Human rhinovirus promotes STING trafficking to replication organelles to promote viral replication

Triantafilou, Martha; Ramanjulu, Joshi; Booty, Lee M.; Jimenez-Duran, Gisela; Keles, Hakan; Saunders, Ken; Nevins, Neysa; Koppe, Emma; Modis, Louise K.; Pesiridis, G. Scott; Bertin, John; Triantafilou, Kathy

doi:10.1038/s41467-022-28745-3

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Published: 17 March 2022

Human rhinovirus promotes STING trafficking to replication organelles to promote viral replication

Nature Communications volume 13, Article number: 1406 (2022) Cite this article

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Abstract

Human rhinovirus (HRV), like coronavirus (HCoV), are positive-strand RNA viruses that cause both upper and lower respiratory tract illness, with their replication facilitated by concentrating RNA-synthesizing machinery in intracellular compartments made of modified host membranes, referred to as replication organelles (ROs). Here we report a non-canonical, essential function for stimulator of interferon genes (STING) during HRV infections. While the canonical function of STING is to detect cytosolic DNA and activate inflammatory responses, HRV infection triggers the release of STIM1-bound STING in the ER by lowering Ca²⁺, thereby allowing STING to interact with phosphatidylinositol 4-phosphate (PI4P) and traffic to ROs to facilitates viral replication and transmission via autophagy. Our results thus hint a critical function of STING in HRV viral replication and transmission, with possible implications for other RO-mediated RNA viruses.

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Introduction

Human rhinoviruses (RVs) are medically important single-stranded RNA viruses that belong to the family Picornaviridae. Originally neglected, as they were considered less virulent and capable only of causing mild common cold symptoms. It is only recently that they have been recognised to be important causative agents of acute upper and lower respiratory infections, wheezing, pneumonia, bronchiolitis, exacerbations of both asthma and COPD^1,2,3,4,5, as well as severe respiratory disease⁶. Together with coronaviruses, RVs are responsible for the majority of upper and lower respiratory tract infections in all age groups^7,8,9 as well as being a major cause of morbidity in infants, young children and the elderly^9,10. In the United States alone, HRV causes 500 million non-influenza viral respiratory infections, resulting in a huge socioeconomic burden with estimated direct costs of USD $17 billion and indirect costs of $22.5 billion annually¹¹.

To date, there are 171 rhinovirus (RV) genotypes recognized and classified into the three species as RV-A (83 types), RV-B (32 types), and RV-C (56 types)^12,13,14. The large number of RV serotypes and their considerable genetic diversity has represented a major obstacle to the development of antiviral agents¹⁵, as well as a successful vaccine. Strategies to combat HRV infections and associated pathology are limited to supportive therapy and novel avenues are much needed. To date, there is no approved antiviral treatment for RV infections.

For HRV viruses to replicate and amplify their genomes, they reorganise intracellular membranes into membranous structures forming phosphatidylinositol 4-phosphate (PI4P) lipid enriched organelles termed replication organelles (ROs)^16,17.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER)-associated membrane protein that has been shown to have many functions including sensing of intracellular DNA via the cGAS-STING pathway, resulting in triggering an interferon (IFN) and inflammatory response^18,19,20,21, as well as inducing autophagy for clearance of cytosolic DNA through a TANK binding kinase (TBK1) independent mechanism²². Although part of the DNA sensing machinery STING has been previously shown to be implicated in responses against RNA viruses²³. It has been shown that cells lacking STING are more sensitive to RNA virus infection and that during those infections, STING plays a role in inhibiting the translation machinery to limit the RNA viral synthesis²⁴. Crosstalk between RNA RLRs, such as RIG-I and MDA5, and STING has also been implied from studies with HIV-1^25,26, HTLV-1²⁷, and Dengue^28,29. To date, no direct interaction of STING with foreign RNA has been reported.

STING activation seems to rely on its location within the cell. When STING localizes to the ER and is bound by stromal interaction molecule 1 (STIM1) it is rendered inactive³⁰. Upon activation, STING traffics from the ER to the ER–Golgi intermediate compartment (ERGIC), where STING-mediated TBK1/Interferon Regulatory Factor 3 (IRF3) signalling occurs³¹. STING activation needs to be regulated and excessive STING signalling is implicated in severe sterile inflammatory conditions^31,32. Thus, STIM1 regulates STING trafficking and activation by directly interacting with STING to retain it in an inactive state on the ER membrane. STIM1 is an ER calcium sensor and when the ER Ca²⁺ stores are depleted, STIM1 moves to the ER–plasma membrane (PM) junction sites, where it binds to calcium release-activated calcium channel protein 1 (CRCM1 (Orai1)) and opens Ca²⁺ release-activated Ca²⁺ (CRAC) channel to allow Ca²⁺ influx³⁰. The small hydrophobic HRV 2B protein targets the ER and the Golgi complex to reduce Ca ²⁺ levels^33,34. Here, we have discovered that during HRV infection, HRV 2B reduces the ER and Golgi complex stored Ca²⁺ forcing STIM1 to leave the ER, thus releasing STING. Once released, STING can bind PI4P via a patch of positively charged residues and is recruited to the RO, inhibiting downstream immune responses. STING can bind to phosphatidylserine (PS) autophagosomal vesicles which provide a cellular exit point for the virus, elucidating a role for STING as a homeostatic regulator between PI4P/PS lipid exchange. This study highlights a critical role for STING in HRV viral replication and transmission, involved in lipid recognition and homeostasis, with possible implications for other RO-mediated viruses, such as coronavirus.

Results

STING is required for HRV replication

The identification of host proteins and pathways exploited by viruses to enhance their infectious cycle has historically provided insight into host–pathogen interactions and possible therapeutic avenues. Using an unbiased genome-wide small interference RNA (siRNA) screen of HRV infection in airway epithelial cells, Tmem173 (STING) was identified as a strong cellular target of HRV, the only gene to have all four siRNAs within the top 30 hits, with two in the top ten³⁵.

To validate the requirement of STING in HRV replication, Tmem173 (hereby referred to as STING) knockouts were generated by CRISPR-Cas9 editing in both BEAS-2B and air-liquid interface primary human airway epithelial (ALI) cells. STING-deficient cells were infected with A and B groups (including major and minor serotypes) such as HRV-A1B, HRV-A2, HRV-A16, HRV-B14, and HRV-B4 as well as other RNA respiratory viruses such as Respiratory syncytial virus (RSV) or Influenza A virus (IFV-A). This confirmed that STING downregulation abrogated HRV infection in all serotypes whereas RSV and IFV-A infection was not affected (Fig. 1a).

**Fig. 1: STING is required for HRV replication.**

HRV group C (HRV-C), a novel, more pathogenic rhinovirus group which has been implicated in childhood acute asthma and causes more severe attacks than other viruses or HRV groups³⁶, was also investigated. Using the same ALI cultures as above, we infected wildtype or STING-deficient cells with HRV-C15 and assessed viral replication by qPCR over time. Our results confirmed that rhinoviruses HRV-C15 replication was also STING dependent (Fig. 1b).

Since our data suggested that STING is crucial for HRV replication in lung-derived cells, we proceeded to investigate STING expression in lung tissue. Profiling of mRNA expression in the GTEx database demonstrated detectable levels of STING across multiple tissue types, with greatest levels observed in lung tissue and blood (Fig. 1c). Expression in lung tissue is consistent with GSK internal microarray data from multiple studies (unpublished), which demonstrate consistent expression in isolated air-liquid-interface (ALI) cultured bronchial epithelial cells. In addition, transcriptomics analysis of STING expression in ALI cultures, demonstrated that STING was significantly upregulated in response to HRV infection (Fig. 1d).

Canonical function of STING is compromised upon HRV infection

It is well documented that in the presence of ligands such as interferon stimulatory DNAs (ISDs) as well as cGAMP, STING becomes activated, followed by TANK-binding kinase 1 (TBK1) activation and IRF3 phosphorylation leading to IFN-β production. Although HRV is an RNA virus, we investigated whether there was interferon-β (IFN-β) production in response to HRV infection in ALI cells. It was shown that ALI cells produced IFN-β in response to ISDs and cGAMP, but also HRV infection (Fig. 2a). However, BX795, a TBK1 inhibitor, as well as RU.521, a cGAS inhibitor, did not inhibit the HRV-induced IFN-β production (Fig. 2a), demonstrating cGAS-STING-independent IFN-β production post HRV infection.

**Fig. 2: STING role in HRV infection is independent of cGAS/STING canonical pathway.**

As STING canonically cannot detect RNA, we assessed the possibility that HRV could be triggering cGAMP production, thus the level of cGAMP after infection with HRV, or with stimulation using ISDs was examined (Fig. 2b). Our data showed there was no production of cGAMP downstream of HRV infection, therefore aligning with inhibitor-obtained data above suggesting cGAS-STING independent IFN-β production.

Our group and others have shown IFN production upon HRV infection is RNA helicase dependent³⁷. To show this, as well as STING-independence, we knocked out MDA5 and RIG-I using CRISPR. Whilst STING-deficient and wildtype cells were able to generate an IFN-response, MDA5/RIG-I KO cells were not (Fig. 2c). Additionally, HRV infection did not induce TBK1 phosphorylation, highlighting the independence from the STING-TBK1 pathway (Fig. 2d). Additionally, ablation of TBK1 or IRF3 had no effect on HRV replication in bronchial epithelial cells as measured by qPCR (Fig. 2e). As expected, these results collectively indicate that RLRs, and not STING, drive the IFN-response to HRV infection. Interestingly, when ALI cells were challenged with HRV followed by cGAMP or ISDs, STING was unable to signal as expected with no TBK1 or IRF3 phosphorylation (Fig. 2f). These results collectively indicate that RLRs, and not STING, are responsible for downstream IFN production following HRV infection in respiratory cells, and more interestingly that STING cannot canonically function following HRV infection.

STING traffics to PI4P-rich viral replication organelles during early infection

One important feature of STING signalling is its trafficking. STING localizes to the ER in the resting state and upon activation, STING moves to the ER–Golgi intermediate compartment (ERGIC), and then to the Golgi where it triggers phosphorylation of TBK1 and IRF3 at the trans Golgi network (TGN)³⁸. STING is also post-translationally palmitoylated at the TGN, as is required for IFN response³⁹.

Studies have confirmed that blocking the ER-Golgi route for STING using brefeldin A inhibits STING-dependent signalling, confirming that ER-Golgi traffic is detrimental for STING activation¹¹. Coupled with our observations of interference by HRV upon traditional STING signalling, we wanted to examine the intracellular behavior of STING upon HRV infection. Cells were infected with HRV, and STING trafficking was monitored by confocal microscopy. Our results showed that STING did not follow the canonical ER to ERGIC route upon infection, unlike stimulation with cGAMP (Fig. 3a).

**Fig. 3: STING traffics to PI4P rich replication organelles.**

Instead, in HRV-infected cells STING was targeted to vesicular structures. Similar to other positive-strand RNA viruses, HRV replicates in ROs forming distinct vesicular clusters from cytoplasmic membranes, composed of host proteins and lipids⁴⁰. We infected bronchial epithelial cells with HRV-A1B and monitored STING trafficking post-infection (p.i.) throughout the virus replicative cycle (6–8 h). Our data showed that at 2–4 h p.i. STING trafficked to the viral ROs, and colocalised with the virus replicating dsRNA as well as the HRV replication protein 3A (Fig. 3b) and PI4P (Supplemental Fig. 1).

To determine the lipid content and composition of the replication organelles we used Coherent Raman scattering microscopy (CRS) techniques. Stimulated Raman scattering (SRS) and Coherent anti-Stokes Raman Scattering (CARS), are complementary to fluorescence modalities. They offer non-invasively intrinsic chemical selectivity without staining, as different molecules have specific and detectable vibrational motion⁴¹.

SRS microscopy is a relatively new approach combining two short-pulsed near-infrared laser sources and by overlaying them both spatially and temporarily at the sample it is possible to coherently probe the vibrational signature of molecules with a spatial resolution on sub-micron length scales. Thus, we used SRS to identify the distribution and identification of lipids in the ROs. Our results showed that STING-positive ROs had an increase in lipid composition, particularly phosphatidylinositol-4-phosphate (PI4P) (Fig. 3c). This is in agreement with previous studies which have shown ROs in picornavirus and hepacivirus are enriched in PI4P⁴².

To test whether other HRV serotypes had the same replication pattern, ALI cultures were infected with HRVC15 and imaged using fluorescence microscopy. The data confirmed as expected that dsRNA from the virus was colocalising with PI4P during the replication cycle (Fig. 3d).

Phosphoinositol-4-kinase IIIα (PI4KIIIα) and phosphoinositol-4-kinase IIIβ (PI4KIIIβ) are responsible for the production of PI4P from phosphatidylinositol (PI) and are important for the replication of other RO-mediated RNA viruses, such as HCoV and poliovirus^42,43,44. To determine whether PI4K was required for HRV infection, we firstly investigated whether PI4KIIIα or PI4KIIIβ co-localize with ROs and the HRV dsRNA. As expected, our data showed colocalization of PI4KIIIβ with the dsRNA associated with the RO upon HRV infection (Supplemental Fig. 1e). To assess this further, we utilised the GSK inhibitors GSK2998533, a PI4KIIIβ antagonist, and GSK268449 and PIK93, PI4KIIIα antagonists, in both confocal microscopy (Supplemental Fig. 1e) and viral replication assays (Supplemental Fig. 1f). GSK3000140 was used as an inactive negative control compound. It was clear that inhibition of PI4KIIIβ strongly reduced colocalization of dsRNA to STING, as well as diminishing viral replication.

In order to determine whether PI4KIIIβ interacts with STING and is required to recruit STING to the replication organelles, we performed fluorescence resonance energy transfer (FRET) studies to determine STING interactions with PI4KIIIβ, PI4P and ER (Fig. 4a, b). STING was shown to interact with PI4KIIIβ and PI4P in the ER 1 h after HRV infection, suggesting that PI4KIIIβ initially interacts with STING in the ER before translocating to the ROs.

**Fig. 4: STING interactions with PI4KIIIβ, PI4P and ER.**

These results demonstrate that HRV infection does not drive STING to travel to the ERGIC nor the TGN, where the palmitoylation of STING typically occurs. Therefore, this aberrant trafficking could explain why its canonical function and subsequent downstream signalling is compromised during HRV infection as observed in Fig. 3. To confirm this hypothesis, we investigated whether palmitoylation of STING still occurred after its relocation to the RO following HRV infection.

To test whether STING is palmitoylated, we utilized the in vitro Acyl-Biotin exchange (ABE) assay to determine the palmitoylation level of STING in HRV infected cells. As expected, it was shown that STING becomes palmitoylated only when cells are stimulated with ISDs, while in HRV infected cells STING is not palmitoylated (Fig. 4d). Thus, confirming that location is important for STING palmitoylation and the localization of STING at the ROs during HRV infection prohibits its palmitoylation and limits its subsequent signalling.

HRV 2B protein reduces ER Ca²⁺ levels and triggers release of STING from the ER

Our data confirmed that HRV selectively forms specialized ROs composed of cellular phosphoinositide lipids and that STING traffics to those ROs during HRV replication. However, the mechanism initiating this trafficking is unclear. In its resting state STING localizes to the ER membrane and it was recently shown by Srikanth et al. that stromal interaction molecule 1 (STIM1), a Ca²⁺ sensor, interacts with STING to retain it to the ER membrane. STIM1, is a Ca²⁺ binding protein which localizes throughout the ER when [Ca²⁺] ER is high, but, after depletion of the ER Ca²⁺ stores, it translocates into junctional areas between the ER and plasma membrane and interacts with the pore subunit of store-operated Ca²⁺ channels, CRCM1, and induces Ca²⁺ entry³⁰.

During HRV infection STING is not ER resident nor bound by STIM1 but instead moves to the replication organelles, implying [Ca²⁺] ER has been depleted allowing the release of STING. The HRV 2B protein is a small hydrophobic protein that has been shown to localize to the ER-Golgi complex and reduce [Ca²⁺] ER levels³⁴. We hypothesized that during viral infection HRV 2B reduces the ER Ca²⁺ stores causing STIM1 to translocate into junctional areas and thus releasing STING.

Investigation of both cytosolic and ER Ca²⁺ levels during HRV infection demonstrated that HRV 2B protein reduces the Ca²⁺ content of the ER (Supplemental Fig. 2). Confocal imaging data also showed that upon HRV 2B protein expression and subsequent ER Ca²⁺ depletion, STIM1 trafficked away from the ER (Supplemental Fig. S2).

Fluorescence resonance energy transfer (FRET) was used to determine STING and STIM1 interactions. FRET can occur over 1–10 nm distances, and effectively increases the resolution of light microscopy to the molecular level. The data confirmed that STIM1 and STING interacted in uninfected cells (Fig. 5a). However, upon HRV infection there was no interaction between STIM1 and STING. This data, therefore confirms that HRV 2B affects the cellular Ca²⁺ homeostasis and triggers STING release from STIM (Fig. 5a, b).

**Fig. 5: STIM1 releases STING upon HRV infection.**

STING binds to PI4P via positively charged amino acids

To elucidate the key drivers for STING recruitment to the RO, we investigated the relationship between STING and different phospholipids during HRV infection. It has been shown that changes in cellular lipid and phospholipid content occur during PV infection⁴⁰, which belongs to the same family as HRV.

Using a lipid array, we found that STING could bind to phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol-4,5-bisphosphate (PIP2), and phosphatidylinositol (PI) thus highlighting possible STING-phospholipid interactions (Fig. 6a). The specificity of STING for phosphatidylinositol phosphates was further verified by measuring FRET interactions with these lipids (Fig. 6b).

**Fig. 6: STING interacts with PI4P via a patch of positive charged amino acids.**

Protein–membrane interaction is regulated by electrostatic interactions between the negatively charged phospholipids of the plasma membrane and the positively charged amino acids in a domain of the membrane-binding protein. Therefore, we hypothesized that STING interacted with these lipids in a similar manner. Using the MOE Site Finder⁴⁵, we identified a region of conserved positively charged amino acids on the α3 helix of STING between 280aa and 295aa that contains four evolutionary conserved positively-charged amino acids (Fig. 6c).

The STING N-terminus affords several sites with high protein–ligand binding (PLB) propensity scores⁴⁶ that could accommodate larger molecules such as a steroid or phosphatidylinositol phosphate. Site finder identified alpha spheres that can occupy protein surface concavities and scored them by proximity to amino acid residues. White and red alpha spheres are near hydrophobic and hydrophilic residues, respectively (Fig. 6c). The residues R294 and R298 (K289 and R293 in human) are proximal to STING’s N-terminal tail and thus less likely to interact with the PI4P phosphate, which could more easily interact with R286 or R289 (corresponding to human R281 or R284, respectively), especially when STING is in an active state (Fig. 6d).

To test whether these residues are involved in PI4P recruitment, we mutated all four to alanine (4posA) or glutamate (4posE). When ectopically expressed in STING deficient huh7.0 cells, we observed using confocal microscopy that both the STING 4posA and 4posE mutants could no longer interact with PI4P (Supplemental Fig. 3a), this was also confirmed using the lipid array strips (Fig. 6a) where STING 4posA and 4posE were shown not be able to bind to PI4P on the strip. These results confirm that STING’s “positive patch” serves as a PI4P binding domain for STING recruitment to the ROs.

Additionally, the use of H-151⁴⁷, a STING antagonist, was able to completely inhibit HRV infection as well as limit the interaction between STING and PI4P (Supplemental Fig. 3c).

This data suggests that upon HRV infection, STING is released from STIM1 due to alterations in calcium homeostasis, which drives the binding of STING to PI4KIIIβ and PI4P and recruitment to the RO. Using both genetic modifications and chemical interventions, it was possible to limit this interaction, stalling STING in the ER and limiting HRV replication (Supplemental Fig. 3d).

Upon completion of replication STING traffics in phosphatidylserine enriched autophagosomes

Recent studies on sea anemone Nematostella vectensis and Xenopus laevis have shown that STING is important for autophagy induction²². Upon binding cGAMP, STING translocates to ERGIC which serves as a membrane source of LC3 lipidation and formation of autophagosomes²². However, we have seen that in HRV infected cells STING does not travel to ERGIC but binds PI4P and traffics to ROs instead.

Multiple studies have shown the accumulation of autophagosomes following picornavirus infection promoting non lytic release of picornavirus autophagosome derived vesicles²¹. Many pathogens are degraded by autophagy, but some subvert autophagy for their own advantage. Other Picornaviridae related to HRV, such as Coxsackievirus B3 (CBV3), induces the autophagosomal pathway but inhibits degradation of autophagic cargo⁴⁸.

HRV infectious cycle is typically from 6 to 8 hours, thus we imaged STING translocation at different times post infection (p.i.) by confocal microscopy. STING trafficked to PI4P rich ROs which became visible at 2–4 h p.i., then between 5 and 8 h p.i. STING sequentially moved from the ROs to autophagosome like vesicles forming large vesicular clusters that appeared after the peak of RNA replication (Supplemental Fig. 4).

We also investigated the timeframe of viral RNA replication as well as viral assembly and release. We infected bronchial epithelial cells with HRV and then at various times post-infection (p.i.) we measured viral RNA as well as infectious virus by plaque assay (Supplemental Fig. 3e, f). Viral replication peaked at 4–6 h p.i., while after the peak between 6 and 8 h p.i. there was an increase in extracellular viral titre, which corresponded with the autophagosome like vesicle formation.

The generation of HRV particles is a multistep process. Once RNA is packaged, the VP0 subunits get cleaved into VP2 and VP4 to generate mature infectious virions. To visualize the assembled capsids, we used an anti-VP2 antibody. Confocal imaging showed that between 5 and 8 h p.i. 80% of assembled virus capsids were in these vesicles as well as the autophagosomal marker LC3 and STING (Fig. 7a).

**Fig. 7: STING travels to phosphatidylserine (PS) autophagosomes.**

We tested for the presence of different phospholipids and the vesicles that appear after genome replication contained mainly negatively charged phosphatidylserine (PS) lipids and were distinct from ROs (Fig. 7b). Therefore, the earliest stages of PI4P induction and membrane remodelling actually represent the RO but these vesicles encase autophagy markers. High throughput fluorescence (HTF) imaging showed PS redistribution leading to more localised and intense spots/nodes of lipids in the PS rich organelles of the infected cells compared to that of the control cells where more uniform lipid distribution was observed (Supplemental Fig. 5). Furthermore, heat maps representing number of detected nuclei as shown also reveals that number of nuclei is noticeably increased at 6 h of post infection. This is expected since nuclei are more prominent in autophagy and HRV forms autophagosome like vesicles (Supplemental Fig. 5). Additionally, using Stochastic Optical Reconstruction Microscopy (STORM) we confirmed the presence of STING and LC3 in these PS rich vesicles that were larger than ROs ranging from 350 to 450 nm (Fig. 7b, c).

In order to determine whether STING plays a role in the virus package and release, ALI cells were given H-151, a STING antagonist, either as a pre-treatment or at the middle of the HRV infectious lifecycle and extracellular HRV was determined at 8 h of post infection. It was shown that when H-151 was administered as a pre-treatment, HRV replication was completely inhibited (Supplemental Fig. 3e, orange line graph) suggesting that STING plays a role in the replication of the virus. However, when H-151 was administered in the middle of the HRV replicative lifecycle and extracellular HRV was assessed 8 h of post infection, it was shown that H-151 could inhibit HRV release to the extracellular space suggesting that STING also plays a role in the virus package and release (Supplemental Fig. 3e, gray line graph).

From our experiments, we propose that two different organelles are induced during HRV infection that serve temporally distinct functions during HRV infectious lifecycle, and STING contributes to formation and function of both.

The membranes of eukaryotic cells vary in lipid composition, a feature essential to organelle identity. It has been shown that oxysterol binding related proteins ORP8 and ORP5 extract and exchange PI4P for PS between two membranes⁴⁹ and it is possible that STING, since it binds PI4P, could mediate the PI4P/PS lipid exchange between organelle membranes thus maintaining membrane lipid homeostasis. It is likely that the autophagosomal like organelle originated from ER derived replication organelle membranes.

Using differential ultracentrifugation followed by density gradient ultracentrifugation, we collected the cell fraction that contained the autophagosome/PS rich vesicles. Magnetic beads conjugated to annexin V was used to further purify the vesicle fraction), since annexin V can bind phosphatidylserine, with clear presence of STING and LC3 with VP2 (Fig. 7).

Screening by western blotting for different SNARE proteins we identified that the Ykt6 protein was also present in these vesicles (Fig. 6d). Ykt6 is a unique R-SNARE protein highly conserved from yeast to man localized to both the cytosol and intracellular membranes. It is an important regulator of the vesicular transport between the ER and the Golgi⁵⁰ and recently has been shown to be implicated in autophagosome biogenesis and fusion⁵¹. Thus, it seems that Ykt6 provides a reserve pool for the fusion and membrane transport that leads to the formation of compartments such as the autophagosome-like vesicles during HRV infection. The presence of SNAP29 and VAMP8 was also detected (Fig. 7d).

Subsequently we examined the distribution of STING and Ykt6 before and after HRV infection using FRET. Following 5 and 8 h p.i. of HRV infection, STING assembled in these autophagosome-like vesicles enriched with PS lipids together with Ykt6, SNAP29, and VAMP8 (Fig. 7e).

STING induces conversion of lipidated LC3 in the autophagosomal like vesicles

To determine the effect of autophagosome on HRV infections and to elucidate the role that STING plays in the formation of this autophagosome like vesicle we looked at LC3 lipidation over the time course of the infectious cycle. Our data demonstrated an elevated conversion of LC3-I to LC3-II (Fig. 7f) between 4–6 h p.i. of the virus infectious cycle suggesting augmented autophagosome formation.

Bronchial epithelial cells infected with HRV were treated with autophagy inhibitor bafilomycinA1 (bafA1), which blocks autophagolysosome formation, as well as 3-Methyladenine (3- MA), an established inhibitor of autophagy. Treatment with bafA1 or 3-MA inhibited viral yield suggesting that autophagy has a role in HRV propagation (Fig. 7g).

Previous studies have shown a role for autophagy in non-lytic release of Picornaviridae. To determine the destination of these non-canonical autophagosomal vesicles, we used the cell impermeable Trypan blue dye between 7–8 h p.i. and found that the plasma membrane remained intact. We proceeded to perform time lapse differential fluorescent microscopy in the presence of Alexa488-Annexin V and observed numerous Annexin V labeled vesicles emerged from the plasma membrane (Supplemental Fig. 6)

We have seen that inhibitors of autophagy reduce viral load, and we propose a model by which these non-canonical autophagosome like vesicles provide a mechanism for nonlytic release of rhinovirus. LC3 was detected and co localized with STING and viral capsid protein VP2 demonstrating that these vesicles contain HRV assembled virions (Fig. 7a).

The use of STING antagonist H-151, which abrogated interactions with PI4P and retains STING in the ER completely inhibited virus production (Fig. 6h) and subsequent autophagosomal vesicle formation. FRET studies in the presence of STING antagonist H-151 showed that there was no interaction between STING and LC3 or STING and virus VP2 thus confirming that STING is essential for autophagosome formation which is used for virus assembly and release during HRV infection (Fig. 7i).

Discussion

Since RVs have long been regarded to be the etiological agent of the common cold, a relatively simple, self-limiting syndrome, their importance as possible causal factor of severe respiratory illness has often been neglected. In the past few decades, the importance of RVs in upper and lower respiratory infections, pneumonias, wheezing, and exacerbations of asthma and COPD has become apparent. Despite its socioeconomic impact the broad variety of RV genotypes has represented a major obstacle in the development of specific vaccines or antiviral agents for RVs. In the current study, STING has been found to be crucial for viral replication across all RV serotypes, demonstrating a very novel therapeutic avenue.

STING sensing and activation is tightly controlled by a series of conformational changes, thresholds of activation as well as its location within the cell. In our study, we have identified STING as a multifaceted receptor and an important unexpected feature in HRV replication of all RV strains, with promise as a therapeutic target. A recent study has also confirmed this, but only for HRV-A and HRV-C, but not HRV-B strains³⁵. This difference could possibly by due to the cell types that the authors have used in their study and the virus tropism, as the authors mainly used Huh (hepatic) cells as well as HeLa (cervical epithelial) cells to confirm their findings, whereas our study utilises biologically relevant cell types such as bronchial epithelial and ALI cells that are more susceptible to all respiratory viruses.

In our study, we observed that upon HRV infection, HRV-2B protein affects the intracellular Ca²⁺ homeostasis thus STING–STIM1 interaction is disrupted (Supplemental Fig. 7), STING exits the ER and is recruited to the viral ROs through an interaction between a patch of positively charged amino acids on STING and PI4P, which allows recruitment of STING to ROs.

Using the MOE Site Finder, we identified a patch of conserved positively charged amino acids on the α3 helix of STING between 280aa and 295aa suggestive of a region where STING interacts with PI4P. In particular, the positive-charged residues R281 or R284, which reside in the polymer interface of STING seem to be crucial for allowing STING to bind PI4P.

Interestingly, these residues have been recently linked to STING-associated vasculopathy with onset in infancy (SAVI)-causing STING mutations^52,53, these mutations promote the upregulation of anti-viral type-I-interferon stimulated genes (ISGs). C206Y, R281Q, and R284S all residing within the polymerization interface of STING and have been reported to be hyperactivation mutants independent of cGAMP or cGAS binding⁵⁴. This region is separate from the aa153–177 sequence that participates in STING dimerization^55,56. It is unlikely then that aa280–295 is involved in dimerization, but instead critical for ER retention or interactions with a partner that regulated STING dimerization and activation^53,54. This could be STIM, which has been shown to be a negative regulator of STING by sequestering it to the ER³⁰.

Based on our findings, we propose that upon HRV infection, HRV-2B protein affects the intracellular ER Ca²⁺ homeostasis thus the STING–STIM1 interaction is disrupted (Supplemental Fig. 7). STING is released and assumes an “active state”. Although STING assumes an active conformation, it does not polymerize to a level that leads to signal transduction, since a certain level of STING has to be occupied by cGAMP in order to achieve canonical activation⁵⁷. The existence of a threshold of activation (all or none behavior⁵⁸) is a central mechanism for STING to distinguish between self and foreign dsDNA, therefore its release from STIM allows it to assume an active state without dimerizing and reaching the threshold for full activation. In this active state, R281 and R284 are exposed and able to bind PI4P, which in turn promotes the migration of STING from the ER to the viral ROs, where it serves as a scaffold.

Since this translocation inhibits STING from performing its canonical function following HRV infection, it is possible that HRV infection may interfere with response against DNA viruses as the c-GAS-STING pathway will be impaired, although other innate immune sensors might compensate DNA sensing in these cases.

Following viral replicative infection, we observe the biogenesis of large autophagosomal like vesicles distinct from ROs with different lipid composition, enriched with phosphatidylserine (PS) lipids and autophagosomal markers which provide a cellular exit of the virus and are important for HRV assembly and transmission. STING traffics to these autophagosomal vesicles after viral replication and it is possible that STING, since it binds PI4P, could mediate the PI4P/PS lipid exchange between organelle membranes thus helping to maintain membrane lipid homeostasis. It is likely that the autophagosomal like organelle originated from ER-derived RO membranes.

This mechanism would fit with the primordial function of the STING-cGAS pathway to induce autophagy. It has been shown that a unique and important feature of the STING pathway is to activate autophagy independently of TBK-1 and interferon induction²². In this mechanism, STING translocates from the ER to the ERGIC which serves as a membrane source for LC3 lipidation and autophagosome biogenesis that target DNA viruses for the clearance from the cytosol to the lysosome. Thus, autophagy induction is an ancient, highly conserved function of the cGAS-STING pathway that is independent of the induction of interferon and inflammatory cytokines—similar to the mechanism that we have observed with HRV.

HRV has evolved to exploit STING and subvert the autophagic pathway and use it as an integral component of the viral life cycle without triggering the interferon or inflammatory response via STING. Our data indicate that HRV can exploit lipid homeostasis contributing to the generation of intracellular membranes and organelles potentially maximising virus replication and spread.

In conclusion our results identify STING as an important facilitator of HRV replication contributing to the assembly and exchange of different lipid microenvironments that leads to the generation of different type of organelles essential for HRV propagation (Fig. 7j).

This non-canonical role of STING has therapeutic potential and is particularly important not only for HRV replication but possibly for other RNA viruses as well, such as Coronaviruses which utilise replication organelles in order to facilitate their replication. In the future, targeting STING may be useful to improve preventive and effective treatment strategies to limit the overall burden of RV disease, and the consequent risk of developing chronic respiratory morbidity and mortality.

Methods

Tmem173 (Sting) tissue expression data

The GTex data presented in this manuscript were obtained directly from the GTEx Portal, release v8 (dbGaP accession number phs000424.v8.p2). The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS.

Acyl-biotin exchange (ABE) assay for palmitoylation

Detection of Protein Palmitoylation by Immunoprecipitation and Acyl-Biotin Exchange (ABE) was performed according to Brigidi et al.⁵⁹. Briefly, STING was immunoprecipitated from protein samples using 1 µg of anti-STING rabbit polyclonal antibody (Thermo Fisher, PA5-23381, 1:100) the complex was pulled using Protein A Sepharose AB beads (Thermo Fisher). To block non-palmitoylated cysteine residues, immune-precipitated STING was treated with 50 mM N-ethylmaleimide (NEM) in RIPA buffer (Sigma Aldrich, R0278) at 4 °C for 2 h on a shaker followed by three washes with RIPA buffer. STING was then treated with hydroxylamine (HAM) buffer to remove palmitate from Cysteine residues (1 M Hydroxylamine, 50 mM tris, 150 mM NaCl, 5 mM EDTA, 0.2% TX100, and pH 7.4) at room temperature for 2 h on a shaker. Then STING was treated with 4 µM of BMCC-Biotin in 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.2% TX100, pH 6.2 for 1–2 h followed by three washes to remove excess biotin. Sixty microliter of reducing protein sample buffer was added and samples were boiled for 2 min to elute STING from beads. Twenty percentage of sample was loaded on to SDS PAGE gel. Membrane was blocked with 5% Bovine serum albumin (BSA) for overnight followed by incubation with streptavidin conjugated with HRP (Thermo Fisher, N100) at 1:30,000 for 60 min and Biotin-streptavidin HRP complexes were visualized by exposing the membrane to ECL and then to X-ray film.

Antibodies

Anti-STING rabbit polyclonal antibody (Invitrogen, PA5-23381, 1:100 IF)), anti-STING goat antibody (SantaCruz, sc241046, 1:100 iF)), J2 mAb English & Scientific Consulting, 1:100 IF. PI4P IgM mAb Echelon, ZP004-2, 1:100 IF), Anti Calreticulin goat polyclonal (ThermoFisher, PA1-33045, 1:100 IF). ERGIC-53 monoclonal antibody (ENZO, OTI1A8, 1:100 IF) as well as ERGIC-53 monoclonal antibody (Sigma-Aldrich, E1031, 1:100 IF). Alexa Fluor-488 or Fluor-546 labeled secondary antibodies against mouse or rabbit IgG or IgM (Thermofisher Scientific, 1:100 IF) were used.

Agonists, antagonists, and lipids

2′3′-cGAMP was purchased from Invivogen. 2′3′-cGAMP (5 µg/ml) (Invivogen, tlrl-nacga23-02) was added to the cells complexed with LyoVec (Invivogen, Lyec-12) in order to aid internalization. ISD (Interferon stimulatory DNA)/Lyovec (Invitrogen, tlrl-isdc, 1 µg) was also used. Membrane lipid strips were obtained from Echelon. The following inhibitors were used: Fura-2 AM, Ca2+ selective fluorescent indicator (Abcam, ab120873), BX795 (TBK-1 inhibitor, Invivogen, tlrl-bx7, 1 µM), RU.521 (cGAS inhibitor, Invivogen, inh-ru521, 500 nM). GSK2998533 (100 nM) & GSK2683449 (100 nM) were provided by GlaxoSmithKline (GSK). PIK93 was also used (Sigma Aldrich, 1 µM)

Cell culture/viruses

Bronchial epithelial cells (BEAS-2B, ATCC cat. Number CRL-9609) were cultured in RPMI medium containing 10% FBS, and 1% non-essential amino acids. Bronchial epithelial cells were seeded in lab-tek 8-well slides (80,000 cells/well) for two days. Once they were ~80% confluent, they were incubated with HRV (MOI:5) in 500 µl of serum-free medium (SFM) for 2 h at 37 °C. Following the stimulation, the supernatant was removed and the cells were washed ×2 with PBS, followed by fixation using 500 µl per well formalin for 15 min at RT.

Air liquid interface (ALIs) of airway epithelial cells were grown on transwells from normal donors (Epithelix, NHBE—Human Bronchial Epithelial Cells, Donor 28060 p3, and Donor 25265COPD Donor 63 p3) and cultured in Pneumacult media (Seeding density: 150,000 cells/well; Age of cells used in experiments post air-lift: 14 days. No differences were observed when cells were infected either through the apical or basolateral membrane suggesting the polarization of the cells had no effect.

Human rhinovirus A2 (HRV-A2) (ATCC, Cat. Number VR-482), HRV-B4 (ATCC, Cat. Number VR-284), HRV-B14 (ATCC, Cat. Number VR-284), HRV-A1B (ATCC, Cat. Number VR-1645), HRV-A16 (ATCC, Cat. Number VR-283) were purchased from the ATCC. In addition, HRV-A1B and HRV-A16 were also purchased from Virapur. Full-length HRV-C (HRV-C15) (GenBank accessionno.GU219984) was synthesized based on the published sequence⁶⁰ as described in ref. ⁶¹.

HRV RNA was quantified using primers in Supplemental Table 1.

These primers are complementary to the 5′ non translated region of HRV. RT-PCRs were performed in triplicate for each sample and quantified on an ABI 7000 real-time PCR system (Applied Biosystems, CA). Data were analysed using GraphPad Prism (GraphPad Software, Inc.).

To generate viral stocks, 95% confluent HeLa-Ohio cells were infected and incubated at 34 °C/5%CO₂ for 3 days. All infected cells underwent freeze-and-thaw cycle three times, cell debris was removed by centrifugation at 500 × g for 5 min at room temperature, supernatant was collected and stored at −80 °C. Virus titre was determined by plaque assay.

Computational modelling of STING-PI4P interactions

Potential PIP4 binding site identified between STING N-terminus and alpha3 helix with MOE Site Finder software (Molecular Operating Environment (MOE), 2019.01; Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2020) utilizing CryoEM STING structures 6NT6 and 6NT7⁶².

A homology model of human STING was constructed using MOE.2019.0101 starting with on chicken STING (6NT7). The C-terminal domain (CTD) from structure 6DXG (internal PSILO code 1ASRC) was superimposed on the homology model. Residues Ile165 to Val343 were removed from the original model and replaced with those of 6DXG. To make a cleaner figure to show the alpha3 helix, the human full length cryoEM STING (6NT5) CTD was replaced with the modelled 6DXG one.

A conformation for PI4P was built starting with a ligand from 3MTC. Missing atoms were added, and a conformational search was carried out in MacroModel in Maestro 2020.1 (MCMM, 100,000 steps, 8 kcal/mol cut-off, other settings default). A low energy conformation of PI4P was then placed in the SiteFinder identified pocket near R281/284 of the full-length human STING homology model. Molecular dynamics was carried out for 100 ns in a 10A box of water using default conditions with Desmond in Maestro 2020.1.

Confocal microscopy

After fixation, the cells were washed ×2 with PBS and were permeabilised using 500 µl per well of PBS/0.02% BSA/0.02% Saponin and labeled with antibodies specific for PI4P, STING, J2 mAb to detect viral dsRNA, ERGIC (1:100) as well as secondary antibodies conjugated to the appropriate fluorophore (1:100). Following the secondary antibody incubation, the cells were washed three times using PBS/0.02% BSA/0.02% NaN_3. Finally all liquid was removed, as well as the plastic inserts. The cells were mounted with Vectashield, covered with a coverslip, and sealed using clear nail varnish.

Cells were imaged on a Carl Zeiss, Inc. LSM710 ELYRA P1 confocal using a 1.4 NA 63× Zeiss objective. The images were analysed using LSM 2.5 image analysis software (Carl Zeiss, Inc.).

No fluorescence was observed from an Alexa 488-labeled specimen using the 594 filters, nor was 594 fluorescence detected using the 488 filter sets.

Quantification of co-localization

In order to quantify the degree of co-localization, we used Costes’ approach (Bolte and Cordelieres, 2006). Coste’s approach, Pearson’s correlation coefficients and p-values were calculated using MBF ImageJ with JACoP (Just Another Colocalisation Plugin) (http://macbiophotonics.ca/). This allows for the calculation of Pearson’s correlation coefficient R(obs), which also accounts for any random overlay of pixels by generating the mean correlation coefficient R(rand) between n images that have identical average pixel intensity to the original images, but a random distribution of pixels. R(obs) is not sensitive to background and has a linear regression range of −1 to 1, with −1 being total negative correlation, 0 being a random correlation and 1 being total positive. In theory, an R(obs) of +1 would represent a perfect pixel correlation between two channels in an image. Values greater than 0.5 are considered significant co-localization. In addition, Costes’ randomisation method calculates the statistical significance of the Pearson’s correlation coefficient. It returns a significance (p-value) expressed as a percentage.

CRISPR STING knockout

The following gRNA sequences were designed (GENscript) to uniquely target genes within the human genome. The gRNA sequences that were used with CRISPR/Cas9 knockout (KO) plasmid, to introduce a DSB for genome editing are shown in Supplemental Table 2.

Bronchial epithelial cells were seeded at a density of 0.8–3.0 × 10⁵ cells/ml in 12-well plates. Fifty nanomolar guide RNA and 2.5 µg plasmid DNA encoding Cas9 were transfected to cells via TransIT-X2 (MIRUS LLC, Madison, USA) following manufacturer’s recommendations.

Generation of STING mutants

Human STING gene subcloned in pUNO1 (Invivogen) was used to generate the STING mutants. Point mutants were generated using standard PCR generated using the Geneart Quickchange Site‐Directed Mutagenesis Kit (Invitrogen) according to the manufacturer’s instructions.

Phosphatidylserine vesicle isolation

Cells were infected with HRV for 30 min and replaced with fresh serum free media for another 6–8 h. Extracellular media then was collected from virus-infected cells. Media was spun down at 500 × g for 5 min (4 °C). Supernatant was collected and spun down again at 5000 × g for 10 min (4 °C). Supernatant and pellet (vesicles) were collected for infection. Collected vesicles and free virus (either collected from supernatant post 5000 × g spin or from directly freeze-thawing [3×] the vesicles) can be used at this stage to infect cells. Further enrichment of PS vesicles from the pellet fraction was done with magnetic isolation using the Annexin V microbead kit as described (Milteny Biotec, CA). The magnetic microbeads lacking Annexin V were used to control for nonspecific binding.

Fura-2/AM measurements

BEAS-2B cells were transfected with 2 µg of p2B-myc using Lipofectin (Invitrogen). After 24 h, 5 × 10⁴ cells were seeded onto 24 mm coverslips and grown for another 16 h before calcium measurements. Alternatively, BEAS-2B cells were infected with HRV1B (MOI 5) for 12 h.

The fluorescent Ca²⁺ indicator Fura-2 was used to measure [Ca²⁺cyt] at the single cell level as described^33,63. Briefly, cells either infected with HRV or transfected with p2B-myc were incubated in medium supplemented with 2.5 µM Fura-2/AM for 30 min, washed with HT buffer to remove the extracellular probe, supplied with preheated HT buffer (supplemented with 1 mM CaCl₂), and placed in a thermostated (37 °C) incubation chamber on the stage of an inverted fluorescence microscope. Fluorescence was measured every 2 s with the excitation wavelength alternating between 340 and 380 nm and the emission fluorescence being recorded at 492 nm. At the end of the experiment, a region free of cells was selected, and one averaged background frame was collected at each excitation wavelength for background correction. To measure the [Ca²⁺] of the stores, the amount of thapsigargin-releasable Ca²⁺ was determined. After recording the cells in Ca²⁺-containing HT buffer, the medium was replaced with Ca²⁺-free HT buffer supplemented with 0.5 mM EGTA. After 10 min, the cells were challenged with 1 µM thapsigargin, and [Ca²⁺]cyt was measured as described above.

Statistical analysis

Data represent the mean ± SD. Differences between groups were analysed using an unpaired student’s t-test or 1-way ANOVA test as required, using GraphPad Prism 7 software, unless indicated otherwise. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For all experiments n number of independent experiments, yielding consistent results.

Reporting summary

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

Data availability

All relevant data are available from the authors. Source data are provided with this manuscript. Source data are provided with this paper.

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Acknowledgements

This work was supported by the GlaxoSmithKline Immunology Network.

Author information

Authors and Affiliations

Immunology Catalyst, Immunology Network, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK
Martha Triantafilou, Lee M. Booty, Gisela Jimenez-Duran, Emma Koppe & Kathy Triantafilou
Cardiff University, Institute of Infection and Immunity, School of Medicine, University Hospital of Wales, Heath Park, Cardiff, CF14, UK
Martha Triantafilou, Gisela Jimenez-Duran & Kathy Triantafilou
Innate Immunity Research Unit, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, 19426, USA
Joshi Ramanjulu, Neysa Nevins, G. Scott Pesiridis & John Bertin
Functional Genomics, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK
Hakan Keles
Adaptive Immunity Research Unit, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK
Ken Saunders & Louise K. Modis

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Martha Triantafilou
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K.T. and M.T. were involved in the conception and design of the work as well as the interpretation; K.T., M.T., L.B., G.J.D., K.S., N.N., and H.K. were involved in the acquisition, and analysis of the data; E.K., J.R., G.S.P., L.K.M., and J.B. provided reagents; M.T. supervised the project and wrote the original manuscript with input from all authors.

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Correspondence to Martha Triantafilou.

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Triantafilou, M., Ramanjulu, J., Booty, L.M. et al. Human rhinovirus promotes STING trafficking to replication organelles to promote viral replication. Nat Commun 13, 1406 (2022). https://doi.org/10.1038/s41467-022-28745-3

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Received: 27 October 2020
Accepted: 18 January 2022
Published: 17 March 2022
DOI: https://doi.org/10.1038/s41467-022-28745-3

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