live attenuated virus vaccines. In addition, this virus mutant might provide a useful model to further elucidate the basic mechanisms of mRNA nuclear export.

FIG. 1

Model of vRNA with the wild-type conserved terminal sequences. The proposed RNA hook model of the vRNA template (49) is shown. The U₆ track (the polyadenylation site) is in boldface.

FIG. 2

Effects of mutations of the U₆ track on CAT expression. Human 293 kidney cells were transfected with four protein expression plasmids (pGT-h-PB1, pGT-h-PB2, pGT-h-PA, and pGT-h-NP) and pPOL-I-CAT-RT with a wild-type polyadenylation signal (lanes 1 to 4), a U₆→A₆ mutation (lanes 5 and 6), or a U₆→UCUACG mutation (lanes 7 and 8). The CAT activities of the cell lysates were analyzed as described in Materials and Methods. Dilution factors of the cell lysates are indicated. Lane 9, mock transfection.

FIG. 3

Poly(U)-tailed NA mRNA is synthesized by the A₆ transfectant virus. (A) RT-PCR assays. Total RNA from cells infected with the wild-type virus (lanes 2, 5, and 8) or the A₆ mutant (lanes 3, 6, and 9) was tested by RT-PCR for vRNA (lanes 2 and 3), poly(U)-tailed NA mRNA (lanes 5 and 6), and poly(A)-tailed NA mRNA (lanes 8 and 9). RT-PCR products were analyzed on 8% native acrylamide gels. The high-molecular-weight bands near the wells are nonspecific PCR products. Lanes 1, 4, and 7 are DNA markers. (B) Poly(U) sequence n = 84) of a cDNA clone derived from poly(U)-tailed NA mRNA. The influenza virus mRNA sequence is indicated. N, either C, A, G, or T.

FIG. 4

The A₆ mutant is attenuated due to the reduction of NA expression. (A) Growth curves of transfectant viruses on MDBK cells. Cells were infected with the wild-type virus (WT) or the A₆ mutant (A₆) at an MOI of 0.01. Infectious particles released into the media at the indicated time points were titrated by plaque assay of MDBK cells. (B) NA activities of wild-type virus and the A₆ mutant. NA activity was expressed as nanomoles of 4-methylumbelliferone released per minute per microgram of viral protein. (C) NA protein expression in infected cells. Cells infected with the A₆ mutant (a and b) or the wild-type virus (c and d) and mock-infected cells (e and f) were tested for NA protein expression. NA protein expression was visualized by fluorescence microscopy with a fluorescein isothiocyanate (FITC)-labelled antibody. Cells were mounted in antibleach medium containing DAPI for DNA staining. The merged images of NA protein expression and cell nuclei (b, d, and f) are shown. Note that not all cells were infected with virus.

FIG. 4

The A₆ mutant is attenuated due to the reduction of NA expression. (A) Growth curves of transfectant viruses on MDBK cells. Cells were infected with the wild-type virus (WT) or the A₆ mutant (A₆) at an MOI of 0.01. Infectious particles released into the media at the indicated time points were titrated by plaque assay of MDBK cells. (B) NA activities of wild-type virus and the A₆ mutant. NA activity was expressed as nanomoles of 4-methylumbelliferone released per minute per microgram of viral protein. (C) NA protein expression in infected cells. Cells infected with the A₆ mutant (a and b) or the wild-type virus (c and d) and mock-infected cells (e and f) were tested for NA protein expression. NA protein expression was visualized by fluorescence microscopy with a fluorescein isothiocyanate (FITC)-labelled antibody. Cells were mounted in antibleach medium containing DAPI for DNA staining. The merged images of NA protein expression and cell nuclei (b, d, and f) are shown. Note that not all cells were infected with virus.

FIG. 5

Primer extension assay for NA-specific vRNA. (A) NA-specific vRNA levels in cells infected with the A₆ mutant (A₆) and the wild-type virus (WT). Total RNAs from infected cells were isolated at the indicated time points p.i. The expected products for NA (259 nt) and NS (196 nt) vRNA are indicated. (B) NA-specific vRNA levels in purified A₆ mutant and wild-type virus. The expected products for NA (259 nt) and NS (196 nt) vRNA are indicated. (C) NA vRNA-to-NS vRNA ratio in cells infected with the A₆ mutant (A₆) or wild-type virus (WT).

FIG. 6

Primer extension assay for NA-specific cRNA and mRNA. (A) NA-specific cRNA and mRNA levels in cells infected with the A₆ mutant (A₆) and the wild-type virus (WT). Total RNA from infected cells was isolated at the indicated time points p.i. The expected products for NA cRNA (142 nt), NA mRNA (152 to 157 nt), HA cRNA (94 nt), and HA mRNA (104 to 109 nt) are indicated. Since influenza virus mRNAs are initiated by capped RNA primers, which are heterogeneous in size, primer extension products of the mRNA are expected to produce a broad band which is 10 to 15 nt longer than that produced by products of cRNA. (B) NA cRNA-to-HA cRNA ratios in cells infected with the A₆ mutant or the wild-type virus. (C) NA mRNA-to-HA mRNA ratio in cells infected the A₆ mutant or the wild-type virus.

FIG. 7

Poly(U)-tailed NA mRNA is predominantly localized in the nucleus. Cells infected with the A₆ mutant (A to C and J to L) or wild-type (D to F and M to O) virus and mock-infected cells (G to I and P to R) were hybridized with NA mRNA-specific (A to I) or poly(U)-tailed NA mRNA-specific (J to R) probes. Nuclei of cells were stained with DAPI (left column). The distribution of the corresponding probe was visualized by fluorescence microscopy with FITC (middle column). The right column consists of the merged images of the other two columns. Note that not all the cells were infected with virus.