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. 2023 Apr 13;12(8):1152.
doi: 10.3390/cells12081152.

Prolonged Primary Rhinovirus Infection of Human Nasal Epithelial Cells Diminishes the Viral Load of Secondary Influenza H3N2 Infection via the Antiviral State Mediated by RIG-I and Interferon-Stimulated Genes

Hsiao Hui Ong  1   2 , Jing Liu  1   2 , Yukei Oo  2 , Mark Thong  3 , De Yun Wang  1   2 , Vincent T Chow  2   4
Affiliations

Prolonged Primary Rhinovirus Infection of Human Nasal Epithelial Cells Diminishes the Viral Load of Secondary Influenza H3N2 Infection via the Antiviral State Mediated by RIG-I and Interferon-Stimulated Genes

Hsiao Hui Ong et al. Cells. .

Abstract

Our previous study revealed that prolonged human rhinovirus (HRV) infection rapidly induces antiviral interferons (IFNs) and chemokines during the acute stage of infection. It also showed that expression levels of RIG-I and interferon-stimulated genes (ISGs) were sustained in tandem with the persistent expression of HRV RNA and HRV proteins at the late stage of the 14-day infection period. Some studies have explored the protective effects of initial acute HRV infection on secondary influenza A virus (IAV) infection. However, the susceptibility of human nasal epithelial cells (hNECs) to re-infection by the same HRV serotype, and to secondary IAV infection following prolonged primary HRV infection, has not been studied in detail. Therefore, the aim of this study was to investigate the effects and underlying mechanisms of HRV persistence on the susceptibility of hNECs against HRV re-infection and secondary IAV infection. We analyzed the viral replication and innate immune responses of hNECs infected with the same HRV serotype A16 and IAV H3N2 at 14 days after initial HRV-A16 infection. Prolonged primary HRV infection significantly diminished the IAV load of secondary H3N2 infection, but not the HRV load of HRV-A16 re-infection. The reduced IAV load of secondary H3N2 infection may be explained by increased baseline expression levels of RIG-I and ISGs, specifically MX1 and IFITM1, which are induced by prolonged primary HRV infection. As is congruent with this finding, in those cells that received early and multi-dose pre-treatment with Rupintrivir (HRV 3C protease inhibitor) prior to secondary IAV infection, the reduction in IAV load was abolished compared to the group without pre-treatment with Rupintrivir. In conclusion, the antiviral state induced from prolonged primary HRV infection mediated by RIG-I and ISGs (including MX1 and IFITM1) can confer a protective innate immune defense mechanism against secondary influenza infection.

Keywords: IFITM1; MX1; RIG-I; human nasal epithelium; innate immune responses; interferon-stimulated genes; rhinovirus persistence; rhinovirus re-infection; secondary influenza virus infection.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Reduction in virus progeny production and expression of IAV NS1 and M1 proteins of secondary H3N2 infection, but not HRV VP2 of HRV-A16 re-infection in hNECs (following primary prolonged HRV infection). (A) Timeline of prolonged primary HRV infection and subsequent secondary H3N2 infection or HRV-A16 re-infection. (B) Infectious virus progeny was quantified (PFU per 100 μL) using plaque assay (n = 7). There were no significant changes in (C) HRV RNA or (D) VP2 protein of HRV re-infection as compared to single HRV infection and controls. (E–H) However, IAV RNAs and relative protein levels (RPL) of NS1 and M1 of secondary H3N2 infection were significantly reduced (n = 5). The relevant band intensities were measured using ImageJ software. The corresponding p-values are shown. The p-values were calculated by comparison with single infection of the respective virus using the non-parametric Mann–Whitney U-test. The data are represented as medians with interquartile values. (I) Representative Western blot images are shown.
Figure 2
Figure 2
Increased protein expression of RIG-I, but not MDA5, during secondary H3N2 infection and HRV-A16 re-infection of hNECs (following primary prolonged HRV infection). The mRNA expression profiles of (A) RIG-I and (B) MDA5 in infected and mock control hNECs (n = 6). Relative protein levels (RPL) of (C) RIG-I and (D) MDA5 in infected and mock control hNECs (n = 5). The relevant band intensities were measured using ImageJ software. Protein levels were normalized to GAPDH housekeeping protein. (E) Representative Western blot images are depicted. The p-value was calculated by one-way ANOVA and non-parametric Kruskal–Wallis test. The data are represented as medians with interquartile values.
Figure 3
Figure 3
Increased expression of MX1 and IFITM1 proteins during secondary H3N2 infection and HRV-A16 re-infection of hNECs (following primary prolonged HRV infection). The mRNA expression profiles of ISGs (A) MX1 and (B) IFITM1 in infected and mock control hNECs (n = 6). Relative protein levels (RPL) of (C) MX1 and (D) IFITM1 protein expression in infected and mock control hNECs (n = 5). The relevant band intensities were measured using ImageJ software. Protein levels were normalized to GAPDH housekeeping protein. (E) Representative Western blot images are shown. The p-value was calculated by one-way ANOVA and non-parametric Kruskal–Wallis test. The data are represented as medians with interquartile values.
Figure 4
Figure 4
Earlier and longer-duration Rupintrivir-mediated inhibition of HRV 3C protease from prolonged primary HRV infection abolished the reduction in live influenza viral load of secondary H3N2 infection. (A) Timeline of early and multi-dose Rupintrivir treatment prior to secondary H3N2 infection of hNECs. (B) Infectious virus progeny was quantified (PFU per 100 μL) using virus plaque assay (n = 6). (C) The mRNA levels (n = 4) and (D) relative protein levels (RPL) (n = 6) of IAV M1 of secondary H3N2 infection, with and without Rupintrivir treatment, in infected and mock control hNECs. Representative Western blot images are shown. The relevant band intensities were measured using ImageJ software. Protein levels were normalized to GAPDH housekeeping protein. The p-value was calculated by one-way ANOVA and non-parametric Kruskal–Wallis test. The data are represented as medians with interquartile values.
Figure 5
Figure 5
Earlier and longer-duration Rupintrivir inhibition of 3C protease from primary prolonged HRV infection led to decreasing trends in protein levels of ISG proteins MX1, IFITM1, and ISG15 during secondary H3N2 infection. The mRNA fold changes (n = 4) and relative protein levels (RPL) (n = 6) of (A,B) MX1, (C,D) IFITM1, and (E,F) ISG15 during secondary H3N2 infection, with and without longer-duration Rupintrivir treatment, in mock and infected hNECs. Representative Western blot images are shown. The relevant band intensities were measured using ImageJ software. Protein levels were normalized to the host GAPDH protein. The p-value was calculated by one-way ANOVA and non-parametric, Kruskal–Wallis test. The data are represented as medians with interquartile values.
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Grants and funding

This research was funded by the National Medical Research Council, Singapore (NMRC/CIRG/1458/2016), and by the Ministry of Education Academic Research Fund Tier 3, Singapore (MOE-000095-01). Funding from the National University of Singapore is also gratefully acknowledged.
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