Hantaan virus can infect human keratinocytes and activate an interferon response through the nuclear translocation of IRF-3
Introduction
Hantaviruses, from the family Bunyaviridae, are trisegmented, single-stranded, negative-sense RNA viruses. The genomes of hantaviruses consist of L, M, and S segments, which encode RNA-dependent RNA polymerase, glycoprotein, and nucleoprotein, respectively (Hepojoki et al., 2012, Vaheri et al., 2013). In nature, hantaviruses are mainly carried by reservoir animals, such as rodents, shrews, moles and even bats, with infections in such reservoir animals being mostly chronic and asymptomatic (Easterbrook and Klein, 2008, Vaheri et al., 2013, Zhang, 2014). Hantaviruses cause two types of infections when transmitted to humans. Hemorrhagic fever with renal syndrome (HFRS) is primarily caused by Hantaan virus (HTNV) in Asia, Seoul virus (SEOV) worldwide, and Puumala virus (PUUV) and Dobrava virus (DOBV) in Europe; these viruses are harbored by Apodemus agrarius, Rattus norvegicus, Myodes glareolus and Apodemus flavicollis, respectively (Jonsson et al., 2010). Of these, PUUV causes a mild form of HFRS that is termed nephropathia epidemica (NE) and results in reduced mortality compared to other forms (Mustonen et al., 2013). Hantavirus pulmonary syndrome (HPS or HCPS), which is caused by Sin Nombre virus (SNV) and related viruses in North America and by Andes virus (ANDV) and related viruses in Latin America, is harbored by Peromyscus maniculatus and Oligoryzomys longicaudatus, respectively (Jonsson et al., 2010, Schountz and Prescott, 2014). All of these pathogenetic hantaviruses are rodent-borne, and the pathogenicity of newly discovered hantaviruses that are harbored by insectivores or bats requires further investigation.
Due to their impact on human health, rodent-borne hantaviruses have been widely studied (Kallio et al., 2009, Pedrosa and Cardoso, 2011, Schountz and Prescott, 2014). As rodents and insectivores infected by hantaviruses always maintain significant levels of virus, which can be transmitted to humans, in vivo, these animals are termed reservoir hosts. Percutaneous transmission within an animal reservoir is highly prevalent (Wesley et al., 2010).
HFRS/HPS infection can be acquired after exposure to aerosolized urine, droppings, or saliva from infected rodents or after exposure to dust from their nests (Hardestam et al., 2008, Jonsson et al., 2010, Xu et al., 1985). Transmission may also occur when infected urine or other biological material is directly introduced into broken skin or onto the mucous membranes of the eyes, nose, or mouth. One study described that transmission could occur through the ingestion of infected rodent-contaminated food or water (Mesic and Almedin, 2008); however, further evidence to support this finding is lacking. Additionally, individuals who work with live rodents can be exposed to hantaviruses through bites from infected rodents. Transmission from one human to another may occur, e.g., ANDV has been reported to be directly transmitted from person to person (Martinez et al., 2005), but this is extremely rare. Moreover, PUUV is reportedly able to be transmitted via the transfusion of platelets or other blood products (Sinisalo et al., 2010).
Type I interferons (IFNs) are proteins with antiviral activity, with IFN-α and IFN-β typically being produced in leukocytes and fibroblasts, respectively (Havell et al., 1975). IFN-β is considered to have a significant role against RNA viral infections. Host pathogen pattern receptors (PRRs) recognize viral RNA or other pathogen-associated molecular patterns (PAMPs), including Toll-like receptors (TLR), retinoic acid-inducing gene I-like receptors (RLRs) and NOD-like receptors (NLRs) (Akira and Takeda, 2004, Lupfer and Kanneganti, 2013, Yoneyama et al., 2004). RIG-I and MDA5 are involved in the assembly of mitochondrial antiviral signaling protein (MAVS or IPS-1, Cardif, VISA) complexes at the mitochondrial membrane (Hou et al., 2011, Seth et al., 2005, Yoneyama et al., 2004). Subsequently, MAVS binds tumor necrosis factor receptor-associated factor 3 (TRAF3) and recruits TBK1/IKKε kinases, which activate nuclear factor κB (NF-κB) and phosphorylate IRF-3 and IRF-7 (Hacker et al., 2011, Oganesyan et al., 2006). IRF-3 and IRF-7 are then translocated to the nucleus to initiate IFN-β mRNA synthesis (Hiscott, 2007). Secreted type I IFNs bind to receptors on neighboring cells; and IFN-stimulated gene factor 3 (ISGF3) translocates to the nucleus, via the JAK-STAT pathway, to initiate the expression of ISGs by binding to IFN-stimulated response elements (ISREs) (Au-Yeung et al., 2013). Previous studies have indicated that TLR3, TLR4 and RIG-I are responsible for sensing HTNV infection and then inducing IFN expression in A549, EVC-304 and Huh7 cells, respectively (Handke et al., 2009, Lee et al., 2011, Yu et al., 2012). Cells pretreated with IFN can prevent pathogenic hantaviruses from establishing successful replication, which indicates that IFN plays a role in controlling hantavirus infections (Matthys and Mackow, 2012). Moreover, the Gn tail of hantaviruses (NY-1V, ANDV and TULV) has been reported to bind TRAF3, thus blocking IRF-3 phosphorylation and inhibiting RIG-I/TBK1-directed IFN-β transcription (Alff et al., 2008, Matthys et al., 2011, Matthys et al., 2014). Thus, the relationship between the IFN system and hantaviruses is complicated.
As a first line of defense, the skin possesses different types of cells and molecular mediators of innate and adaptive immunity (Nestle et al., 2009). Keratinocytes, comprising the majority of the epithelial layer of the skin, have been found to serve as a replication site for a number of vector-borne viruses, including West Nile virus (WNV) (Hoover and Fredericksen, 2014, Lim et al., 2011), dengue virus (DENV) (Bustos-Arriaga et al., 2011, Surasombatpattana et al., 2011) and Chikungunya virus (CHIKV) (Puiprom et al., 2013), which were previously thought to be transmitted by direct infestation into subcutaneous veins via ticks, mosquitos, mites and other arthropod vectors. Although members of the Hantavirus genus have not been proven to be transmitted by vectors, viruses from other genera of Bunyaviridae have preferential relationships with the arthropods of only one or two families, such as Bunyavirus with mosquitoes (Culicidae), Uukuvirus and Nairovirus with ticks (Ixodidae and Argasidae), and Phlebovirus with sand flies (Psychodidae) and mosquitoes (Labuda, 1991). Furthermore, hantavirus-specific RNA was detectable in mites (Houck et al., 2001) but not in ticks (Wojcik-Fatla et al., 2011, Wojcik-Fatla et al., 2013). Hantavirus can infect many different cell types, such as endothelial cells, epithelial cells, dendritic cells (DC), mast cells and monocytes (Manigold and Vial, 2014). As keratinocytes may play important roles in dermal viral transmission, we addressed whether keratinocytes, which can serve either as a replication site for viruses or in the recruitment of macrophages and DCs to the site of infection, are able to support hantavirus replication. As normal immortalized keratinocytes, HaCaT cells are widely used in research associated with keratinocytes (Aksoy et al., 2014, Maas-Szabowski et al., 2001, Puiprom et al., 2013). In this study, we first demonstrated that HTNV is capable of infecting the HaCaT keratinocyte cell line. We then explored the innate immune responses against HTNV in these cells and found that HTNV can induce the secretion of high levels of interferon (IFN)-β and the nuclear translocation of IRF-3. The levels of a large number of ISGs, including Mx1, OAS1 and IFIT2, were found to be correlated with the level of IFN-β. Moreover, the nuclear translocation of STAT1 was also detected in HTNV-infected HaCaT cells.
Section snippets
Cells and viruses
HaCaT cells were purchased from the China Center for Type Culture Collection (CCTCC) at Wuhan and cultured in DMEM/F-12 medium (HyClone, Logan City, UT, USA) supplemented with 10% fetal bovine serum (HyClone), 100 units/ml penicillin and 100 μg/ml streptomycin at 37 °C with 5% CO2. The HTNV 76-118 strain was stored in our lab, and the HTNV virus stock was prepared by infecting monolayers of Vero E6 cells in 75-cm2 flasks at a dose (0.5 ml) of approximately 5 × 106 LD50 of HTNV. At three days
HaCaT cells are susceptible to HTNV infection
Due to a paucity of historical data regarding whether the human keratinocyte cell line HaCaT is permissive to hantavirus, HaCaT cells were challenged with Hantaan virus (HTNV) strain 76-118 at an MOI of 1. Subsequently, the amount of intracellular viral nucleoprotein (NP) was assessed by western blotting at different time points post-infection. As shown in Fig. 1A, HTNV NP was detected in HaCaT cells challenged with virus at 6 h and 24 post-infection (hpi), whereas it was not detected in
Discussion
Endothelial cells are considered a primary site of viral replication and the major target of hantavirus infection in vivo. However, hantaviruses can also infect monocytes, macrophages, mast cells, dendritic cells (DCs), epithelial cells and vascular smooth muscle cells (vSMC), which demonstrates their remarkably broad cell tropism (Guhl et al., 2010, Markotic et al., 2007, Marsac et al., 2011, Raftery et al., 2002, Temonen et al., 1995, Zaki et al., 1995). As HaCaT cells are a widely used
Author Contributions
Conceived and designed the study: W.Y., Y.L., and F.Z. Performed the study: W.Y., Y.X., Y.W., Y.D., Q.X. and M.C. Analyzed the data: W.Y. Contributed reagents/materials/analysis tools: L.Y., L.Z., L.C., X.W., and Z.X. Wrote the manuscript: W.Y. Edited the manuscript: Y.L. and F.Z.
Acknowledgments
We are grateful to Dr. Steve Goodbourn for providing the IFN-β promoter reporter plasmid, pIFΔ(−116) lucter, and to Dr. Wu Min for the pISRE-luc-cis reporter plasmid. This work was supported partially by grants from the Major State Basic Research Development Program of China (No. 2012CB518905), the National Major Infectious Diseases Prevention and Control Special Issues (2013ZX10004609-002), and the National Natural Science Foundation General Program of China (No. 30970148, 30972590 and 31370193
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All authors contributed equally to this work.