Expanding the tolerance of segmented Influenza A Virus genome using a balance compensation strategy

Abstract

Reporter viruses provide powerful tools for both basic and applied virology studies, however, the creation and exploitation of reporter influenza A viruses (IAVs) have been hindered by the limited tolerance of the segmented genome to exogenous modifications. Interestingly, our previous study has demonstrated the underlying mechanism that foreign insertions reduce the replication/transcription capacity of the modified segment, impairing the delicate balance among the multiple segments during IAV infection. In the present study, we developed a "balance compensation" strategy by incorporating additional compensatory mutations during initial construction of recombinant IAVs to expand the tolerance of IAV genome. As a proof of concept, promoter-enhancing mutations were introduced within the modified segment to rectify the segments imbalance of a reporter influenza PR8-NS-Gluc virus, while directed optimization of the recombinant IAV was successfully achieved. Further, we generated recombinant IAVs expressing a much larger firefly luciferase (Fluc) by coupling with a much stronger compensatory enhancement, and established robust Fluc-based live-imaging mouse models of IAV infection. Our strategy feasibly expands the tolerance for foreign gene insertions in the segmented IAV genome, which opens up better opportunities to develop more versatile reporter IAVs as well as live attenuated influenza virus-based vaccines for other important human pathogens.

Fig 1. Pan-handle stabilizing mutations at the 3’-NCR enhances the transcription/replication of influenza vRNA.

(A) A schematic overview of the dual-template reporter constructs and the cell-based dual-template RdRp assay. (B) Presentation of two of the proposed vRNA promoter structure models. #, indicates the pan-handle stabilizing mutation site. (C) Normalized ratio of firefly to Renilla luciferase (Fluc/Rluc) at the vRNA, mRNA as well as protein expression levels. ***, p<0.001, students’ t test.

Fig 2. Compensatory enhancement restores the replication kinetics and virulence of a reporter influenza PR8-NS-Gluc virus.

(A) Construction of modified NS-Gluc segment. *, replication enhancing mutations. (B) Relative replication efficacies of NS-derived vRNAs normalized to M. (C) In vitro replication kinetics of wildtype and modified influenza viruses. (D and E) Female BALB/c mice were infected with 100 TCID₅₀ of indicated viruses, and the body weight (D) and survival (E) were monitored for 14 days. (F) Relative genome copies to virus titer ratio of the parental PR8-WT and recombinant IAVs PR8-NS-Gluc, PR8-NS^CE1-Gluc and PR8- NS^CE2-Gluc viruses. Indicated viruses derived from MDCK were titrated and extracted for vRNAs followed by quantification of segment M by qPCR. The ratio of genome copies to virus titer were then evaluated. The standard deviations were calculated based on three replicates. *, p<0.05; ***, p<0.001; ns, no significance; students’ t test.

Fig 3. Compensatory enhancement augments the expression of reporter genes.

(A) Kinetics of reporter luciferase expression during virus replication in vitro. (B) Normalized Gluc activity to virus titers of identical time points (relative Gluc activity/viral titer) for original PR8-NS-Gluc and mutated PR8-NS^CE1-Gluc viruses. (C) Adaptation of the reporter viruses as an in vitro high-throughput screening approach. CV, coefficient of variation; S/N, signal-to-noise ratio. (D) Kinetics of reporter luciferase expression during virus replication in mice. (E) Ex-vivo imaging of excised mice lungs. (F) The signals of ex-vivo imaging derived from mice lungs infected by reporter PR8-NS-Gluc and PR8-NS^CE1-Gluc viruses.

Fig 4. Generation and characterization of a recombinant IAV expressing Fluc gene.

(A) Schematic representation of the natural NS segment and modified NS encoding the Fluc reporter gene. SD/SA—Splice donor/acceptor sites. (B) Relative replication efficacies of NS-derived vRNAs normalized to M. (C) MDCK cells were infected with PR8-NS^CE1-Fluc or PR8-NS^CE2-FLuc viruses from serial passage experiments in eggs (passages 1 to 5) at an MOI of 0.01. At 24 h.p.i. cells were harvested, and luciferase assays were performed. (D) Comparation of the in vitro replication kinetics of PR8-WT and reporter PR8-NS^CE2-Fluc viruses. (E) PR8-NS^CE2-Fluc infection induces severe body weight loss and lethal disease in mice.

Fig 5. Establishment of an in vivo imaging mouse model of IAV infection.

(A) Mice were infected with 1,000 TCID₅₀ of PR8-NS^CE2-Fluc virus and the bioluminescence was monitored daily for 9 days. (B) Bioluminescence imaging data corresponding to the time course of PR8-NS^CE2-Fluc infection. (C) In a separate experiment, in vivo imaging was performed and mice were then euthanized for determination of viral load in lungs. The bioluminescence density and viral load from individual animals were plotted against each other from time courses of PR8-NS^CE2-Fluc infection. (D) PR8-NS^CE2-Fluc infected mice were treated with 10–30 mg/kg/day of oseltamivir phosphate or vehicle only, and the in vivo imaging was carried out on days 2 and 5. (E) Data correspond to the in vivo imaging. *, p<0.05; **, p<0.01; students’ t test.

Fig 6. Establishment of an in vivo imaging mouse model of IAV subtype H3N2 infection.

(A) Comparation of the in vitro replication kinetics of wildtype X31 and reporter X31-NS^CE2-Fluc viruses. (B) The lethality of wildtype X31 and reporter X31-NS^CE2-Fluc viruses in mouse models. (C) Mice were infected with series doses of X31-NS^CE2-Fluc virus and the bioluminescence was monitored at days 1, 2, 3, 5, and 8 post challenge. (D) The kinetics of bioluminescence imaging in mice infected with varies doses of X31-NS^CE2-Fluc virus. Inset, linearization analysis of the bioluminescence signals at day 2 post challenge to challenge doses.

Fig 7. Representative diagram of the molecular mechanism underlying attenuation of reporter IAVs and the proposed “balance compensation” strategy for directed optimization.

(A) The NS segment was modified with foreign insertions, resulting in reduced replication/transcription of the NS-derived vRNA and impaired balance of the multiple segments. Subsequently, a large proportion of progeny virions are NS-null and non-infectious. (B) For directed optimization of the reporter IAV, proper compensatory enhancement was incorporated during initial construction. The replication/transcription of NS-derived vRNA was specifically increased, while rebalance of the segmented genome could be achieved, restoring the wildtype-like fitness. Of note, the multiple segments in wildtype PR8 virus infected cells were shown in equal molar ratio for conceptual illustration only.