Most influenza a virions fail to express at least one essential viral protein

Abstract

Segmentation of the influenza A virus (IAV) genome enables rapid gene reassortment at the cost of complicating the task of assembling the full viral genome. By simultaneously probing for the expression of multiple viral proteins in MDCK cells infected at a low multiplicity with IAV, we observe that the majority of infected cells lack detectable expression of one or more essential viral proteins. Consistent with this observation, up to 90% of IAV-infected cells fail to release infectious progeny, indicating that many IAV virions scored as noninfectious by traditional infectivity assays are capable of single-round infection. This fraction was not significantly affected by target or producer cell type but varied widely between different IAV strains. These data indicate that IAV exists primarily as a swarm of complementation-dependent semi-infectious virions, and thus traditional, propagation-dependent assays of infectivity may drastically misrepresent the true infectious potential of a virus population.

Fig 1

Low-multiplicity IAV infection results in cells expressing an incomplete set of viral proteins. (A) MDCK cells were infected with PR8 at MOIs of 5 and 0.04. At 7 h p.i. cells were fixed, permeabilized, and assessed for HA (human MAb 70-1F02), NA (rabbit pAb), and NS1 (mouse MAb 1A7) expression by IF. Images were acquired using a 40× objective and are representative of six independent experiments. (B) MDCK cells were infected with PR8 harvested from infected mouse lung at an MOI of 0.04 based on TCID₅₀ titer. At 7 h p.i., cells were assessed for HA, NA, and NS1 expression by IF as described for panel A. Images were acquired using a 63× objective and are representative of four independent experiments. Scale bar, 20 μm.

Fig 2

Incomplete viral protein expression is not observed during low-multiplicity VSV infection. MDCK cells were infected with VSV at an MOI of <0.01. At 5 h p.i. cells were fixed, permeabilized, and assessed for expression of the viral G (mouse MAb 8G5-F11) and N (rabbit pAb) proteins by IF. Images were acquired using a 63× objective and are representative of two independent experiments. Scale bar, 10 μm.

Fig 3

The majority of infectious IAV virions express an incomplete set of viral proteins. MDCK cells were infected in triplicate with PR8 at an MOI of 0.03. Two hours later, neutralizing anti-HA MAb (H17-L2; 10 μg/ml) was added to cultures. At 16 h p.i., cells were harvested, fixed/permeabilized, stained against HA (mouse MAb H36-26), NA (mouse MAb NA2-1C1), NP (mouse MAb HB-65), and NS1 (mouse MAb 1A7), and then run on an LSR II flow cytometer. (A) Representative dot plots from mock- and PR8-infected MDCK cells assessed for HA and NA expression. (B) Representative NS1 and NP expression histograms of the populations defined in panel A based on HA and NA expression. (C) Infected cells (based on expression of one or more of the viral proteins examined) were classified based HA, NA, NP, and NS1 expression patterns, and the percentage of the total infected cell pool that each of these populations made up was then calculated. Values represent the means of three infection replicates and are representative of five independent experiments. (D) MDCK cells were infected with PR8 at MOIs of 0.01, 1, and 10 and then analyzed as described for panel A.

Fig 4

An incomplete viral protein expression pattern is seen in multiple target cells. MDCK, Vero, and A549 cells were infected in triplicate with PR8 at an MOI of 0.03. Two hours later, neutralizing anti-HA MAb (H17-L2; 10 μg/ml) was added to cultures. At 16 h p.i. cells were harvested, fixed/permeabilized, stained against HA (mouse MAb H36-26) and NA (mouse MAb NA2-1C1), and then run on an LSR II flow cytometer. (A) Infected cells (based on expression of one or more of the viral proteins examined) were classified based on HA and NA expression patterns. (B) The percentage of the total infected cell pool that each of these populations made up was then calculated. Data represent the means ± standard errors of the means of three infection replicates and are representative of two independent experiments.

Fig 5

The majority of infectious IAV virions are incapable of producing infectious progeny. Confluent monolayers were grown on coverslips, infected with the indicated viruses at an MOI of <0.01, and then overlaid with 0.9% agarose. After 15 h, cells were fixed, permeabilized, and then stained against the indicated viral proteins. (A) MDCK cells were infected with PR8 and then stained against HA (mouse MAb H36-26), NP (rabbit pAb), and M1/M2 (goat cross-reactive pAb) (all false-colored white). The confocal image was generated with a 40× objective by tiling z-stacks and is representative of 12 independent experiments. Scale bar, 100 μm. (B) MDCK and Vero cells were infected with PR8 and then stained against HA (mouse MAb H36-26) and NP (rabbit pAb). Between 100 and 400 infection events on each coverslip were counted by eye as either abortive (1 to 2 adjacent, lone infected cells) or productive (3 or more adjacent infected cells), and the percentage of infection events that were productive was calculated. Each data point represents the value generated from a single coverslip, and the bars represent the means. (C) MDCK cells were infected with stocks of PR8 grown in eggs and MDCK cells, lung homogenates from PR8-infected mice, and nasal wash from PR8-infected guinea pigs and assessed as described for panel B. Data are representative of two independent experiments.

Fig 6

The efficiency of propagation-competent virus production differs widely between different viral genotypes. (A) A/Udorn/307/72 (Udorn) and A/California/07/09 (Cal) were grown in embryonated hen eggs and used to infect MDCK cells at MOIs of 0.01 to 0.03. At 7 (Udorn) or 16 (Cal) h p.i., cells were fixed, permeabilized, and assessed for HA (mouse MAb H14-A2), NP (rabbit pAb), and M1/M2 (goat cross-reactive pAb) (for Udorn) or HA (human MAb 70-1F02), NA (rabbit pAb), and NS1 (mouse MAb 1A7) (for Cal) expression by IF. Images were acquired using a 40× objective and are representative of three independent experiments. (B) Confluent monolayers of MDCK cells were grown on coverslips, infected with the indicated viruses at an MOI of <0.01, and then overlaid with 0.9% agarose. Fifteen hours later, cells were fixed, permeabilized, and then stained against HA (human MAb 70-1F02) and NP (rabbit pAb). Between 100 and 400 infection events on each coverslip were counted by eye as either abortive (1 to 2 adjacent, lone infected cells) or productive (3 or more adjacent infected cells), and the percentage of infection events that were productive was calculated. Each data point represents the value generated from a single coverslip, and the bars represent the means. Data are representative of four independent experiments, using multiple stocks of each virus grown in eggs and MDCK cells.