Human telomerase reverse transcriptase supports respiratory syncytial virus replication

Huynh, HoangDinh; Wu, Chien-Ting; Kahn, Jeffrey S.

doi:10.1038/s42003-026-09958-4

Human telomerase reverse transcriptase supports respiratory syncytial virus replication

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Published: 10 April 2026

Volume 9, article number 807 (2026)
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Communications Biology

Human telomerase reverse transcriptase supports respiratory syncytial virus replication

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HoangDinh Huynh¹,
Chien-Ting Wu² &
Jeffrey S. Kahn^1,2

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Abstract

Respiratory syncytial virus (RSV), discovered in 1956 and identified in children in 1957, is the major respiratory pathogen of infants and children under the age of 5 worldwide. RSV remains a significant challenge, despite recent advancements of vaccine development and monoclonal antibody prophylaxis. While specific antiviral agents have shown success as therapeutics for many other viruses, viral mutations inevitably develop, making these therapeutic interventions ineffective due to viral resistance. This unavoidable obstacle warrants alternative approaches to targeting host cellular factors that are essential for viral replication regardless of viral mutations. Our goal was to assess the feasibility of this approach. In doing so, we sought to determine the cellular enzymes and functions that are required for RSV replication. Here, we demonstrate that inhibiting human telomerase reduced or abolished viral protein production. Further, we showed that RNA helicase eIF4A is essential for RSV protein and progeny production. Lastly, targeting nuclear transport receptor reduces RSV RNAs and proteins synthesis, suggesting a role of host nucleus for viral replication. Overall, the inhibition of virus replication-dependent host functions may be an effective means to combat viral infections and would sidestep the inevitable resistance that emerges with virus-specific strategies.

Introduction to Respiratory Syncytial Virus

Respiratory syncytial virus infection and novel interventions

Article 12 July 2023

A Rapid Yeast-Based System for Modifying Respiratory Syncytial Virus

Introduction

Since its isolation from children with lower respiratory tract disease in 1957, respiratory syncytial virus (RSV) has been recognized as the major respiratory pathogen of infants and children under the age of 5 worldwide¹. RSV infection in children is nearly unescapable with most children infected within the first few years of life; severe life-threatening acute lower respiratory tract infection (LRTI) represents the most worrisome sequelae of RSV infection. In 2019, there were ~33 million cases of acute LRTI globally in children under 5 years of age with 3.6 million hospitalizations, and 101,400 deaths due to RSV². In addition, RSV globally accounts for approximately 466,000 hospitalizations with 33,000 deaths in older adults (≥60 years) per year in high-income countries³. In the United States, it is estimated that between 60,000 and 160,000 hospitalizations and 6000–10,000 deaths occur annually in elderly or high-risk adults⁴. Thus, RSV infection represents a substantial burden on healthcare. There are 2 recognized subgroups of RSV, A and B, that can be distinguished by serological and genetic methods. Both subgroups circulate during each seasonal epidemic^5,6. RSV strains identified in one location may be similar to or quite distinct from strains identified in similar or previous different years, pointing to the complex epidemiology of this pathogen.

RSV a member of the Pneumoviridae family⁷ is an enveloped virus, with a non-segmented single-stranded, negative sense RNA genome⁸. The viral genome is approximately 15,000 bases in length and encodes 11 proteins with short intergenic distances or overlapping reading frames⁹. The viral genome encodes three surface proteins: fusion (F), attachment (G), and short hydrophobic (SH)¹⁰. Viral attachment is dependent on the F protein, which, as the name implies, functions as a mediator of fusion between the viral envelope and cell membrane and is the viral factor that creates the typical syncytia observed in cell culture¹¹. The viral ribonucleocapsid consist of viral genome, nucleoprotein (N), phosphoprotein (P), polymerase (L) and transcription factor M2–1¹², which is encased with envelope-associated matrix (M) protein¹³. The viral polymerase is thought to be a component of the infectious viral particle, initiating transcription of viral genes upon infection and uncoating via RNA-dependent, RNA polymerase activity.

Although the virus was originally discovered in chimpanzees in 1956¹⁴, and isolated from symptomatic children in 1957¹⁵, the management of RSV had changed little until 2023. Antiviral agents for RSV infections have poor effectivity and toxicity profiles and are rarely used in the management¹⁶. Recent advancements in vaccine and monoclonal antibody development, exploiting the pre-fusion form of the RSV fusion F protein, offers additional strategies to protect infants from RSV infection and disease via maternal vaccination¹⁷. Despite these advancements, one foreseeable obstacle is the development of viral mutations^18,19 that will result in variants that evade the vaccine-induced immunity. Indeed, resistance mutations were identified in RSV F protein from RSV-infected infants that received nirsevimab²⁰. Further, it is unlikely that, even with the most effective vaccines, RSV will be eliminated. Therefore, alternative antiviral approaches should be considered.

Once inside the host, RSV utilizes specific host intracellular pathway(s) or machinery for viral replication¹⁹, and these pathways are potential targets for alternative antiviral therapy. Interfering with specific host cellular factors required for RSV gene expression, protein synthesis or progeny virus production could mitigate viral replication and this strategy could potentially be used as effective antiviral in the clinical setting. Since this approach does not target the virus directly, it would be highly unlikely that any viral mutation would result in resistance. Consistent with this notion, we have previously shown that the mechanistic target of rapamycin (mTOR) signaling plays an important role in RSV replication and growth²¹. mTOR signaling is known to play important role in many fundamental cellular processes, including energy metabolism, lipogenesis, autophagy, lysosome biogenesis, cytoskeletal organization, cell growth and survival²². In this present study, we evaluate the role of a specific pathways downstream of mTOR signaling in the RSV replication cycle. In addition, we explore novel cellular functions that may serve as potential targets of antiviral strategies.

Results

RSV viral protein synthesis does not require host 5’ mRNA cap protein eIF4E function but requires eIF2α

Eukaryotic initiation factor 4E (eIF4E) is known as m⁷G cap recognition factor²³ and is a site of translational control by eIF4E-binding proteins (4E-BPs), a target regulated by mTOR signaling²². To evaluate the role of eIF4E on RSV viral proteins synthesis, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system^24,25 was employed to eliminate eIF4E in human A549 cells, a standard cell line is known permissive for RSV viral replication. eIF4E-negative CRISPR A549 cells were infected with RSV strain NH409A²¹, and western blotting assay was performed at 24 h post-infection (hpi). Reduction of host eIF4E protein (Fig. 1A, C) does not significantly impact RSV viral nucleoprotein N (Fig. 1F) and fusion F1 (Fig. 1G) proteins expression. However, reduction of eIF4B (Fig. 1A, D), a co-factor involved 5’mRNA cap-dependent ribosome recruitment²⁶, significantly reduced RSV viral proteins (Fig. 1F, G). The reduction of viral protein synthesis was confirmed by CRISPR eIF2α (Fig. 1A, E–G), a subunit of translation initiator factor eIF2 protein complex which binds to Met-tRNA_i and modulates the global step of protein synthesis initiation²⁷. Our previous work has shown both RSV and human coronaviruses (HCoV) utilized mTOR signaling for viral replication²¹. To evaluate whether these factors are unique for RSV protein synthesis, HCoV OC43 strain was tested. In contrast to RSV, eliminating eIF4E nearly abolished HCoV OC43 spike S protein synthesis (Fig. 1B, H). As opposed to RSV, reduction of eIF4B does not negatively impact HCoV viral protein expression. Like RSV, CRISPR eIF2α abolished HCoV S protein production. These observations suggest RSV viral protein synthesis is cap eIF4E-independent, and RSV has distinct mechanism from HCoV in regards to viral protein synthesis.

Inhibition of host RNA helicase eIF4A abolishes RSV viral protein synthesis and virus progeny

Reduction of eIF4B protein level has significant negative impact on RSV viral protein production (Fig. 1). eIF4B is also known as an accessory factor for eIF4A, an RNA helicase²⁸. To evaluate whether human RNA helicase has any impact on RSV replication, eIF4A was targeted by the CRISPR/Cas9 system. Reduction of eIF4A protein (Fig. 2A–C) nearly completely abolished RSV viral protein production (Fig. 2D, E). To confirm the observations using the CRISPR system, RSV-infected A549 cells were treated (2 hpi) at varying doses with episilvestrol, a specific eIF4A inhibitor²⁹ for 22 h (Fig. 2F, G). At 10 nM, episilvestrol completely abolished RSV viral proteins expression (Fig. 2H, I). Functionally, reduced viral protein levels also correlated with decrease in viral production of virus progeny (Fig. 2J). However, pharmacologic inhibition of eIF4A does not negatively impacted the expression of RSV viral mRNAs non-structural protein 2 (NS2) (Fig. 2K), nucleoprotein N (Fig. 2L), phosphoprotein P (Fig. 2M), matrix protein M (Fig. 2N), glycoprotein G (Fig. 2O), fusion protein F (Fig. 2P), and large polymerase subunit L (Fig. 2Q), indicating that viral protein synthesis and replication and generation of infectious progeny virus is inhibited downstream of viral RNA transcription.

To further explore the effect of helicase activity reduction in the viral lifecycle, both coronavirus OC43 and influenza were studied in an identical manner. Both CRISPR knockdown and episilvestrol treatment did not inhibit OC43 spike S protein synthesis (Fig. 2R, T), which is not surprising as coronaviruses, whose genomes are positive sense, encodes its own helicase³⁰. Curiously, CRISPR and episilvestrol-treated (at 3nM) cells showed a statistically significant increase in spike protein synthesis (Fig. 2S, U). Influenza, like RSV, contains a negative sense RNA genome, and like RSV is not known to encode a helicase. Influenza protein synthesis was also sensitive to episilvestrol treatment (Fig. 2AA–CC). Like RSV, episilvestrol treatment does not negatively impact the expression of influenza viral mRNAs (Fig. 2DD–JJ). Taken together, the targeting of host helicase may be an effective means of inhibiting viral protein synthesis and replication for negative strand RNA viruses that do not encode a helicase activity.

Viral RNA synthesis occurs in the presence of an RSV polymerase inhibitor

The RSV RNA-dependent, RNA polymerase, encoded by the L gene, is essential for RSV viral replication³¹. However, the mRNA expression of L polymerase is negligibly low during infection (Fig. 2Q); yet viral transcription of other genes is sufficiently robust (Fig. 2K–P). This raised a question as to whether there are alternative mechanism to enhance or amplify viral RNA transcription and replication. To test this concept, A549 cells were pre-treated at varying doses with small molecule ALS-8112, a nucleoside analog inhibitor of RSV L polymerase³², for 20 h. Pre-treated A549 cells were then infected with RSV, and assays were performed at 24 and 48 hpi with ALS-8112, known to have 29 h half-life in culture medium³³, kept in the media throughout the processes. At 1 μM, ALS-8112 nearly abolished viral proteins expression at 24 h (Fig. 3A–C); however, the level of viral proteins was elevated at 48 h (Fig. 3D–F) relatively to vehicle. At a higher dose of 10 μM of ALS-8112, viral proteins were completely abolished at both 24 and 48 h; however, viral mRNAs expression levels rose significantly at 48 h as compared to 24 h (Fig. 3G–M, ANOVA; Supplementary Fig. 1A–G, t-test), suggesting a novel cellular factor plays a role in viral RNA synthesis complementing RSV L polymerase.

Human telomerase is involved in RSV replication

Our data suggest that a novel cellular RNA-dependent, RNA polymerase activity plays a role in the transcription of RSV genes (Fig. 3), therefore, we hypothesized that eliminating or inhibiting this novel factor, which functions as a RNA-dependent RNA polymerase (RdRp), would negatively impact viral RNA transcription, protein synthesis and replication. Human telomerase reverse transcriptase (hTERT) is the only known mammalian protein that has RdRp activity^34,35. To test its role in RSV viral replication, hTERT was genetically knocked down in A549 cells by stable lenti-viral short-hairpin RNA (shRNA) (Fig. 4A, B). Knockdown of hTERT drastically reduced RSV viral fusion protein F1 expression (Fig. 4C, D). To confirm this observation, A549 cells were infected with RSV for 2 hours and subsequently infected cells were treated with varying doses with BIBR-1532, a selective hTERT inhibitor³⁶. At 10 μM, BIBR-1532 exerted significant inhibition of viral proteins expression at 24 h (Fig. 4E–G, Supplementary Fig. 2A–C), and further decreased at 48 h (Fig. 4H–J, Supplementary Fig. 2D–F) relatively to vehicle. At 30 μM of BIBR-1532, viral proteins were not detectable; similar to ALS-8112 however, viral mRNA expression levels were significantly elevated at 48 h compared to 24 h (Fig. 4K–Q, ANOVA; Supplementary Fig. 3A–G, t-test). In contrast, BIBR-1532 treatment did not exerted negative impact on influenza viral RNA expression (Supplementary Fig. 4A–I) suggesting that the viral RNA-dependent, RNA polymerase, not known to be inhibited by BIBR-1532, was responsible for the uptick in the levels of RSV viral gene transcripts. In all, these data suggest that hTERT could function as RdRp, possibly involved in viral genome transcription and replication, while maintaining its presumed nuclear localization.

**Fig. 4: Human telomerase functions as viral RNA-dependent RNA polymerase.**

Therapeutic inhibition of RSV L polymerase and hTERT blocks RSV replication

Given the apparent redundancy of both RSV L polymerase and hTERT functioning as RdRp for viral RNA synthesis, we then predicted that therapeutic inhibition of both enzymatic activities at once would block RSV viral replication. To evaluate this therapeutic potential, A549 cells were infected with RSV for 2 h and then treated with either ALS-8112, BIBR-1532, or a combination of both inhibitors. At 24 h, 1 μM ALS-8112 alone blocked the synthesis of viral proteins relative to vehicle while 10 μM BIBR-1532 had a less robust negative impact on viral protein levels (Fig. 5A–C). A combination of ALS-8112 and BIBR-1532 treatment pheno-copied ALS-8112 alone. At 48 hpi, however, ALS-8112 monotherapy resulted in elevated protein levels as compared to BIBR-1532 monotherapy (Fig. 5D–F). A combination of ALS-8112 and BIBR-1532 abrogated viral proteins synthesis, indicating an additive effect, if not synergistic, of the inhibition of RSV L polymerase and hTERT (Fig. 5A–F). Treatment with either ALS-8112 or BIBR-1532 alone resulted in an increase in viral mRNA levels at 48 h as compared to 24 hpi (Fig. 5G–M, ANOVA; Supplementary Fig. 5A–G, t-test; Supplementary Fig. 6A, B). However, viral RNA transcription at 48 hpi was dramatically reduced in dual treated cells as compared to cells treated with either inhibitor, indicating the additive effect of L polymerase and hTERT activity on viral RNA transcription. To test whether high dose of ALS-8112 and BIBR-1532 would block or abolish viral mRNA synthesis, 10 μM ALS-8112 or 30 μM BIBR-1532 were used. Treatment with either ALS-8112 or BIBR-1532 alone moderately elevated viral mRNA levels at 48 h compared to 24 hpi (Fig. 5N–T, ANOVA; Supplementary Fig. 6C, D; Supplementary Fig. 7A–G, t-test). However, a combination of ALS-8112 and BIBR-1532 blocked viral mRNA synthesis. Functionally, reduction of viral protein synthesis and mRNAs also correlated with decrease in production of virus progeny (Fig. 5U, V). One potential issue in this series of experiments is the use of A549 cells, which are derived from a lung cancer cell line. Reactivation of telomerase activity is a known hallmark in many cancers³⁷ and our findings may simply reflect a cell-specific phenomenon. To address the concern of cell-line specific hTERT-related effects, primary human small airway epithelial cells (HSAEC) were obtained to validate the role of hTERT on RSV replication using low dose (Fig. 6A–M) and high dose (Fig. 6AA–MM) of inhibitors. Our data show that treatment of primary respiratory epithelial cells with either an hTERT inhibitor or RSV polymerase inhibitor, or both, dramatically reduce viral RNA production. Taken together, these data confirm the redundancy of RSV L polymerase and hTERT functions as RdRp for viral RNA synthesis and replication.

**Fig. 6: Therapeutic effects of telomerase inhibitor for RSV RNA replication in primary human small airway epithelial cells.**

RSV viral proteins interact with eIF2α kinases, and localize to the nucleus

Under stress conditions, including viral infection, the cellular response typically demands translational repression of protein biosynthesis through regulation of eIF2α by eIF2α kinases³⁸. Consistent with this notion, reduction of eIF2α protein is correlated with diminishing viral protein synthesis (Fig. 1). To test the possibility of whether RSV proteins could interfere with the activity of eIF2α during viral infections, non-infected and RSV infected cell lysates were subjected to immunoprecipitation targeting eIF2α protein kinases, including double-stranded RNA-dependent (PKR) and PKR-like ER kinase (PERK). Immunoprecipitation of PKR resulted in the detection of eIF2α at both 24 and 48 hpi, representing the positive control (Fig. 7A). As anticipated, RSV viral protein F1 was not detected. However, we could detected both RSV N and P proteins at 48 hpi. Similarly, immunoprecipitation of PERK could also detect both viral proteins N and P (Fig. 7B). To confirm the co-binding of viral proteins N and P with eIF2α kinases, RSV-infected cell lysates were immunoprecipitated with either anti-IgG control, anti-PKR (Fig. 7C), or anti-PERK (Fig. 7D) antibodies and evaluated for the present of viral proteins. Both RSV N and P were detected in immunoprecipitants using anti-PKR or anti-PERK antibodies. These results suggested that viral proteins N and P could redundantly interfere the regulations of eIF2α by eIF2α kinases during RSV infections through physical interactions, in conjunction with phosphatase PP2A³⁹. Given PKR is resident in cytoplasm and nucleus⁴⁰, we hypothesized that viral N and P proteins may localize to the nucleus. To test this, 48 hpi cells were fractionated with different concentration of non-ionic detergent NP-40 (Fig. 7E). As expected, most of the cytoplasmic protein eIF2α⁴¹ was extracted with 0.075% NP-40, and further extracted with 0.5% detergent (Fig. 7F). Similarly, viral surface glycoprotein F was not detected in the nuclei fractions. On the other hand, both viral proteins N and P were detected in the cytoplasmic and nuclei fractions. The cellular fractionation techniques were verified using lamin B1⁴², a scaffolding component of the nuclear envelope, and cytoplasmic and nuclear β-actin⁴³. To confirm whether both viral proteins N and P could localize to the nucleus, immunofluorescence was performed 24 hpi using confocal microscopy. Both N and P proteins were visualized in the cytoplasm and nucleus of infected cells (Fig. 7G), although their nuclear localization was observed in only approximately 5% of infected cells. This suggests these viral proteins could redundantly function as moderators of innate antiviral cellular responses, in addition to being structural and regulatory component of ribonucleocapsids, respectively.

**Fig. 7: RSV viral proteins interact with eIF2α kinases, and nuclear localizations.**

Pharmacologic inhibition of nuclear Importins reduce RSV RNAs and proteins synthesis

It has been known RSV replicates in the cytoplasm⁴⁴ and can replicate in cells devoid of nuclei⁴⁵. Our data indicate telomerase, a resident protein of the nucleus⁴⁶, plays a role in RSV replication (Figs. 4–6). This, and the localization of RSV N and P proteins to the nucleus (Fig. 7), suggest a possibility that viral RNA could localize to the nucleus given RSV N and P proteins are components of the viral ribonucleocapsid. To test this, 24 hpi cells were fractionated into cytosolic (soluble) and nuclei (insoluble) fractions. Whole genome of viral mRNAs expression was evaluated. Given RSV replicates in the cytoplasm, we expected to detect the majority if not all of viral mRNAs in the cytosolic fraction. In contrary to our hypothesis, the viral mRNAs expression were significantly higher in the nuclei compared to cytosolic fraction at 24 hpi (Fig. 8A). Further, viral mRNAs were enriched in the nuclei compared to cytosolic fraction at 4 hpi (Fig. 8B). One potential explanation of this observation was that the nuclear fraction also included inclusion bodies (IB), cytoplasmic “factories” of viral replication and assembly. To address this issue, we employed the use of nuclear transport inhibitors as an alternative approach. Specifically, cells were pre-treated with nuclear transport receptor importin-β inhibitor, Importazole⁴⁷, or nuclear exportin 1 (XPO1/CRM1) inhibitor, KPT-330 (clinically approved Selinexor)⁴⁸ for 20 h before RSV infection. Infected cells were fractionated into cytosolic and nuclei fractions at 24 hpi. Total (Fig. 8C), cytosolic (Fig. 8D), and nuclei (Fig. 8E) mRNAs expression were evaluated. As expected, ALS-8112 or BIBR-1532 treated cells significantly reduced viral mRNAs in the total, cytosolic and nuclei fractions compared to vehicle as controls. Unexpectedly, Importazole treatment also significantly reduced viral mRNAs expression in all fractions. KPT-330 treatment modestly reduced viral mRNAs level in the cytosol, while slightly enriching the levels in the nuclei fraction. Consistent with mRNA expression (Supplementary Fig. 8A–F), Importazole treated cells also reduced viral proteins expression at 24 (Fig. 8F–I), and 48 hpi (Fig. 8J–M). The reduction of viral proteins is recapitulated with Ivermectin (Fig. 8N–Q; Supplementary Fig. 9A–H), FDA approved for importin αβ1 inhibitor^49,50. Treatment with SUMO-Activating Enzyme (SAE) inhibitor, ML-792⁵¹ does not have a negative impact on viral proteins expression, indicating Importazole and Ivermectin were specific (Supplementary Fig. 10A–D). Together, these data suggesting host nucleus is important for RSV replication, a novel finding which further confirmed the role of hTERT in RSV replication. Additionally, these data also suggest viral RNA could localize to the nucleus, and one mechanism is through nuclear transport receptors.

**Fig. 8: Inhibition of nuclear importins reduce RSV RNAs and proteins synthesis.**

Discussion

Our studies uncovered the novel biological importance of host/pathogen interactions during RSV infection. Specifically, RSV utilizes specific host intracellular pathways for viral replication and viral protein synthesis, and these pathways are potential targets for antiviral therapy. Therefore, interfering with specific host cellular function(s) required for RSV protein synthesis would mitigate or eliminate viral growth, regardless of viral mutations. Consistent with this notion, we have shown the mechanistic target of rapamycin (mTOR) signaling plays an important role in RSV replication and growth²¹. mTOR is a serine/threonine protein kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family²². mTOR kinase interacts with several proteins to form two distinct complexes, known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 phosphorylates two immediate downstream effectors p70S6 kinase 1 (S6K1) and eIF4E Binding Protein (4E-BP) to promote protein synthesis. Phosphorylation of 4E-BP induced the release of eIF4E, the only known eukaryotic initiation factor to bind to 5’ cap mRNA and this interaction subsequently allows protein translation to occur^27,52,53.

The non-segmented, negative-strand RNA viruses are classified into three families: Rhabdoviridae, Pneumoviridae and Filoviridae. Most research on NNS RNA virus 5’ mRNA capping has been based on the characterization of vesicular stomatitis virus (VSV), a member of the family Rhabdoviridae, from in vitro reticulocyte and wheat germ translation systems⁵⁴. RSV is a member of the family Pneumoviridae (previously a subfamily within the Paramyxoviridae⁵⁵) and RSV mRNA capping by viral polymerase is proposed with its caps at the 5’ ends biochemically indistinguishable from cellular caps. This is based on the assumptions that the mechanisms of synthesis of the cap for RSV and other NNS RNA viruses are conserved³¹. If this were the case, eliminating the cellular host 5’ mRNA cap binding protein eIF4E would inhibit viral protein synthesis. Reduction of eIF4E protein by CRISPR does not exert significant negative impact on RSV viral protein translation (Fig. 1A). However, reduction of cellular eIF4E almost abolished spike S protein synthesis of HCoV OC43, non-segmented, positive-strand enveloped RNA virus (Fig. 1B). This observation is in line with the finding that disruption of the binding of pathogenic coronavirus SARS-CoV-2 mRNA and eIF4E suppressed viral replication⁵⁶, suggesting that HCoV viral 5’ mRNA cap closely resembles the human form while RSV viral cap is distinct. Alternatively, eIF4E-independent protein synthesis also suggests RSV possibly utilizes an alternative mechanism for viral replication, given RSV mRNA caps indistinguishable from cellular caps.

On the other hand, activated S6K1 kinase (immediately downstream of mTORC1) phosphorylates and promotes the degradation of Programmed Cell Death 4 (PDCD4), inhibitor of eIF4B⁵⁷. Activated eIF4B, a positive regulator of the 5’ cap binding eIF4F complex⁵⁸, promotes and enhances 5’ cap-dependent translation initiation. One would predict that eliminating eIF4B would exhibit a similar phenotype as eIF4E-negative environment for RSV and HCoV viral protein translation. Unexpectedly, reduction of eIF4B protein significantly reduced RSV viral proteins; whereas reduction of eIF4B does not have negative impact on HCoV S protein level, suggesting eIF4B functions independent of eIF4E in term of viral protein synthesis, and RSV and HCoV utilize different mechanisms of protein translation. eIF2, comprises of α, β, and γ subunits, is known to bind to Met-tRNA_i, and is considered as rate-limiting or first step in cellular translation initiation⁵⁹. As expected, CRISPR of eIF2α inhibited both RSV and HCoV viral proteins, suggesting eIF2 can be considered as a generalizable translational therapeutic target.

eIF4F complex is comprised of eIF4E, eIF4G, and eIF4A²⁸. eIF4G, a scaffolding protein, interacts with Polyadenylate-Binding Protein (PABP) to mediate the circularization of cellular mRNA and protein translation stability⁶⁰. Reduction of eIF4G protein by CRISPR significantly reduced RSV viral protein synthesis (Supplementary Fig. 11A–E); whereas no significant impact is observed for HCoV protein level (Supplementary Fig. 11F, G). On the other hand, the reduction of PABP protein exerted significant negative impact on both RSV (Supplementary Fig. 12A–D) and HCoV (Supplementary Fig. 12E, F) viral protein translation, suggesting 3’ mRNA tail is important for viral replication, and PABP is a potential novel therapeutic target for viral infection. Currently, however, there is no specific small molecule inhibitor for eIF4G or PABP available to evaluate the potential value of therapeutics that target these molecules.

Although the editing efficiency by CRISPR is not achieved to desired level, 55–62% reduction of eIF4A1 protein almost abolished RSV viral protein synthesis (Fig. 2A–E); whereas reduction of eIF4A1 protein enhanced HCoV S protein (Fig. 2R, S). The off-target effects of CRISPR/Cas9 system⁶¹ maybe a possible confounding factor; the incomplete reduction of eIF4A1 protein exerted a drastic impact on viral protein levels and this is likely due to the effect of overlapping sequence of guide RNA for all 3 isoforms versus specific recognition of anti-eIF4A1 mAb. Pharmacologic inhibition of eIF4A with episilvestrol, a derivative of silvestrol⁶² inhibited RSV viral replication (Fig. 2F–J); whereas, HCoV-infected cells treated with episilvestrol showed enhanced viral protein (Fig. 2T, U), indicating off-target effects by CRISPR are minimal. On the other hand, the inhibition of eIF4A with episilvestrol does not exerted negative impact on RSV viral mRNA expression (Fig. 2K–Q). In hindsight, this could be explained by RSV viral negative-sense genome. Once entered into the cytoplasm of the host, the viral polymerase transcribes positive-sense viral mRNAs from negative-sense genome as template^63,64, thus generating double-stranded RNA (dsRNA) as intermediates by negative sense RNA viruses⁶⁵. Consequently, the presence of RSV dsRNA is recognized by melanoma differentiation-associated gene 5 (MDA5)^66,67,68.

eIF4A family is comprised of 3 isoforms. eIF4A3 is known to localize to the nucleus, whereas eIF4A1 and eIF4A2 are cytoplasmic proteins with eIF4A1 most abundantly expressed⁶⁹. eIF4A is one of the best-characterized DEAD-box, RNA-dependent ATPase and RNA helicase^70,71. In the presence of eIF4B, eIF4A functions as a processive helicase for unwinding mRNA secondary structure and duplex of RNA^72,73,74. Further, there is no evidence indicates RSV encodes RNA helicase in its genome, suggesting eIF4A mediates subsequent viral protein translation through the unwinding of dsRNA intermediates. Thus, these observations also confirm the role of eIF4B in RSV viral replication. To our knowledge, this is the first study to report RSV required host eIF4A RNA helicase for viral protein synthesis and replication. Our data also explains why interfering of host eIF4A or eIF4B does not exhibited negative impact on HCoV viral protein synthesis due to its single-stranded, positive sense genome, which is readily gained access by ribosomes and the fact that coronaviruses encode a helicase³⁰. Given the generation of viral dsRNA intermediates that result from negative-sense templates, we hypothesized that other negative, single-stranded RNA viruses would also utilize eIF4A RNA helicase as a platform for replication. Indeed, segmented, single-stranded, negative sense, enveloped RNA virus influenza H1N1/A/NWS/33-infected A549 cells treated with episilvestrol displayed diminishing influenza viral nucleoprotein (NP) and neuraminidase (NA) protein levels (Fig. 2AA–CC) without reduction of viral mRNAs expression (Fig. 2DD–JJ), a similar characteristic to RSV. We hypothesize that the inhibition of RNA helicase activity would not impact viral RNA synthesis early during infection, but rather, pharmacological inhibition of helicase activity would impact ribosome access resulting in reduced protein expression (see Fig. 2). In agreement with our data, inhibition of eIF4A with hippuristanol, a polyhydroxysteroid, reduced influenza viral mRNAs translation⁷⁵. Cumulatively, these observations suggest eIF4A can be considered as a generalizable translational therapeutic target for negative-strand RNA, and possibly dsRNA viruses.

We observed that the mRNA expression of RSV L polymerase was negligibly low (Fig. 2Q); yet, the transcription of other viral genes was sufficiently robust (Fig. 2K–P), which prompted an re-evaluation of ALS-8112, a known RSV antiviral³² on viral RNA synthesis and replication. In agreement with previous reports^32,76, 10 μM, ALS-8112 given prophylactically abolished RSV viral protein expression levels at both 24 and 48 hpi (Fig. 3A–F). The inhibition of viral proteins by ALS-8112 is correlated with the reduction of mRNA expression both prophylactically (Fig. 3G–M), and therapeutically (Supplementary Fig. 13A–M) in a dose-dependent manner. Given that RSV L polymerase is thought to be solely responsible for viral RNA synthesis, complete inhibition of L polymerase activity or L mRNA expression should abolish viral mRNAs expression across the genome. At 10 μM of ALS-8112, the viral proteins expression are blocked, and no significant changes of the polymerase L mRNA expression is observed for 24 and 48 hpi (Fig. 3); however, all non-L viral mRNA levels were significant elevated at 48 hpi compared to 24 hpi (Supplementary Fig. 1, t-test), suggesting that a novel or host factor can synthesize viral RNAs in addition to RSV L polymerase. Human TERT, is the only known mammalian protein that can function as a cellular RdRp³⁴. If hTERT plays a substantial role in generation of viral RNA or dsRNA, interfering hTERT function would have a negative impact on viral replication. Genetic knockdown and pharmacologic inhibition of hTERT resulted in diminishing levels of viral proteins and correlated with reduction of viral mRNAs expression (Fig. 4). Given hTERT appears to play a role in the synthesis of viral RNAs, the simultaneous impairment of both hTERT and viral L polymerase would completely block viral RNA synthesis. As expected, therapeutic inhibition of viral L polymerase and hTERT abolished viral RNA and protein synthesis (Figs. 4–6). It has been reported that hTERT inhibitor BIBR-1532 at 40 μM for 48 h did not have significant toxicity on A549 cells, and no toxicity at 20 μM for 6 days⁷⁷. This is consistent with our observations for 10 and 30 μM treatment. The selectivity of BIBR-1532 was also assessed with various DNA and RNA polymerases, including HIV reverse transcriptase⁷⁸. Consistent with these observations, BIBR-1532 did not exerted negative effects on influenza RNA expression (Supplementary Fig. 4). It is highly unlikely that a viral genomic mutation was present in the clinical strain used in these experiments (4th passage directly from clinical samples) that resulted in partial resistance to ALS-8112 as it took at least 32 in vitro passages of RSV in presence of ALS-8112 to generate noticeable mutations of L gene³².

Under cellular stress condition, the phosphorylation of serine 51 on eIF2α by a family of four well-characterized serine-threonine kinases, PERK, PKR, general control non-derepressible-2 (GCN2), and heme-regulated inhibitor (HRI) occurs to inhibit the translation of mRNAs⁴⁰. Some viruses, such as dsRNA viruses, induce cellular and ER stress, thereby triggering the activations of PKR and PERK, respectively, which in turn limits viral mRNAs translation. Indeed, RSV has been shown to sequester PKR away from eIF2α by binding of the kinase to the RSV N protein³⁹, which is consistent with our data (Fig. 7A, C). In addition to these observations, we showed not only RSV N but P protein could also bind to PKR. Moreover, we showed both RSV N and P proteins could also bind to PERK, a new finding (Fig. 7C, D), supporting the multifaceted interfering of eIF2α activity, given eIF2 an important first step in cellular translation initiation and innate immune response. RSV N protein is known as nucleocapsid structural component while P is regulatory protein of ribonucleocapsids⁶⁸. It has been shown RSV ribonucleocapsids concentrated in the cytoplasmic inclusion bodies (IB)^{79,80,81,82,83}. We have noticed both N and P proteins presented in the nuclei fraction (Fig. 7F). It is possible that N protein could co-sediment with nuclei due to the high molecular weight of nucleocapsid. On the other hand, RSV F protein is not presented in the nuclei fraction due to its high molecular weight trimeric form⁸⁴, indicating it is unlikely for P protein (~35 kDa) to co-sediment with nuclei. To confirm these observations, N and P proteins were visualized by confocal microscopy⁸⁵. As expected, majority of RSV N and P proteins are detected in the cytoplasm (Fig. 7G). However, we noted approximately 5% of RSV N and P proteins could also be detected in the nucleus of infected cells. Low level of nuclear localization was observed possibly due to various factors, including sensitivity of assay, m.o.i, and time. As we have shown in Fig. 7A, we could detect the interaction of viral proteins with host kinase PKR at 48, but not at 24 hpi (m.o.i = 0.2) for Western Blot. . On the other hand, high expression of viral proteins also increased background for immunofluorescence; thus, 24 hpi of m.o.i = 0.1 was performed on Fig. 7Gfor better visualization. These observations support the notion that these viral proteins could redundantly interfere the eIF2α kinases in the cytoplasm and nucleus. The localization of the viral N and P proteins to the nucleus suggests that these proteins may play a role in anti-innate immune responses due to the generation of dsRNA during viral replication, which may complement the activities of the non-structural NS1 and NS2 proteins^68,86. Being structural components, as is the case of the N protein in the viral particle, the RSV M protein has also been identified in the nucleus^87,88,89 further suggesting dual roles of RSV structural proteins.

Our findings suggest that viral RNA imported into the nucleus may play an important role in RSV genome replication or transcription, perhaps complementing the functions of the cytoplasmic IBs. Once inside the cytoplasm of the host, the presence of viral RNAs is recognized by host pattern recognition receptors (PPRs), such as RIG-1 and MDA-5, leads to the activation of interferon and immune responses⁹⁰. Viral IBs are known as aggregates of viral components and various of host cellular factors that form in the cytoplasm and can serve as sites for viral replication⁹¹; while viral nuclear import is known as the shuttling of viral components into the nucleus from cytoplasm through nuclear pore complexes (NPC) via host nuclear transport receptors, such as importins⁹². Although viral IBs has been observed in many negative-strand RNA virus (NSV) infections, including rabies, vesicular stomatitis, respiratory syncytial virus, measles, Nipah, Ebola, Marburg, and influenza A viruses⁹¹, viral components (proteins or RNAs) of those viruses also have been detected in the nuclei of infected cells^92,93,94,95. It is conceivable that the sequestration of the host PPRs to viral IBs may help the virus evade the host immune responses while the localization of the viral components, especially viral RNAs, to the nucleus may help viral replication and the avoidance of the recognition of the cytosolic sensors. Hence, multifaceted strategies could be applied as novel therapeutic methods for infections.

It has been shown IBs are a major site for RSV viral RNA synthesis⁸³. We noticed viral mRNAs are enriched in the nuclei compared to cytosolic fractions (Fig. 8A, B). Given cytoplasmic IBs are the major site for viral RNA synthesis and could possibly co-sediment with nuclei during fractionation. We consider this as a possible explanation for our findings though the absence of RSV F in the nuclear fraction does not support this hypothesis. If it were true that IBs were part of the nuclear fraction and RSV replication did not involve the nucleus, one would expect inhibiting nuclear transport activities should have no impact on viral mRNA or protein synthesis. Our data showed inhibiting nuclear transport receptor with Importazole not only exerted significant negative impact on viral mRNAs expression but also viral proteins synthesis, a novel finding (Fig. 8). The inhibition of RSV viral protein synthesis by Importazole was recapitulated with Ivermectin, similar to the reduction of viral RNAs by hTERT inhibitor BIBR-1532, supporting an important role of host nucleus for RSV replication that is under-appreciated.

Effective approaches to RSV require multifaceted efforts, including vaccinations and antiviral drugs. Despite the advancements of the understanding of the molecular biology of RSV, suitable, authentic and pliable animal models for human RSV are still lacking. The common animals used for RSV infection are cotton rats, mice, ferrets, guinea pigs, hamsters, chinchillas, neonatal lambs, and non-human primates, including African green monkey and macaques⁹⁶. However, these animals are semi-permissive for human RSV replication in part due to relatively large doses of experimental infection and/or lack of clinical signs of disease. The chimpanzee, on the other hand, is the only animal that is known fully permissive to human RSV replication⁹⁷. Due to the high cost and rising ethical concerns, the use of chimpanzees in preclinical trials still remains a challenging aspect. One potential approach would be to observe the extent of clinical RSV infection in cancer patients who are receiving telomerase inhibitor treatment. BIBR-1532, a small molecule, is a direct selective noncompetitive inhibitor of hTERT³⁶. However, BIBR-1532 suffers poor pharmacokinetics properties, thus hindering it from proceeding to clinical trials⁹⁸. On the other hand, Imetelstat (Rytelo^®, Geron), a complementary oligonucleotide inhibitor that act on human telomerase RNA component hTERC, is the only inhibitor FDA-approved (in June 2024) to treat Myelodysplastic syndromes (MDS)⁹⁹. Thus, Imetelstat may provide some insights into an association between hTERT inhibition and RSV disease.

It has been reported that hTERT could interact with RNA component of the mitochondrial RNA-processing endoribonuclease (RMRP), a complex that could function as RdRp³⁴. Cellular RdRps are known play important role in gene silencing through siRNAs and Dicer-dependent manner in various organisms, including mammals, plants, nematodes, fruit flies, and fungi³⁵. In fission yeast, Rdp1 is a known RdRp homolog to hTERT. It is well known NSV, including RSV, viral mRNAs transcription starts at the 3’ end of the genome³¹. RNA viruses initiate minus-strand RNA synthesis by two different mechanisms: de novo and primer-dependent initiation¹⁰⁰. Although de novo initiation, known as primer-independent, has been suggested for RSV¹⁰¹, the primer-dependent initiation can be divided into two groups. One type is the binding of primers to complementary template RNA, including the oligonucleotide primers and the 5’ end cap-snatch of cellular mRNA primers¹⁰⁰. The other type is the 3’ terminus of the template RNA that folds back as a hairpin loop, referred as back-priming^34,35. If RSV utilizes back-priming initiation for viral RNA synthesis by L polymerase or hTERT, then the RNA sequences at the 3’ end genome or 5’ end antigenome would be able to form hairpin loop. Consensus viral RNA sequences³¹ were predicted for hairpin loop or secondary fold by RNAstructure software¹⁰². Consistent with the hypothesis, back-priming structures is found at the 3’ end genome and 5’ end antigenome (Supplementary Fig. 14), in line with the notion that RSV L polymerase could utilizes back-priming mechanism as an alternative for viral RNA initiation and synthesis¹⁰¹. Given the initiation of viral RNA synthesis start from the 3’ end of the genome, we speculate back-priming is a possible alternative pathway utilize by RSV in vivo, and viral replication could occur in the nucleus due to known localization of hTERT⁴⁶.

Understanding the interactions of RSV and host cellular machinery would help devise novel antiviral strategies. In summary (Fig. 9), the significance and novelty of our current study include the following: (1) RSV viral protein synthesis is independent of cellular 5’ mRNA cap protein eIF4E, an implication of novel viral protein synthesis; (2) RNA helicase eIF4A is required for RSV and influenza viral protein synthesis, a novel translational therapeutic target for negative sense and dsRNA viruses; (3) hTERT could functions as viral RNA-dependent RNA polymerase, providing novel mechanism of RSV viral RNA synthesis and replication and; (4) a role of nucleus for RSV RNAs and proteins synthesis, a novel therapeutic implication of nuclear transport receptors. In all, our current study show targeting of host factors may be a useful antiviral therapeutic strategy regardless of viral genomic mutation, and a proof-of-concept for potential therapeutic treatment for other and emerging RNA viruses.

**Fig. 9: Schematic of RSV replication.**

Methods

Viruses and cells

RSV clinical isolates were obtained from RSV-infected individuals, as described previously, in New Haven, CT¹⁰³ and Dallas, TX¹⁰⁴, (IRB reference #10517 and #STU 012014-052, respectively). Collection and use of clinical isolates followed all institutional requirements and guidelines, were consistent with policies and regulations for the use of patient derived materials and approved by the respective Institutional Review Boards. The need to obtain informed consent was waived by the Institutional Review Board (IRB) of Yale University and the University of Texas Southwestern Medical Center as we were using left over materials that were de-identified. Isolates were plaque-purified and concentrated by HEp-2 cells. Plaque titration and working stocks prepared by 0.5% methylcellulose overlays of infected A549 cells for 3 days before being stained as described previously^21,104. Viral stocks were prepared with a low inoculum with multiplicity of infection (m.o.i) of 0.01 to minimize the production of defective interfering (DI) particles. Further, the number of passages of virus in cell culture was limited to prevent the potential viral adaption to cell culture. All specimens from which virus were obtained were submitted as part of routine care. Only left-over material was used for viral propagation. Collection of specimens from the Clinical Virology Laboratory at Yale-New Haven Hospital was approved by the Yale University Human Investigations Committee. The RSV isolates from Dallas, Texas were propagated from a de-identified clinical specimen obtained from the Clinical Microbiology Laboratory at Children’s Medical Center, Dallas. Collection and use of clinical isolates followed all institutional requirements and guidelines and was consistent with policies and regulations for the use of patient derived materials. Human coronavirus (HCoV) OC43 (ATCC VR-1558), and human H1N1 influenza virus A/NWS/33 (ATCC VR-219) were obtained from American Type Culture Collection (Manassas, VA).

A549 (CCL-185) cells were obtained from ATCC and cultivated in F-12 Kaighn’s modification media with 10% fetal bovine serum. Primary human small airway epithelial cells (PCS-301-010) was cultured in airway epithelial cell basal medium (PCS-300-030) that supplemented with bronchial epithelial cell growth kit (PCS-300-040) were all obtained from ATCC. RSV viral titers were determined using a plaque assay^21,104. Plaques were detected with an immunohistochemical staining technique with a primary anti-RSV Fusion protein antibody (palivizumab, MedImmune, Gaithersberg, MD) and a secondary HRP conjugated anti-human antibody (Jackson ImmunoResearch Laboratories, Inc., West grove, PA).

Chemicals, CRISPR, shRNA plasmids, and antibodies

Episilvestrol (HY15359), ALS-8112 (HY12983), BIBR-1532 (HY17353), Importazole (HY101091), and ML-792 (HY108702) were obtained from Medchemexpress, KPT-330 (B14645) from Apexbio, and Ivermectin (J62777M) from Thermo Scientific Chemicals were dissolved in DMSO as manufacturer instructions. Equal volume of DMSO served as vehicle (veh).

pLentiCRISPR v2 eIF4E, eIF4B, eIF2α, eIF4A, eIF4G, PABP, and empty vector (GenScript); Scramble shRNA, and shTERT (Santa Cruz Biotechnology) plasmids were packaged by lentiviral plasmids pMD2.G (12259) and psPAX2 (12260) (Addgene). Briefly²¹, CRISPR or shRNA plasmid together with pMD2.G and psPAX2 were transfected into 293 T cells. Media supernatants which contained viruses were collected after 72 h of transfection, and used to infect A549 cells. Infected-A549 cells were screened with puromycin (1.5 µg/mL) after 2 days of infection. Western blots were performed to evaluate protein expression. All western blots/images are pre-cut. All uncropped or unedited gel images are provided in Supplementary Figs. 16–37.

The following antibodies were used: 1:1000 dilution of primary rabbit eIF4E, eIF4B, eIF2α, eIF4A1, eIF4G1, PABP1, PKR, PERK, β-actin antibodies (Cell Signaling Technology); mouse RSV N, P 3-5-18 antibodies (Novus Biologicals); human RSV F1 antibody (palivizumab) (MedImmune); rabbit H1N1 influenza NP, NA, coronavirus spike S antibodies (Invitrogen) were detected with 1:10,000 dilution of secondary goat anti-rabbit HRP antibody (Thermo Fisher Scientific), horse anti-mouse HRP antibody (Cell Signaling Technology), or rabbit anti-human HRP antibody (Jackson ImmunoResearch Laboratories).

RSV, HCoV OC43, influenza H1N1 infection and protein detection

Overall, 1.5 × 10⁵ cells per well of a 12-well plate of A549 cells were infected with RSV (m.o.i. of 0.2), HCoV OC43 (m.o.i of 0.5), or influenza H1N1/A/NWS/33 (m.o.i. of 0.3). After 120 min of infection, the inoculum was removed, the cells were washed with serum free media, and fresh F-12 Kaighn’s modification media containing 5% FBS with or without indicated drugs was added to the infected monolayers. The cells were directly lysed with laemmli sample buffer (Bio-rad Laboratories) 24 or 48 h post-infection. Western blotting was performed with SDS-PAGE gels. Chemiluminescence signals were captured by Cytiva Amersham ImageQuant 800 or Bio-rad Laboratories ChemiDoc MP Imaging System. Relative protein quantifications were analyzed by ImageJ¹⁰⁵. All western blots/images are pre-cut. All uncropped or unedited gel images are provided in Supplementary Fig. 16-37.

RNA extraction, reverse transcription and quantitative PCR

A549 cells were infected with RSV (m.o.i. of 0.2), or influenza H1N1/A/NWS/33 (m.o.i. of 0.3). Total, cytoplasmic, and nuclear RNAs were extracted at 24 or 48 h post-infection by TRIzol reagent (Invitrogen). First-strand of total cDNAs were synthesized with random primers by high capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative PCR (qRT-PCR) was performed with PowerUp SYBR green (Applied Biosystems) and CFX96 thermal cycler (Bio-rad). Quantitative PCR primer sequences are provided in Supplementary Fig. 15.

Cell lysis, immunoprecipitations, and fractionations

Immunoprecipitations were performed as previously described¹⁰⁶. Briefly, A549 cells were infected with RSV at m.o.i. of 0.2 for 24 or 48 h. Cells rinsed twice with cold PBS were lysed in cold lysis buffer (40 mM HEPES, [pH 7.4], 1 mM NaF, 1 mM pyrophosphate, 1 mM glycerophosphate, 0.1% NP-40, and one tablet of EDTA-free protease inhibitors [Pierce] per 10 mL). The soluble fractions of cell lysates were isolated by centrifugation at 12,000 x g for 10 min at 4 °C. For immunoprecipitation, rabbit anti-PKR, -PERK, or -IgG control (Cell Signaling Technology) antibodies were added to the lysates and incubated with rotation for 1.5 h at 4 °C. A volume of 20 µL of resuspended Protein A/G Plus-Agarose (Santa Cruz Biotechnology) was then added, and the incubation continued for additional 2 h. Immunoprecipitates were gently washed twice with low salt wash buffer (40 mM HEPES, [pH 7.4], 100 mM NaCl, 1 mM NaF, 1 mM pyrophosphate, 1 mM glycerophosphate, 0.1% NP-40, and one tablet of EDTA-free protease inhibitors per 10 mL), and twice with cold PBS. Immunoprecipitated proteins were denatured by the addition of 20 µL of Laemmli buffer (Bio-Rad), resolved by 8% SDS-PAGE, and analyzed by western blotting with secondary goat anti-rabbit HRP antibody (Thermo Fisher Scientific), horse anti-mouse HRP antibody (Cell Signaling Technology), or rabbit anti-human HRP antibody (Jackson ImmunoResearch Laboratories). Cytoplasmic and nuclear protein extraction was previously described¹⁰⁶. Briefly, A549 cells were infected with RSV at m.o.i. of 0.2 for 48 h. Cells were washed once with cold PBS, removed with enzyme-free dissociation buffer (Gibco), and pelleted by centrifugation. Cells were resuspended with Cytoplasmic Extract (CE) buffer (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 0.075% NP-40, 1 mM DTT, 1 mM PMSF, and one tablet of EDTA-free protease inhibitors per 10 mL, adjusted to [pH 7.6]), and incubated on ice for 4 min. The soluble fractions of cell lysates were collected by 4 °C centrifugation at 3000 rpm for 5 min. The insoluble fractions were then resuspended with CE + 0.5% NP-40, and incubated on ice for 10 min. The soluble fractions of lysates were isolated by centrifugation at 3000 rpm for 10 min. Nuclei were pelleted by centrifugation and washed twice with CE buffer without NP-40. Nuclei pellets were resuspended in Nuclear Extract (NE) buffer (20 mM Tris Cl, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM PMSF, 25% glycerol, and one tablet of EDTA-free protease inhibitors per 10 mL, adjusted to [pH 8]), pass through 30-gauge needle 10 times, and incubated on ice for 45 min with intermittent vortex. Supernatants were collected after centrifugation at 12,000 rpm for 5 min, and subjected to western blot analysis. For RNA fractionations, cells rinsed twice with cold PBS were lysed in cold lysis buffer (40 mM HEPES, 1 mM pyrophosphate, 0.8% NP-40). The soluble fractions of cell lysates were isolated by centrifugation at 3000 rpm for 10 min at 4 °C. Nuclei pellets were washed twice with cold lysis buffer (40 mM HEPES, 1 mM pyrophosphate, 0.8% NP-40). Total, cytoplasmic, and nuclear RNAs were extracted by TRIzol reagent (Invitrogen). First-strand of total cDNAs were synthesized with random primers by high capacity cDNA reverse transcription kit (Applied Biosystems).

Immunofluorescence confocal microscopy

Immunofluorescence confocal microscopy was carried out as previously described⁸⁵. Briefly, A549 cells cultured on coverslips were infected with RSV at m.o.i of 0.1. At 24 h post-infection, cells were fixed with 4% paraformaldehyde (PFA) in PBS at 4 °C for 20 min, followed by three washes with PBS. Samples were then blocked with 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) in immunofluorescence (IF) buffer (3% BSA and 0.4% saponin in PBS; for all other applications: 3% BSA and 0.1% NP-40 in PBS) at room temperature for 1 h. Cells were incubated with 1:600 dilution of primary antibodies in IF buffer overnight at 4 °C, followed by three washes with IF buffer. Fluorescently labeled 1:1000 dilution of secondary antibodies were applied at room temperature for 1.5 h, followed by nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 5 min at room temperature and three washes with IF buffer. Coverslips were mounted onto glass slides using Fluoromount-G (0100-01, SouthernBiotech), and images were acquired using an AX R Confocal system (Nikon).

Statistical analyses

The biological data was log-transformed to obtain normal distribution. Shapiro-Wilk test was used to test normality. Brown-Forsythe test was used to test equal variances among different groups. When the log-transformed data do not pass the normality test, (1) Wilcoxon Rank Sum test was used to test difference in median between two groups; (2) Kruskal-Wallis test was performed to test the difference in median among 3 or more groups. Dunn’s method was used to adjust p value for multiple comparisons between test groups and control group (Vehicle, Vector, or Scramble). When the log-transformed data pass the normality test, (1) two-sample t-test was used to test difference in mean between two groups; 2a) standard one-way ANOVA was performed to test difference in mean among 3 or more groups if data also pass equal variance test; 2b) Welch’s one-way ANOVA was used to test difference in mean among 3 or more groups if data do not pass equal variance test. Dunnett method was used to adjust p value for multiple comparisons between test groups and control group (Vehicle, Vector, or Scramble). The adjusted p values were reported for multiple comparisons. The data were displayed in original scale (without transformation) in figures. Biological data for “No infection” were presented in the figures, but they were not included for statistical tests. All statistical analyses were performed by Graphpad Prism 10.6.1 software. The significance levels were designated as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, **** for p < 0.0001 and n.s. for nonsignificant (p > 0.05).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The numerical source data for the graphs in this study are provided in the Supplementary Data. All other data are available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by the Department of Pediatrics and The Sarah M. & Charles E. Seay Chair in Pediatric Infectious Diseases, The University of Texas Southwestern Medical Center, Dallas, Texas. C.T.W supported by R01 grant (R01AI184984).

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Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA
HoangDinh Huynh & Jeffrey S. Kahn
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA
Chien-Ting Wu & Jeffrey S. Kahn

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HoangDinh Huynh
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Chien-Ting Wu
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Jeffrey S. Kahn
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H.D.H. and J.S.K. conceptualized the study, designed the experiments. H.D.H. and C.T.W. conducted experiments. H.D.H., C.T.W., and J.S.K. performed data curation and analysis; H.D.H. prepared all of the figures. H.D.H. and J.S.K. wrote the manuscript. All authors reviewed and approved the manuscript.

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Correspondence to Jeffrey S. Kahn.

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Communications Biology thanks Xiaoyong Bao, Yuqing Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Kaliya Georgieva. A peer review file is available

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Huynh, H., Wu, CT. & Kahn, J.S. Human telomerase reverse transcriptase supports respiratory syncytial virus replication. Commun Biol 9, 807 (2026). https://doi.org/10.1038/s42003-026-09958-4

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Received: 27 May 2025
Accepted: 19 March 2026
Published: 10 April 2026
Version of record: 15 June 2026
DOI: https://doi.org/10.1038/s42003-026-09958-4
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Human telomerase reverse transcriptase supports respiratory syncytial virus replication

Abstract

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Introduction

Results

RSV viral protein synthesis does not require host 5’ mRNA cap protein eIF4E function but requires eIF2α

Inhibition of host RNA helicase eIF4A abolishes RSV viral protein synthesis and virus progeny

Viral RNA synthesis occurs in the presence of an RSV polymerase inhibitor

Human telomerase is involved in RSV replication

Therapeutic inhibition of RSV L polymerase and hTERT blocks RSV replication

RSV viral proteins interact with eIF2α kinases, and localize to the nucleus

Pharmacologic inhibition of nuclear Importins reduce RSV RNAs and proteins synthesis

Discussion

Methods

Viruses and cells

Chemicals, CRISPR, shRNA plasmids, and antibodies

RSV, HCoV OC43, influenza H1N1 infection and protein detection

RNA extraction, reverse transcription and quantitative PCR

Cell lysis, immunoprecipitations, and fractionations

Immunofluorescence confocal microscopy

Statistical analyses

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

Supplementary information

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