AXL-Mediated Productive Infection of Human Endothelial Cells by Zika VirusNovelty and Significance
Rationale: The mosquito-borne Zika virus (ZIKV) is now recognized as a blood-borne pathogen, raising an important question about how the virus gets into human bloodstream. The imminent threat of the ZIKV epidemic to the global blood supply also demands novel therapeutics to stop virus transmission though transfusion.
Objective: We intend to characterize ZIKV tropism for human endothelial cells (ECs) and provide potential targets for intervention.
Methods and Results: We conducted immunostaining, plaque assay, and quantitative reverse transcription-polymerase chain reaction of ZIKV RNA to evaluate the possible infection of ECs by ZIKV. Both the African and the South American ZIKV strains readily infect human umbilical vein endothelial cells and human ECs derived from aortic and coronary artery, as well as the saphenous vein. Infected ECs released infectious progeny virus. Compared with the African strains, South American ZIKV isolates replicate faster in ECs and are partially cytopathic, suggesting enhanced virulence of these isolates. Flow cytometric analyses showed that the susceptibility of ECs positively correlated with the cell surface levels of tyrosine-protein kinase receptor UFO (AXL) receptor tyrosine kinase. Gain- and loss-of-function studies further revealed that AXL is required for ZIKV entry at a postbinding step. Finally, small-molecule inhibitors of the AXL kinase significantly reduced ZIKA infection of ECs.
Conclusions: We identified EC as a key cell type for ZIKV infection. These data support the view of hematogenous dissemination of ZIKV and implicate AXL as a new target for antiviral therapy.
Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family.1 First isolated from a febrile rhesus macaque in 1947 in Uganda, ZIKV has not been recognized as a major viral pathogen until ZIKV infection in pregnant women in the Americas was confirmed as the cause of microcephaly and other birth defects seen in neonates.2,3 Indeed, studies in mouse models confirmed ZIKV can be transmitted from the pregnant mother to the fetus.4–6 In addition, ZIKV may be transmitted during sex7–9 or a blood transfusion.10 To date, ZIKV has been detected in the human central nervous system,11,12 blood, saliva,13 semen,14 and urine,15 suggesting the virus has developed mechanisms to reach multiple tissues. The Centers for Disease Control has now placed ZIKV on the list of blood-borne pathogens. The detection of ZIKV in blood further raises serious concerns about the risk of transfusion-related transmission and, in particular, sparking fear about the potential for severe outcomes in at-risk recipient populations.
Editorial, see p 1149
In This Issue, see p 1145
Conceptually, ZIKV may be carried into bloodstream by infected macrophages or dendritic cells after local mosquito bites.16 Here, we report that ZIKV directly infects human endothelial cells (ECs) and disseminates infectious progeny virus. Specifically, both the African and the South American ZIKV strains readily infect primary human ECs isolated from umbilical veins, the aorta, the coronary arteries, and the saphenous veins, leading to the release of infectious virus. The candidate receptor of entry seems to be the tyrosine-protein kinase receptor UFO (AXL) receptor tyrosine kinase (RTK) because specific small molecular inhibitors of AXL blocked ZIKV infection. Our results support a model that ZIKV spreads through infected ECs and bypasses the barriers that would otherwise restrict viral infection.
A detailed Methods section is provided in the Online Data Supplement.
The interior surfaces of vascular and lymphatic vessels are lined with endothelium, forming an interface between circulating blood or lymph in the lumen and the rest of the vessel wall. ECs are also major components of the blood–brain barrier and part of the placental blood barrier, preventing circulating virus from entering the brain and the fetal tissues, respectively. To investigate whether ZIKV directly infects ECs, we cultured the immortalized human blood–brain barrier EC line hCMEC/D3 (human cerebral microvascular endothelial cell line), which is a well-characterized blood–brain barrier cell system.17 The African MR766 and IbH 30656 (IbH) ZIKV strains (ZIKVAF), as well as the South American ZIKV isolates (ZIKVSA) PRVABC59 (PRV) and FLR, but not Dengue virus (DENV-2 strain Thailand 16681), readily infected hCMEC/D3 cells and produced infectious virus with titers comparable to that of the C6/36 cell line (Online Figure I).
To determine the extent of ZIKV EC infection, we used cultures of human ECs at low passages (passage numbers <7) isolated from umbilical veins (human umbilical vein endothelial cells [HUVECs]), aorta (human aortic endothelial cells), coronary artery (human coronary artery derived endothelial cells), and saphenous vein (human saphenous vein derived endothelial cells; Online Figure II). For comparison, we also obtained human lymphatic ECs (Online Figure III). Low-passage ECs were inoculated with the MR766 and IbH 30656 (IbH) ZIKV strains, and the PRV and FLR. Both ZIKVAF and ZIKVSA strains, but not DENV2, infected all vascular ECs from multiple donors, with HUVECs being significantly most susceptible (Figure 1A through 1E). Notably, ZIKV entry into vascular ECs also led to productive infection and the release of infectious progeny virus (Figure 1B and 1D). Although the ZIKVAF strains MR766 and IbH caused minimal morphological change, infection of HUVEC cells with the 2 ZIKVSA isolates induced significant cell death, suggesting enhanced virulence of these isolates (Figure 2A). The PRV isolate also formed noticeably larger plaques on plaque assays (Figure 2B). Indeed, when the kinetics of viral RNA replication was measured, the ZIKVSA isolates displayed faster growth rates (Figure 2C and 2D). Although we cannot completely rule out that the African isolates have been cultured for decades in the laboratory and hence adapted to be less cytopathic, our observation is consistent with a recent report that pups born from a Brazilian ZIKV (ZIKVBR) isolate-infected SJL pregnant mice displayed abnormalities resembling the microcephaly seen in humans.4 These findings highlight the differences between the original ZIKVAF strain, which causes no or very mild symptoms, and the circulating ZIKVSA strains that seem to be more pathogenic. The fact that HUVECs are most susceptible and the partial cytopathic effect of ZIKVSA isolates on these cells also suggest the potential involvement of ECs during vertical transmission of ZIKV.
Because vascular ECs form physical barriers with tight junctions between cells, we conducted confocal microscopy and Western blotting to evaluate the effects of ZIKV infection on endothelium integrity. Transendothelial electric resistance was measured for the assessment of endothelial barrier function. Infection by ZIKV did not directly disrupt the tight junctions or the barrier function of HUVEC or hCMEC/D3 cells (Online Figure IV). Therefore, we reason that the loss of endothelium integrity probably only occurs when significant cell death is caused by ZIKV infection.
ZIKV is known to utilize multiple cell surface receptors, including DC-SIGN, AXL, Tyro3, and TIM-1, to gain entry with a major role for the RTK AXL.16 A recent single-cell expression analysis revealed that the candidate entry receptor AXL is highly expressed in neural stem cells and ECs in developing human cortex.18 To probe into the potential involvement of AXL, we first performed flow cytometric analysis and determined the AXL expression levels on low-passage cultures of human ECs. Notably, although most ECs express AXL, HUVECs seem to express AXL to the highest level, followed by human aortic endothelial cells and human saphenous vein–derived endothelial cells (Figure 3A). By contrast, human coronary artery–derived endothelial cells expressed little AXL. Although we cannot completely exclude potential donor variability, the cell surface expression levels of AXL positively correlate with cellular susceptibilities to ZIKV (Figures 1E and 3A). AXL is an RTK that transduces signals from the extracellular matrix into the cytoplasm by binding to the vitamin K–dependent protein growth arrest-specific 6 (Gas6) gene.19 To explore the role of AXL in the observed ZIKV infection, we conducted 3 sets of experiments. First, a polyclonal antibody that recognizes the extracellular portion of human AXL blocked the entry of virus into HUVECs and hCMEC/D3 cells (Figure 3B). Notably, addition of the antibody did not reduce the attachment of ZIKV to cells (Figure 3C) and was unable to block ZIKV infection if added 3 hours after the infection had been initiated (Figure 3D). These data suggest that AXL is a ZIKV entry factor required at a late stage during entry. Second, ectopic expression of AXL in 293T cells promoted ZIKV infection without enhancing virus binding (Figure 3E, 3F, and 3H). By contrast, the kinase-dead AXL, which carries a K567R mutation that destroys an ATP-binding site and inhibits Axl phosphorylation and signaling,20–22 is significantly impaired its ability to confer permissiveness to 293T cells (Figure 3F and 3H). These observations reinforced the idea that AXL promotes ZIKV entry at a postbinding step and also imply that AXL-mediated signaling is needed for ZIKV entry. Finally, 2 known inhibitors of AXL phosphorylation, Cabozantinib23 and R428,24 significantly impaired ZIKA infection of hCMEC/D3 and the HUVECs in a dose-dependent manner (Figure 4A through 4D). By contrast, RTK inhibitors Sunitinib malate and Sorafenib had none or marginal inhibition at one micromolar (Online Figure V). These results indicate that AXL RTK activity is potentially important to its function in ZIKV entry. The kinetics of R428-mediated inhibition, generated from a time-of-addition experiment, showed that the compound remained inhibitory even when added at 1 hour after the virus was added but drastically lost its effect if added at 2 hours after infection had been initiated (Figure 4E). Therefore, R428 interferes with a postbinding process during ZIKV entry. Of note, treatment of HUVECs with AXL inhibitors neither altered AXL cell surface expression (Figure 4F) nor reduced cell viability (Online Figure VC).
With the imminent threat of the ZIKV epidemic to pregnant women and to the global blood supply, this timely study provides mechanistic understanding of ZIKV tropism and pathogenicity. The significance of our findings is 3-fold: (1) human ECs are likely one of the principal cell types of ZKIV infection. In vivo, the release of infectious ZIKV by ECs would conceivably facilitate hematogenous dissemination of the virus and bypass the barriers that would otherwise restrict viral infection and thus reach tissues where viruses typically cannot reach. Such a route allows the virus to rapidly enter or leave the bloodstream and potentially contributes to the intrauterine and transfusion-mediated ZIKV transmission (Online Figure VI). Although several human placental cell types, including cytotrophoblasts, epithelial cells, fibroblasts, and Hofbauer macrophages, were reportedly permissive to ZIKV,25,26 Miner et al5 recently found that ZIKV-infected Ifnar–/– pregnant mice show signs of vascular damage in the placenta and fewer fetal blood vessels, evidencing ZIKV infection of fetal ECs in vivo. (2) The ZIKVSA isolates replicate faster in ECs than the ZIKVAF strains. Although the ZIKVAF strains MR766 and IbH have been around for many years and might have adapted more through continuous passages in cultures, faster replication kinetics could contribute to the enhanced virulence of ZIKVSA isolates. We speculate that the partial cytopathic effect of the ZIKVSA isolates may trigger vascular changes in vivo, from severe placental vascular damage and a reduction in fetal blood vessels early in pregnancy to hemorrhagic retinopathy and torpedo maculopathy.27 In this regard, it would be highly interesting to assess the prevalence of vascular complications among ZIKV-infected individuals. (3) ZIKV tropism for ECs positively correlates with cell surface levels of AXL. Despite previous studies have implicated a role of AXL in ZIKV entry, our results clearly demonstrated that AXL functions at a postbinding step, where its catalytic activity is required. Several AXL inhibitors, including Cabozantinib and R428 that are currently in clinical trials for anticancer activities, may serve as unique antiviral therapeutics that suppress ZIKV infection of ECs.
We thank Dr Matthew Kappes for critical reading of this article. Zika Virus, MR766, NR-50065 and IbH 30656, NR-50066 were obtained through BEI Resources, the National Institute of Allergy and Infectious Diseases (NIAID), and the National Institutes of Health (NIH) as part of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) program. The following reagents were obtained through BEI Resources, NIAID, NIH: Zika virus, PRVABC59, NR-50240 and FLR, NR-50183.
T. Wang designed the overall experiments. L. DeLalio prepared the primary human endothelial cells. S. Liu and T. Wang performed the experiments and analyzed the data. S. Liu, B. Isakson, and T. Wang wrote the article. All authors read and approved the final article. All authors have provided the corresponding author with written permission to be named in the article.
Sources of Funding
This study was sponsored by National Institutes of Health grant R01DK088787 (to T. Wang), R01HL088554 (B.E. Isakson). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.
In August 2016, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.98 days.
Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive observations, but have a relatively narrow scope. Less comprehensive than Regular Articles but still scientifically rigorous, BURCs present seminal findings that have the potential to open up new avenues of research. A decision on BURCs is rendered within 7 days of submission.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.116.309866/-/DC1.
- Nonstandard Abbreviations and Acronyms
- endothelial cells
- human umbilical vein endothelial cells
- receptor tyrosine kinase
- tyrosine-protein kinase receptor UFO
- African Zika virus stains
- Zika virus
- South American Zika virus strains
- Received August 27, 2016.
- Revision received September 10, 2016.
- Accepted September 19, 2016.
- © 2016 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Besides infecting the developing fetal brain, Zika virus has also been recognized as a blood-borne pathogen.
Endothelial cells are major components of the blood–brain barrier and part of the placental blood barrier, preventing circulating virus from entering the brain and the fetal tissues, respectively.
What New Information Does This Article Contribute?
Low passage human endothelial cells can be readily infected by Zika virus of the African and South American lineage and release infectious progeny virus.
South American Zika virus isolates replicate faster in human endothelial cells and are partially cytopathic.
The receptor tyrosine kinase AXL is required for Zika virus entry of endothelial cells at a postbinding step.
The endothelium is the key cellular barrier between the blood and interstitial space. We find that Zika virus use of the receptor tyrosine kinase AXL allows for entry into endothelium, in particular human umbilical vein endothelium. This work demonstrates that endothelial cells are key targets for ZIKA virus and could be a novel pharmacological target. Critically, this work (1) strongly implies that screening of the stored blood supply should be a priority because of the direct contact between blood and endothelium and (2) could explain the presence of the virus in embryos, in utero, and in stored blood.