Influenza virus inhibits respiratory syncytial virus (RSV) infection via a two-wave expression of interferon-induced protein with tetratricopeptide (IFIT) proteins

Influenza viruses and respiratory syncytial virus (RSV) are respiratory viruses that primarily circulate worldwide during the autumn and winter seasons. Seasonal surveillance shows that RSV infection generally precedes influenza. However, in the last four winter seasons (2016-2020) an overlap of the morbidity peaks of both viruses was observed in Israel, and was paralleled by significantly lower RSV infection rates. To investigate whether the influenza virus inhibits RSV we performed coinfection of Human cervical carcinoma (HEp2) cells or mice with influenza and RSV and we observed that the influenza inhibited RSV growth, both in vitro and in vivo. Mass spectrometry analysis of mouse lungs infected with influenza identified a two-wave pattern of protein expression upregulation, which included members of the interferon-induced protein with tetratricopeptide (IFITs) family. Interestingly, in the second peak of upregulation, influenza viruses were no longer detectable in mouse lungs. We also observed that knockdown and overexpression of IFITs in HEp2 cells affected RSV multiplicity. In conclusion, influenza infection inhibits RSV infectivity via upregulation of IFIT proteins in a two-wave modality. Understanding of the interaction between influenza and RSV viruses and immune system involvement will contribute to the development and optimization of future treatment strategies against these viruses. Author Summary Respiratory syncytial virus (RSV) and influenza viruses are both respiratory viruses associated with morbidity and mortality worldwide. RSV is usually detected in October, with a clear peak in December, whereas influenza virus arrives in November and peaks in January. In the last four seasons, influenza infection overlapped with that of RSV in Israel, which resulted in decreased morbidity of RSV suggesting that influenza virus inhibits RSV infection. To identify the mechanism responsible for the influenza inhibition of RSV we performed experiments in culture and in mice. We observed that influenza infection results in two wave modality of inhibition of RSV infection. Using mass spectrometry perfornmed on lungs from infected mice we show that influenza infection induces the expression of (IFIT) family of proteins which also showed a two-wave modality. Using knockdown and overexpression experiments we showed that indeed the IFTIs inhibits RSV infection. Our study provides new insights on the interaction between influenza and RSV viruses and immune system involvement and contribute to the development of future treatment strategies against these viruses.

associated with morbidity and mortality worldwide. RSV is usually detected in October, with 48 a clear peak in December, whereas influenza virus arrives in November and peaks in January. 49 In the last four seasons, influenza infection overlapped with that of RSV in Israel, which 50 resulted in decreased morbidity of RSV suggesting that influenza virus inhibits RSV 51 infection. To identify the mechanism responsible for the influenza inhibition of RSV we 52 performed experiments in culture and in mice. We observed that influenza infection results in 53 two wave modality of inhibition of RSV infection. Using mass spectrometry perfornmed on 54 lungs from infected mice we show that influenza infection induces the expression of (IFIT) 55 family of proteins which also showed a two-wave modality. Using knockdown and 56 overexpression experiments we showed that indeed the IFTIs inhibits RSV infection. Our 57 study provides new insights on the interaction between influenza and RSV viruses and Respiratory syncytial virus (RSV) and influenza viruses are both respiratory viruses 62 associated with significant morbidity and mortality worldwide(1). RSV is a leading pathogen 63 causing acute lower respiratory tract infection (ALRTI)(2) in all age groups(3, 4) primarily 64 infants and young toddlers(5, 6), while influenza virus affect all age groups(7). In Israel, RSV 65 is usually detected in October, with a clear peak in December, whereas influenza virus arrives 66 in November and peaks in January (8). Previous surveillance demonstrated a close temporal 67 relationship between circulating influenza virus and RSV, where influenza epidemics usually 68 occur when RSV infections subside(9). On the other hand, in the last four seasons, influenza 69 infection overlapped with that of RSV in Israel, which resulted in decreased morbidity of 70 RSV suggesting cross-immunity between influenza virus and RSV infection(10). We assume 71 that changes in the arrival time of one virus can affect the arrival time and morbidity pattern 72 of the other.

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The activity of both innate and adaptive immune systems against both viruses is very 74 well documented(11 ,2). Innate immunity against these viruses includes, among others, 75 interferon stimulating genes (ISGs) pathway, which trigger the expression of the interferon-76 induced protein with tetratricopeptide (IFIT) family of proteins (12,13). In humans, IFIT1-3 77 proteins form a complex with each other to inhibit translation of viral mRNA molecules (14).

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IFIT1 acts as a sensor that identifies specific viral single-stranded RNAs and selectively 79 inhibits viral protein synthesis without affecting host cell protein synthesis(15). While IFIT1 80 is known to specifically recognize the viral mRNA(15), it has been suggested that IFIT2 and 81 IFIT3 facilitate the binding of the IFIT1:2:3 complex structure to the viral mRNA(17 ,16).

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Less in known about the function of IFI44. The present work investigated the role of IFIT1-3 83 and IFI44 proteins in the cross-immunity effect observed between influenza and RSV.

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Samples obtained from both hospitalized and non-hospitalized patients in Israel 2009-87 10, showed that influenza virus A/H1N1pdm09 virus that year was present in the country 88 during the summer season and peaked in July and November 2009 ( Fig 1A). In contrast, in  Fig 1B) and January ( Fig 1C). Surprisingly, from the winter of 2016-17 onwards, both RSV 92 and influenza co-circulated in October and both peaked around January (Fig 1B and C). this 93 early circulation of influenza in the winters of 2016-20 coincided with a lower morbidity of 94 RSV than previous seasons , and also coincided with later peak of RSV in January.

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Overall, RSV morbidity percentages were significantly lower (p<0.0005) as compared to the 96 two preceding winter seasons (Fig 1B and 1C), suggesting that influenza infection affecting 97 RSV infection rate.

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To test whether influenza virus presence can indeed inhibit RSV infection rate, HEp2 100 cells were infected with RSV or with influenza A virus, followed by RSV; the number of 101 RSV copies was then determined at various time points. When applied alone, RSV copy 102 numbers increased with time from infection (24, 48, 72, 96 and 144 hours) (Fig 2), however, 103 when first infected with influenza A, cells produced statistically significant fewer RSV copy 104 numbers at all tested time points (Fig 2).

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Influenza infection inhibits RSV in vivo in a two-wave modality 106 To test whether influenza infection inhibits RSV in vivo, mice were infected with 107 influenza A/PR8/1934 ("Influenza A") and then infected with RSV ( Fig 3A). Influenza A virus 108 was detected in mouse lungs until day 6 post-infection ( Fig 3B). In contrast, RSV was not

Two-wave elevation of anti-viral proteins following influenza infection 115
To understand the two-wave effect of influenza on RSV, a mass spectrometry analysis 116 was performed on healthy mice and on mice infected with influenza A. A total of 10 proteins 117 demonstrated a two-wave behavior of upregulation (Fig 4) and are known to have antiviral 118 activity (Table 1)

Protein names Function
Eif2ak2 Inhibits viral replication via phosphorylation of the alpha subunit of eukaryotic initiation factor. Gbp1 Promote oxidative killing and deliver antimicrobial peptides to autophagolysosomes. Phf11 Inhibitor of prototype foamy virus (PFV) replication.

Ifi44
Exhibits an antiviral activity against hepatitis C virus.

Ifit1
Acting as a sensor of viral single-stranded RNAs and inhibiting expression of viral messenger RNAs.

Ifit2
Distinguish between self and non-self mRNAs by the host during viral infection. Ifit3 IFN-induced antiviral protein which acts as an inhibitor of viral processes,and viral replication. Ifitm3 Inhibits the entry of viruses to the host cell cytoplasm. Oas3 dsRNA-activated antiviral enzyme which plays a critical role in cellular innate antiviral response. Zbp1 Participates in the detection by the host's innate immune system of DNA from viral.

IFIT members inhibit RSV infection 126
To assess the role of IFIT1-3 and IFI44 protein in inhibition of RSV infection, each 127 gene was individually silenced in Hep2 cells. Silencing was confirmed by Western blotting 128 (Fig 6A-D). Silencing of each of the four proteins resulted in increased RSV infection as 129 compared to wild type cells (Fig 6E). In parallel, RSV infection of IFIT1-3 or IFI44-

Statistical analysis
302 T-test was applied to evaluate the differences in percent positivity between the 303 compared groups. A p value < 0.05 was considered statistically significant. All analyses were 304 performed using IBM® SPSS® Statistics software (Version 23) and Excel software 305 (Microsoft®).

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This work was performed in partial fulfillment of the requirements for the Ph.D degree 307 of Yaron Drori, Sackler Faculty of Medicine, Tel Aviv University.