Specific residues of the influenza A virus hemagglutinin viral RNA are important for efficient packaging into budding virions

Abstract

A final step in the influenza virus replication cycle is the assembly of the viral structural proteins and the packaging of the eight segments of viral RNA (vRNA) into a fully infectious virion. The process by which the RNA genome is packaged efficiently remains poorly understood. In an approach to analyze how vRNA is packaged, we rescued a seven-segmented virus lacking the hemagglutinin (HA) vRNA (deltaHA virus). This virus could be passaged in cells constitutively expressing HA protein, but it was attenuated in comparison to wild-type A/WSN/33 virus. Supplementing the deltaHA virus with an artificial segment containing green fluorescent protein (GFP) or red fluorescent protein (RFP) with HA packaging regions (45 3' and 80 5' nucleotides) partially restored the growth of this virus to wild-type levels. The absence of the HA vRNA in the deltaHA virus resulted in a 40 to 60% reduction in the packaging of the PA, NP, NA, M, and NS vRNAs, as measured by quantitative PCR (qPCR), and the packaging of these vRNAs was partially restored in the presence of GFP/RFP packaging constructs. To further define nucleotides of the HA coding sequence which are important for vRNA packaging, synonymous mutations were introduced into the full-length HA cDNA of influenza A/WSN/33 and A/Puerto Rico/8/34 viruses, and mutant viruses were rescued. qPCR analysis of vRNAs packaged in these mutant viruses identified a key region of the open reading frame (nucleotides 1659 to 1671) that is critical for the efficient packaging of an influenza virus H1 HA segment.

FIG. 1.

Characterization of the HA-MDCK cell line. Stable expression of the HA glycoprotein in MDCK cells was assessed in comparison to the parental MDCK cell line. (A) Immunofluorescence using an HA-specific monoclonal antibody (2G9). Nuclei were stained using DAPI (4′,6′-diamidino-2-phenylindole). (B) Western blot using polyclonal anti-PR/8 antiserum. The blot was also probed using monoclonal anti-actin (Sigma) as a loading control.

FIG. 2.

Rescue and characterization of HA vRNA-deficient influenza A viruses with complementing GFP/RFP packaging constructs. (A) Schematic representation of the packaging constructs tested. Each construct consists of a GFP/RFP flanked by the terminal untranslated regions (black) along with 45 3′ and 80 5′ nucleotides (gray) required for efficient incorporation derived from the WSN HA vRNA. (B) Immunofluorescence of HA-MDCK cells infected with wt WSN, deltaHA, and HA(45)GFP-GFP(80) viruses. Infected cells were detected using an anti-M1 monoclonal antibody (mAb). Nuclei of cells were stained using DAPI. (C) Kinetics of growth of the following HA vRNA-deficient viruses in HA-MDCK cells infected at an MOI of 0.001 and titrated by plaque assay on HA-MDCK cells: wt WSN (wt), deltaHA (dHA), HA(45)GFP(80) (GFP), HA(45)GFP-GFP(80) (GFP/GFP), and HA(45)RFP(80) (RFP). Results represent two independent infections titrated in duplicate.

FIG. 3.

Mutational analysis of the packaging regions of the WSN HA vRNA. (A) Schematic representation of the regions of synonymous mutations (white boxes) introduced into the WSN HA vRNA. (B) Synonymous nucleotide changes introduced for each construct; the upper line shows the parental WSN virus HA sequence, with nucleotide changes presented in bold in the lower lines. The numbering of nucleotides is based on the positive-sense RNA sequence.

FIG. 4.

Mutational analysis of the packaging regions of the PR/8 HA vRNA. (A) Schematic representation of the regions of synonymous mutations (white boxes) introduced into the PR/8 HA vRNA. (B) Synonymous nucleotide changes introduced at the 3′ and 5′ ends of the vRNA for each construct; the upper line shows the parental PR/8 virus HA sequence, with nucleotide changes presented in bold in the lower lines. The numbering of nucleotides is based on the positive-sense RNA sequence.