Highly conserved regions of influenza a virus polymerase gene segments are critical for efficient viral RNA packaging

Abstract

The genome of the influenza A virus is composed of eight different segments of negative-sense RNA. These eight segments are incorporated into budding virions in an equimolar ratio through a mechanism that is not fully understood. Two different models have been proposed for packaging the viral ribonucleoproteins into newly assembling virus particles: the random-incorporation model and the selective-incorporation model. In the last few years, increasing evidence from many different laboratories that supports the selective-incorporation model has been accumulated. In particular, different groups have shown that some large viral RNA regions within the coding sequences at both the 5' and 3' ends of almost every segment are sufficient for packaging foreign RNA sequences. If the packaging regions are crucial for the viability of the virus, we would expect them to be conserved. Using large-scale analysis of influenza A virus sequences, we developed a method of identifying conserved RNA regions whose conservation cannot be explained by population structure or amino acid conservation. Interestingly, the conserved sequences are located within the regions identified as important for efficient packaging. By utilizing influenza virus reverse genetics, we have rescued mutant viruses containing synonymous mutations within these highly conserved regions. Packaging of viral RNAs in these viruses was analyzed by reverse transcription using a universal primer and quantitative PCR for individual segments. Employing this approach, we have identified regions in the polymerase gene segments that, if mutated, result in reductions of more than 90% in the packaging of that particular polymerase viral RNA. Reductions in the level of packaging of a polymerase viral RNA frequently resulted in reductions of other viral RNAs as well, and the results form a pattern of hierarchy of segment interactions. This work provides further evidence for a selective packaging mechanism for influenza A viruses, demonstrating that these highly conserved regions are important for efficient packaging.

FIG. 1.

Mutational analysis of the highly conserved region of the WSN PB2 vRNA. (Left) Schematic representations of the regions of synonymous mutations (white boxes) introduced into the WSN PB2 vRNA. nt, nucleotide; ORF, open reading frame. (Right) Synonymous nucleotide changes introduced for each construct. The upper line is the parental WSN virus PB2 sequence, and nucleotide changes are presented in bold on the lower line. The numbering of nucleotides is based on that of the positive-sense RNA sequence.

FIG. 2.

Mutation of single highly conserved codons of the WSN PB2 vRNA. Synonymous nucleotide changes were introduced for each mutated codon. The upper line is the parental WSN virus PB2 sequence, and nucleotide changes are presented in bold on the lower line. Numbering of residues and changes introduced are based on the report by Gog et al. (11). mut, mutation.

FIG. 3.

Mutational analysis of the short highly conserved region of the WSN PB1 vRNA. (Top) Schematic representations of the region of synonymous mutations (white boxes) introduced into the WSN PB1 vRNA. nt, nucleotide; ORF, open reading frame. (Bottom) Synonymous nucleotide changes introduced. The upper line is the parental WSN virus PB1 sequence, and nucleotide changes are presented in bold on the lower line. The numbering of nucleotides is based on that of the positive-sense RNA sequence.

FIG. 4.

Mutational analysis of the highly conserved region of the WSN PA vRNA. (Left) Schematic representation of the regions of synonymous mutations (white boxes) introduced into the WSN PA vRNA. nt, nucleotide; ORF, open reading frame. (Right) Synonymous nucleotide changes introduced for each construct. The upper line is the parental WSN virus PA sequence, and nucleotide changes are presented in bold on the lower line. The numbering of nucleotides is based on that of the positive-sense RNA sequence.

FIG. 5.

Mutational analysis of the conserved region of the PR/8 PA vRNA. (Left) Schematic representations of the regions of synonymous mutations (white boxes) introduced into the PR/8 PA vRNA. (Right) Synonymous nucleotide changes introduced at the 3′ and 5′ ends of the vRNA for each construct. The upper line is the parental PR/8 virus PA sequence, and nucleotide changes are presented in bold on the lower line. The numbering of nucleotides is based on that of the positive-sense RNA sequence.