Influenza A virus nucleoprotein selectively decreases neuraminidase gene-segment packaging while enhancing viral fitness and transmissibility

Abstract

The influenza A virus (IAV) genome is divided into eight distinct RNA segments believed to be copackaged into virions with nearly perfect efficiency. Here, we describe a mutation in IAV nucleoprotein (NP) that enhances replication and transmission in guinea pigs while selectively reducing neuraminidase (NA) gene segment packaging into virions. We show that incomplete IAV particles lacking gene segments contribute to the propagation of the viral population through multiplicity reactivation under conditions of widespread coinfection, which we demonstrate commonly occurs in the upper respiratory tract of guinea pigs. NP also dramatically altered the functional balance of the viral glycoproteins on particles by selectively decreasing NA expression. Our findings reveal novel functions for NP in selective control of IAV gene packaging and balancing glycoprotein expression and suggest a role for incomplete gene packaging during host adaptation and transmission.

Keywords: genome packaging; genome segmentation; host adaptation; influenza virus; nucleoprotein.

Fig. 1.

NP modulates virion glycoprotein balance through epistatic control of NA expression. (A) Relative protein content of HA, NA, and M1 in purified particle preparations. P < 0.0001 for differences between all NA ratios for PR8_WT and PR8_NP:F346S (one-way ANOVA, Tukey test). Each data point represents a comparison of independent stocks of PR8_WT and PR8_NP:F346S grown at the indicated MOIs. (B) NA activity for purified PR8_NP:F346S normalized to average HAU or HA or M1 protein as a percentage of the PR8_WT control, presented as mean ± SEM; P < 0.0001 for differences between all NA ratios compared with the matched wild-type control (one-way ANOVA, Tukey test). (C) Relative viral protein expression as determined by geometric mean fluorescence intensity (GMFI) on MDCK cells infected at an MOI <0.05 with PR8_WT or PR8_NP:F346S that stained positive for the indicated viral proteins. PR8_NP:F346S values are presented as the mean percentage of PR8_WT ± SEM of three technical replicates. (D) Relative copy numbers of HA and NA vRNA and mRNA in FACS-sorted, singly infected HA⁺NA⁺ cells presented as mean ± SEM, as determined by strand-specific quantitative real-time RT-PCR (*P < 0.05; **P < 0.01; ***P < 0.001; t test).

Fig. 2.

NP controls NA expression in vivo. (A and B) Viral protein expression in nasal turbinate cells collected from guinea pigs (n = 2) 48 h after i.n. infection with 10³ TCID₅₀ of PR8_M1:V166M or PR8_{M1:V166M+NP:F346S} viruses as determined by flow cytometry. (A) Comparison of NP, HA, and NA expression within NP⁺HA⁺NA⁺ cells. (B) The mean per-cell fluorescence ratios for the indicated stains were calculated for NP⁺HA⁺NA⁺ cells. Data represent the mean ± SEM (*P < 0.05; t test). (C) Ratio of NA activity (determined using MUNANA substrate) and HA content measured by ELISA in nasal wash from animals (n = 3) infected with 10³ TCID₅₀ of PR8_M1:V166M or PR8_{M1:V166M+NP:F346S} viruses. Data are presented as mean ± SEM (***P < 0.001 *P < 0.05; t test). (D) Nasal wash titers from guinea pigs infected i.n. with 500 TCID₅₀ of the indicated viruses (n = 3). Data are presented as mean ± SEM.

Fig. 3.

NP selectively reduces relative NA gene-segment packaging. (A) Representative flow cytometry plots of viral protein expression in cells infected at an MOI <0.05 with equal HAEU. (B) The population expression pattern illustrated as the percentage of cells in A that expressed detectable levels of the indicated proteins. Data represent mean ± SEM of three technical replicates. (C) Relative HA, NA, and M vRNA content in purified PR8_NP:F346S virus preparations determined by quantitative RT-PCR as a percentage of the matched PR8_WT controls. Data are presented as mean ± SEM (*P < 0.05; t test). (D) Relative abundance of vRNA segments as determined by quantitative PCR in nasal wash of PR8_{M1:V166M+NP:F346S}-infected guinea pigs (n = 2) 48 hpi as a percent of values from PR8_M1:V166M-infected guinea pigs. Data are presented as mean ± SEM (*P < 0.05; t test). (E) NP expression in MDCK cells infected at an MOI of 0.01 TCID₅₀ per cell with PR8_WT or PR8_NP:F346S.

Fig. 4.

SI particles contribute to replication through multiplicity reactivation. (A) Mean TCID₅₀ and SI particle output per HA⁺NP⁺NA⁺NS1⁺ producer cell by 8–9 hpi. Data are pooled from two independent experiments, and each point represents the value obtained from a single culture. P = 0.0885 for TCID₅₀; P = 0.0406 for SI particles; Mann–Whitney test. (B) Virus output (in TCID₅₀/mL) from MDCK cells infected for 8 h with PR8_WT or PR8_NP:F346S presented as the fold increase over output at an MOI of 0.01 for three stocks grown in parallel at each MOI ± SEM. Interaction between strain and MOI: P = 0.0308 by two-way ANOVA.

Fig. 5.

The frequency of viral protein coexpression increases with MOI. MDCK cells were infected for 9 h with PR8_M1:V166M or PR8_{M1:V166M+NP:F346S} at the indicated MOIs, and the percentage of NP⁺ cells that also express HA and NA was determined by flow cytometry. (A) Representative dot plots. (B) Mean ± SEM of two replicates per condition.

Fig. 6.

Coinfection is common in the guinea pig upper airway during IAV infection. (A and B) Guinea pigs were infected i.n. with 10³ TCID₅₀ (or 10⁶ for the 9 hpi time point) of PR8_M1:V166M or PR8_{M1:V166M+NP:F346S}. Nasal wash and turbinates were collected 9 and 48 hpi. (A) Representative dot plots showing NP⁺ MDCK cells following ex vivo infection at an MOI <0.05 with 48 hpi nasal wash or NP⁺ nasal turbinate cells collected at 9 or 48 hpi. (B) The percentage of 48 hpi NP⁺ nasal turbinate cells that also were HA⁺NA⁺ was determined and compared with MDCK cells infected at an MOI <0.05 with nasal washes from the same animals. Data represent the mean ± SD of two animals per virus. (C) Relative expression of HA, NP, NA, and NS1, as determined by flow cytometry, in HA⁺NP⁺NA⁺ Vero cells following infection with PR8_WT at an MOI of 10 HAEU per cell alone or with increasing doses of PR8_NoNA. Data represent the mean percentage change in ratios of the indicated proteins compared with PR8_WT alone ± SEM for three technical replicates.