Evidence for segment-nonspecific packaging of the influenza a virus genome

Abstract

The influenza A virus genome is composed of eight negative-sense RNA segments (called vRNAs), all of which must be packaged to produce an infectious virion. It is not clear whether individual vRNAs are packaged specifically or at random, however, and the total vRNA capacity of the virion is unknown. We have created modified forms of the viral nucleoprotein (NP), neuraminidase (NA), and nonstructural (NS) vRNAs that encode green or yellow fluorescent proteins and studied the efficiency with which these are packaged by using a plasmid-based influenza A virus assembly system. Packaging was assessed precisely and quantitatively by scoring transduction of the fluorescent markers in a single-round infectivity assay with a flow cytometer. We found that, under conditions in which virions are limiting, pairs of alternatively tagged vRNAs compete for packaging but do so in a nonspecific manner. Reporters representing different vRNAs were not packaged additively, as would be expected under specific packaging, but instead appeared to compete for a common niche in the virion. Moreover, 3 to 5% of transduction-competent viruses were found to incorporate two alternative reporters, regardless of whether those reporters represented the same or different vRNAs - a finding compatible with random, but not with specific, packaging. Probabilistic estimates suggest that in order to achieve this level of dual transduction by chance alone, each influenza A virus virion must package an average of 9 to 11 vRNAs.

FIG. 1.

Quantitative assay for influenza virus vRNA packaging. (A) Immunofluorescence flow cytometry of MDCK cells infected with plasmid-derived influenza virus virions. Cultured 293T cells were transfected with the 17-plasmid assembly system supplemented with 2 μg of NP-Y, with 2 μg of NP-G, with 1 μg each of NP-Y and NP-G, or with 2 μg of the control plasmid pHH21. Supernatants harvested 48 h later were used to infect MDCK cells, which were themselves harvested and analyzed by flow cytometry 10 h postinfection. Each point represents one of 20,000 viable MDCK cells from each population, analyzed for green (GFP) and yellow (YFP) fluorescence, each expressed on an exponential scale. The four quadrants of each plot represent (clockwise from lower left) nonfluorescent, yellow fluorescent, dual-colored, and green fluorescent MDCK cells, respectively. Data are from one representative experiment. (B) Quantitative transduction data for viruses prepared with the indicated amounts of various reporter plasmids. Each bar indicates the numbers of yellow, green, and dual-colored transductants obtained per 20,000 MDCK cells. Data are mean ± standard error of the mean for triplicate transfections in a single experiment. Statistically significant differences between yields from homologous reporters were observed in some individual experiments, as here, but were not reproducible (see Table 2).

FIG. 2.

Saturable packaging and transduction of reporter vRNA. (A) Dose-dependent expression of reporter transcripts. 293T cells were transfected with the indicated amounts of NP-Y reporter vector along with either all 17 standard plasmids (at left) or all except the wild-type (wt) NP vRNA vector (at right). Cellular RNA harvested 48 h posttransfection was probed for positive-sense reporter transcripts (mRNA and cRNA) by RPA. Control cells were transfected either with a GFP-mRNA expression vector (EGFP-N1) or with pHH21. (B) Virions isolated from supernatants of the same cells (whose transfections included the wild-type NP vRNA vector) were assayed for MDCK-transducing activity at 10 h postinfection by flow cytometry. (C) Total plaque-forming activities of the supernatants in panel B, assayed in MDCK monolayers.

FIG. 3.

Reporter vRNAs are comparably and independently expressed in 293T cells. (A) RPA of reporter-specific negative-sense RNA (vRNA) in 293T cells transfected 48 h previously with 1 μg of each indicated reporter plasmid along with the 17-plasmid assembly system. (B) Reporter expression in 293T cells 48 h after transfection with 1 μg of each indicated reporter type either alone (alone) or in combination with an NA- or NP-based competitor plasmid encoding RFP (+ comp). Levels of vRNA were assayed by RPA with a probe that detects GFP and YFP but not RFP sequences and were quantified by phosphorimaging. Each of the five reporters was tested alone (in quadruplicate) and with each of the two competitors (in duplicate); data shown are mean ± standard error of the mean, pooled for homologous reporters. At right, titration of one representative cellular RNA sample to determine the sensitivity of the assay.

FIG. 4.

Frequency of dual transductants. Dual transductant MDCK cells are expressed as a percentage of total transductants for combinations of homologous (Hom) and nonhomologous (Non-Hom) reporters. Data were pooled from three independent experiments.