Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus
© The Author(s). 2017
Received: 22 November 2016
Accepted: 24 March 2017
Published: 31 March 2017
Two lineages of Zika virus (ZIKV) have been classified according to the phylogenetic analysis: African and Asian lineages. It is unclear whether differences exist between the two strains in host cell permissiveness, this information is important for understanding viral pathogenesis and designing anti-viral strategies.
In the present study, we comparatively studied the permissive spectrum of human cells for both the African (MR766) and Asian strains (PRVABC59) using an RNA in situ hybridization (RISH) to visualize RNA replication, an immunofluorescence technology, and a western blot assay to determine viral protein production, and a real-time RT-PCR to examine viral RNA multiplication level. The experiments were undertaken in the condition of cell culture.
We identified several human cell lines, including fibroblast, epithelial cells, brain cells, stem cells, and blood cells that are susceptible for the infection of both Asian and African strains. We did not find any differences between the MR766 and the PRVABC59 in the permissiveness, infection rate, and replication modes. Inconsistent to a previous report (Hamel et al. JVI 89:8880–8896, 2015), using RISH or real-time RT-PCR, we found that human foreskin fibroblast cells were not permissive for ZIKV infection. Instead, human lung fibroblast cells (MRC-5) were fully permissive for ZIKV infection. Surprisingly, a direct interaction of ZIKV RNA with envelop (E) protein (a structure protein) was demonstrated by an RNA chromatin immunoprecipitation (ChIP) assay. Three binding sites were identified in the ZIKV RNA genome for the interaction with the E protein.
Our results imply that the E protein may be important for viral RNA replication, and provide not only the information of ZIKV permissiveness that guides the usage of human cells for the ZIKV studies, but also the insight into the viral RNA-E protein interaction that may be targeted for intervention by designing small molecule drugs.
KeywordsZika virus (ZIKV) RNA in situ hybridization (RISH) Permissiveness RNA Chromatin Immunoprecipitation (ChIP) RNA replication
Zika virus (ZIKV) has recently drawn worldwide attention due to the recent outbreaks that are temporally and spatially consistent with the increased occurrence of congenital microcephaly and Guillain–Barré syndrome (GBS) in the Americas. Recently, an increasing number of strains of ZIKV have been isolated from more than 60 countries since its first isolation in Uganda in 1947 [1, 2]. Phylogenetic studies classified ZIKV into Asian and African lineages [3, 4]. For unknown reasons, the Asian lineage ZIKV has been linked to recent epidemics and most likely the congenital microcephaly and GBS while African strains cause milder symptoms. The recent cases of microcephaly and GBS linked to ZIKV are mostly, if not all, caused by the strains of Asian lineage . However, more experimental evidence is needed to support the hypothesis that ZIKVs from the two lineages are different in viral replication, pathogenesis, transmission, and cell permissiveness.
The first ZIKV was isolated from a monkey, and it is known that ZIKV can be transmitted by being bitten by the infected Aedes species of mosquito [6, 7] or sexually between humans [8, 9]. The known primary hosts of ZIKV include human, monkey, and mosquito. During the evolution of ZIKV, the virus may have developed new molecular relationships with factors of the host cells. Only a few human cells are known to be permissive for ZIKV replication including an epithelial cell line (A549), neural stem cells , and a skin fibroblast cell line . It remains unknown whether other cell lines are permissive for the infection of ZIKV.
Little is known regarding the interaction of ZIKV proteins and RNA with the host or viral factors although the interactions may determine the fate and/or efficiency of infection, pathogenicity, transmission, and epidemic potential of the ZIKV. It therefore remains important to determine the spatial relationship between the viral proteins and RNA replication. Of equal importance is their temporal relationship, whether the viral RNA replication occurs before protein production.
Belonging to family Flaviviridae, ZIKV contains a positive single stranded RNA (ssRNA) genome with a size around 11 k nucleotides (nt). After infecting permissive cells, the ZIKV genome is translated into a precursor protein (a polyprotein) at a size of about 3424 amino acid (Aa). The precursor protein is then co- and post-translationally processed by viral and cellular proteases into 3 structural and 7 non-structural (NS) proteins. Viral replication has been demonstrated in mitochondria and endoplasmic reticulum (ER) . Viral RNA replication has not been characterized for ZIKV. In the present study, we determined the viral permissiveness in different human cell lines using anti-viral antibody, real-time RT-PCR, and RNA in situ hybridization (RISH), then we examined the interaction between the viral RNA and E protein by an RNA ChIP (Chromatin Immunoprecipitation) assay.
Cell lines, Tissue Culture, and Viruses
Cell Permissiveness of Zika Virus (ZIKV)
Host cell infected
Human fibroblast cells
HFF (ATCC® SCRC-1041™)
MRC-5 (ATCC® CCL-171™)
BJ (ATCC® CRL-2522™)
Human Epithelial cells
ARPE-19 (ATCC® CRL-2302™)
A549 (ATCC® CCL-185™)
HT1080 (ATCC® CCL-121™)
Hep-2 (ATCC® CCL-23™)
HEK 293T(ATCC® CRL-1573™)
Human Endothelial cells
SLK (AIDSRP, cat# 9402)
Human blood cells
CEM/CD4 T cell (AIDSRP, cat# 117)
THP-1 (AIDSRP, cat# 9949)
PBMC (ATCC® PCS-800-011™)
Other Human cells
U-251MG (SIGMA 09063001)
Neural Stem cell
SK-N-SH (ATCC® HTB-11™)
Vero (ATCC® CCL-81™)
COS7 (ATCC® CRL-1651™)
NIH3T3 (ATCC® CRL-1658™)
Anti-Giantin (ab24586) for visualizing Golgi body, anti-Cox IV (ab16056) for showing mitochondria, and anti-Calreticulin (ab196156) for examining endoplasmic reticulum (ER) were purchased from Abcam (Cambridge, MA). The anti-ZIKV envelope antibody was generated from the hybridoma cell line, D1-4G2–4-15 (ATCC® HB-112™) and anti-ZIKV serum was produced from ZIKV-infected mice in our laboratory.
Western blot assay
Viral and cellular proteins in the whole cell lysate (WCL) samples were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10 to 20 μg loaded in each lane using Novex NuPAGE SDS-PAGE Gel System purchased from ThermoFisher Scientific.), transferred to nitrocellulose membranes (Amersham Inc., Piscataway, NJ), and blocked with 5% nonfat milk for 60 min at room temperature. Membranes were incubated overnight at 4 °C with primary antibody followed by incubation with a horseradish peroxidase-coupled secondary antibody (Amersham Inc.) and detection with enhanced chemiluminescence (Pierce, Rockford, Ill.), according to standard methods. Membranes were stripped with stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.8), washed with 0.1% PBS-Tween 20, and used to detect additional proteins.
RNA isolation and real-time RT-PCR
Following instructions of the manufacturers, total RNA was isolated using Aurum™ Total RNA Mini Kit (Bio-Rad, Cat# 732–6820). To quantitatively examine the RNA level of ZIKV from the infected cells, real-time RT-PCR was undertaken using the SsoAdvanced™ Universal SYBR Green Supermix kit (Bio-Rad, Hercules, CA). The primers for ZIKV were: forward- 5′-AAATACACATACCAAAACAAAGTGGT-3′ and reverse- 5′-TCCACTCCCTCTCTGGTCTTG-3′; and the primers for beta-actin (as control) were: forward- 5′ -GGTTCCGATGCCCTGAGGCTC-3′ and reverse- 5′-ACTTGCGGTGCACGATGGAGG -3′. 0.5–2 μg of total RNA and 250 nM of sense and antisense primers (amplifying the RNA fragment in NS5 location) were used in a final 10 μl volume containing 100 ng of cDNA for all samples except for PBMC, which had 25 ng of cDNA. PCR reactions consisted of 40 cycles with the following optimal conditions: 95 °C for 30 s followed by a two-step PCR reaction of 95 °C for 15 s and 60 °C for 30 s. All samples were run in technical triplicates, and the data were collected and recorded by the CFX Manager software (Bio-Rad). The data was analyzed using 2ΔΔCq = (Cq,Target-Cq,Actin)Time X - (Cq,Target-Cq,Actin)Time 0 to obtain the fold change in expression via the 2ΔΔCq method Time 0 was used as the calibrator of the relative quantification of product generated in the exponential phase of the amplification curve for real-time RT-PCR. A melting temperature curve analysis was obtained by measuring (after the amplification cycles) the fluorescence during a period of warming from 65 to 95 °C.
RNA Chromatin immunoprecipitation (ChIP) assay
Primers for the ZIKV RNA ChIP assays
Immunocytochemistry and fluorescence in situ hybridization
Cells grown on coverslips, and immunostaining was performed after fixation with 1% paraformaldehyde (10 min at room temperature) and permeabilization in 0.2% Triton (20 min on ice) by sequential incubation with primary and Texas red (TR) -labeled secondary antibodies (Vector Laboratories, Burlingame, Calif.) for 30 min each (all solutions in PBS). For simultaneous detection of ZIKV RNA, cells were first immunostained for cellular or viral proteins and then treated for 1 h at 37 °C with RNase-free DNase I (Roche, Indianapolis, Ind.; 200 U/ml in PBS containing 25 mM MgCl2). After refixation in 4% paraformaldehyde (10 min at room temperature), samples were equilibrated in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), dehydrated in ethanol (70, 80, and 100% ethanol for 3 min each at −20 °C), air dried, and incubated overnight at 37 °C with the hybridization mixture. To detect RNA, only the probe DNA was denatured at 94 °C for 5 min. After hybridization, samples were washed at 37 °C with 55% formamide in 2× SSC (twice for 15 min each), 2× SSC (10 min), and 0.25× SSC (twice for 5 min each). Hybridized probes were labeled with FITC-avidin (Vector Laboratories; 1:500 in 4× SSC plus 0.5% BSA), and signals were amplified by using biotinylated anti-avidin (Vector Laboratories; 1:250), followed by another round of FITC-avidin staining. Finally, cells were equilibrated in PBS, stained for DNA with Hoechst 33,258 (0.5 μg/ml), and mounted in Fluoromount G (Fisher Scientific, Newark, Del.).
Probe preparation-Nick translation
The plasmid pZIKVMR766 containing the whole ZIKV cDNA was used to be labeled with biotin-11-dUTP by nick translation. The DNase concentration for nick translation was adjusted to yield probe DNA 200 to 500 bp in length. Probe DNA was dissolved at 10 ng/μl in Hybrisol VII (Oncor, Gaithersburg, Md.) containing 100 ng of salmon sperm DNA (Gibco-BRL), 1 μg of yeast tRNA (Sigma), and 0.5 mg of cot1 DNA (Gibco-BRL)/μl.
Cells were examined with a Leica TCS SPII confocal laser scanning system. Two or three channels were recorded simultaneously and/or sequentially and controlled for possible breakthrough between the fluorescein isothiocyanate and Texas Red signals and between the blue and red channels.
ZIKV MR766 and PRVABC59 have a similar permissiveness spectrum in human cell lines
Phylogenetic studies based on viral nucleotide and amino acid sequences classify Zika viruses into African and Asian lineages [3, 4, 15]. ZIKV MR766 was isolated from Uganda in 1947 and is the representative of African strains . PRVABC59 strain was isolated in 2015 from a Puerto Rican patient who was infected by a Brazilian strain . According to the phylogenetic studies, PRVABC59 stands for an Asian strain . We were curious about whether they have a different permissiveness spectrum in cell lines of human, monkey and mouse.
Several methods were employed to determine the permissiveness of ZIKV in different types of cell lines as listed in Table 1. First, we performed immunofluorescence assay (IFA) combined with RNA in situ hybridization (RISH). We infected cells with MR766 or PRVABC59 (PR) for 24 h at an MOI of 0.5. After fixation with 1% paraformaldehyde, the cells were permeabilized and immunostained for viral E protein with anti-E protein antibody in red. After refixation with 4% paraformaldehyde, the cells were hybridized with biotin-labeled DNA probe made by Nick translation  from a plasmid carrying the whole DNA derived from ZIKV genomic cDNA that was synthesized by the Genescript Inc. (Piscataway, NJ). The RNA was stained in green with FITC-avidin. The cell nuclei were shown in blue using DAPI staining. If the infection rate was greater than 70%, it is set as “++++” in both IFA (red) and RISH (green). The method for defining the infection rate was referenced to that used for cytomegalovirus infection  and described in the legend of Table 1.
First, as shown in Additional file 1: Figure S1, we infected Vero cells with MR766 strain or PRVABC59 strain at the same MOI (0.5) for 24 h. The cells were fixed for immunostaining with anti-E protein in red and RISH for RNA in green. After repeating the experiments independently for 3 times, as can be seen in the Additional file 1: Figure S1, there is no differences regarding the infection rate, RNA replication pattern and E protein production between the two strains.
Inconsistent with another report , we found that ZIKV had different replication patterns in lines of human fibroblast cells. To examine the permissiveness of ZIKV in human fibroblast cells, we infected a human foreskin fibroblast (BJ, ATCC) and an HFF used in our laboratory previously for cytomegalovirus infection, and MRC-5 (human lung fibroblast). MRC-5 (Fig. 1d) are permissive for ZIKV infection, particularly the MRC-5 showed a better infection rate for ZIKV than Vero cells. However, the HFF in our laboratory and the BJ cell line from ATCC were not permissive for viral infection, which is different from the report from Hamel et al. that HFF was found to be completely permissive . The permissiveness of ZIKV in human lung fibroblast cells (MRC-5) and in lung epithelial cell (A549) creates a new hypothesis that the ZIKV may infect and replicate in the human lung.
Mosquitos transmit ZIKV by direct contacting blood of the ZIKV carriers. We wondered whether ZIKV could grow in blood cells. We selected three kinds of blood source cells or blood cells (CEM/CD4 T cell, THP-1 monocyte, and PBMC). We found that PBMC is permissive for ZIKV infection (Fig. 1e), however the ZIKV cannot replicate in the T cell line and monocytes by the methods tested in our experiments. We, at this point, do not know which type of the blood cells are permissive for ZIKV infection. The observation that ZIKV infects PBMC is important because it suggests a mechanism for how ZIKV viremia is maintained. The maintenance of viral load in blood is important for ZIKV transmission by mosquitos .
The major medical problem caused by ZIKV infection is that it interferes with fetal development by causing microcephaly [19, 20]. ZIKV is infectious to the neural stem cells . Here, we found that SK-N-SH, a neuroblastoma cell line that displays epithelial morphology, is also highly permissive for ZIKV infection (Fig. 1g). The effects of ZIKV infection on the stem cell proliferation has been studied for neural stem cells [10, 12, 21, 22], but it has not been reported from other type of cells in brain. We tested the permissiveness of ZIKV on a glioblastoma cell line (U-251MG) which is from a brain tumor. We found that U-251MG is highly permissive for ZIKV infection (Fig. 1f). The viral E protein and RNA production were shown to be comparable to these in Vero cells.
In addition, we verified that Cos7 cell line is permissive for ZIKV infection (Fig. 1h). Two cell lines from mouse (NIH3T3 and MEF-mouse embryo fibroblast) did not support ZIKV infection.
RNA replication or viral protein production was further demonstrated in permissive cells by real-time RT-PCRs or western blots
Viral protein is detected ahead of viral replicative RNA by IFA and RISH
Generally, at the early time of viral infection, some viral proteins need to interact with cellular proteins to form a restricted compartment that is utilized by the the virus for viral DNA/RNA replication. For most RNA viruses, RNA replication occurs in the cytoplasm . If proteins of ZIKV are important for viral RNA replication and forms a pre-replication compartment, the viral proteins should be produced before RNA replication occurs. To demonstrate the presumption, we performed an IFA to examine viral E protein production and a RISH to examine viral RNA replication with a time course of infection in Vero cells.
Viral E protein directly interacts with viral RNA
After infected with ZIKV MR766 for 24 h at an MOI of 0.5, the Vero cells were fixed and sonicated to shear the viral genome to a range of 250–500 nt. The RNA-protein complexes were then pulled down by anti-ZIKV antibody-conjugated beads. After careful washing, the beads were then eluted, and the eluate (containing RNA, DNA, and proteins) was treated with proteinase K and DNase I (RNase free). The RNA was finally precipitated and used for RT-PCR. We performed the end point RT-PCR (Fig. 5b) and real-time RT-PCR (Fig. 5c). As can be seen in Fig. 5b, the PCR products showed a strong band in the 2nd, 6th and 11th lanes. The 2nd (nt 135–294) and 6th (nt 734–899) lanes respond the nucleotides within the first 1051 nt of viral genome, while the 11th (nt 10,474–10,644) lane reflects the nucleotides in the 3′ UTR of viral genome. The real-time RT-PCR shown in Fig. 5c is consistent with the results of the regular RT-PCR: the 3 peaks are correspondent to the 3 stronger bands seen in the Fig. 5b. Therefore, our RNA ChIP assay results demonstrated that the viral E protein interacts with viral replicative RNA. At least three E protein-binding sites were identified in the ZIKV MR766 RNA genome.
ZIKV RNA co-localizes with ER, and is probably associated with Golgi apparatus and mitochondria
During the evolution of the ZIKV, it appears that the ZIKV underwent a pathogenic differentiation: Asian lineage ZIKV has been linked to recent epidemics and high likely the congenital microcephaly and GBS while African strains cause milder symptoms. However, how the ZIKV infection results in the neural diseases is not fully understood. The knowledge of the molecular biological interactions of the ZIKV with host are lacking, although it is critical to develop anti-ZIKV strategies. Although it is believed that ZIKV infection can be transmitted via Aedes species mosquito biting and/or sexually, other transmission route may exist . Viral transmission is related to cell permissiveness for infection. Here, we performed comparative studies of cell permissiveness for ZIKV African strain (MR766) and Asian strain (PRVABC59). Both strains infect a wide range of human cell types. No significant difference was found in terms of RNA replication and viral protein production in those cells between the two strains. Further studies are needed to figure out whether they have differences in viral RNA replication and infection at in vivo level.
Viral permissiveness may relate viral replication to viral transmission and spreading. For example, one of the spreading routes of ZIKV is by Aedes mosquito biting [6, 7]. The most substance the mosquito obtains from the ZIKV-carrying host is blood, which is also the source of mosquito infection. We tested endothelial cell for the infection of ZIKV and found that the endothelial cells are not permissive for ZIKV infection. Importantly, our IFA, RISH, and real-time RT-PCR experiments demonstrated that ZIKV productively infects the PBMC (Figs. 1 and 2, Table 1). Although we do not know yet which type of blood cells are permissive for ZIKV infection, our finding that ZIKV infects PBMC suggests that PBMC is the source of blood virus and is important for maintenance of viral level in the blood.
It has been reported that a great number of viral particles of ZIKV were detected in brain tissue and fluid . It has been reported that ZIKV productively infects neural stem cell [10, 12, 21, 22]. Another important cell line that supports ZIKV infection is U-251MG (Figs. 1 and 2, Table 1). U-251MG is a glioblastoma cell line and derived from brain. Our results showed that ZIKV replicates in U-251MG productively, which may suggest another factor that enhances the ZIKV pathogenesis in brain. Further studies using a mouse model will be necessary to confirm our hypothesis that ZIKV infects different types of cells in brain tissue to affect the development of the fetal brain.
ZIKV is a member of the family Flaviviridae. It contains a positive and single stranded RNA genome. Viral protein can be produced immediately after viral entry, which is consistent with our finding that viral protein was detected before viral RNA replication can be seen (Fig. 4). Flaviviral genome replication requires three steps: 1) negative strand RNA synthesis, 2) positive strand RNA synthesis, and 3) 5′-RNA capping and methylation. Before viral RNA replication, a pre-replication complex needs to be formed [26, 27]. The pre-replication complex contains both viral and cellular proteins. Some of these proteins may be able to bind to viral RNA. Our RISH and IFA results showed that the viral replicative RNA localizes in the cytoplasm as a domain that overlaps with viral E protein, forming the viral RNA replication domain (Fig. 1). In this study, RNA ChIP assays demonstrated that viral proteins directly interact with viral RNA (Fig. 5). At least 3 binding sites have been identified in the viral genome for the viral E protein binding (Fig. 5). This results is surprising because E protein is a structural protein of ZIKV and functions as an initiator of viral infection through interacting with cell receptor. However, we found that E protein presents continuously in the viral replication compartments. The finding that E protein interacted with viral RNA implies that E protein may play roles in viral replication. This is important because the protein-RNA interaction may be an effective target for designing the intervention drugs against viral RNA replication.
Lastly, we revealed that the viral replication occurs in ER (Fig. 6C1–C3), which is consistent with the report for other flaviviral RNA replication [26, 27]. Although the RNA replication compartment is not colocalizing with mitochondria or Golgi apparatus, the matrix protein of mitochondria is shown surrounding the RNA domain as shown by the arrow in the Fig. 6A1-A3. A similar observation was also shown for Golgi apparatus in the Fig. 6B1-B3. This information suggests that the cellular organelles may play different roles in ZIKV viral replication.
In summary, we employed different techniques to determine the ZIKV infection permissiveness in human cells, the two strains have a similar permissiveness in human cells. More experiments are needed to reveal the differences between Asian and African strains because it is important to know why only the strains derived from Asian linage cause microcephaly. We revealed that ZIKV can replicate to a high titer in human lung fibroblast cells, which imply that ZIKV can replicate in the lung. PBMC is permissive for ZIKV replication, which is important because the information may explain why the mosquito is the transmitter for ZIKV. We also discovered that viral protein directly interacts with viral genome via binding to different sites of the viral genome. We will test whether the protein-RNA interaction can be a target for designing drugs to intervene the viral replication and infection.
Our results that E protein of ZIKV interacts with viral RNA imply that the E protein may be important for viral RNA replication. Our results provide not only the information of ZIKV permissiveness that guides the usage of human cells for the ZIKV studies, but also the insight into the viral RNA-E protein interaction that may be targeted for intervention by designing small molecule drugs. Our future studies will be focused on how the ZIKV infection permissiveness is determined by virus-host interactions.
- NS proteins:
Peripheral blood mononuclear cell
RNA in situ hybridization
single stranded RNA
Whole cell lysate
We acknowledge the instrument support of the PSM Molecular Biology Core Laboratory. This study was supported by an American Cancer Society grant (RSG-090289-01-MPC) (Q.T.), NIH/NIAID SC1AI112785 (Q.T.), National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number G12MD007597 (Q.T.), and NIH/NCI grant P20CA192989 (X.P.).
An American Cancer Society grant (RSG-090289-01-MPC) (Q.T.), NIH/NIAID SC1AI112785 (Q.T.), National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number G12MD007597 (Q.T.), and NIH/NCI grant P20CA192989 (X.P.).
Availability of data and materials
The dataset and materials supporting the conclusions of this article can be made freely available to any scientist wishing to use them for non-commercial purposes.
WH and QT wrote the article. WH, NA and QT generated the figures. WH performed the IFA and ChIP assay, NA performed the real-time RT-PCR, LAO performed the western blot. XP tested the viral titer. MT and KSJ tested the cell state. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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