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Human metapneumovirus uses endocytosis pathway for host cell entry
Molecular and Cellular Probes xxx (2016) 1e7
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Original research article
Human metapneumovirus uses endocytosis pathway for host cell entry Hui Yang, Hao He, Bin Tan, Enmei Liu, Xiaodong Zhao*, Yao Zhao** Chongqing Key Laboratory of Child Infection and Immunity, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children’s Hospital of Chongqing Medical University, Chongqing, 400014, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 March 2016 Received in revised form 15 June 2016 Accepted 15 June 2016 Available online xxx
Human metapneumovirus (hMPV) is a prevalent pathogen worldwide and causes various respiratory infections. Although it is a critical pathogen in pediatric patients, it is unclear how it enters host cells. In this study, we focused on hMPV cell entry using two kinds of cell lines (Vero E6 and LLC-MK2), which are most commonly used for isolating and propagating for hMPV, and we used fluorescent dyes to label the virus particles and monitored how they enter the host cell in real time. We found that endocytosis was the predominant pathway by which hMPV entered host cells. When the virus particles were traced inside host cells, we found that a low intracellular pH was needed for intracellular fusion in LLC-MK2 cells. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Human metapneumovirus Cell entry Endocytosis Low pH
1. Introduction Since 2001, when human metapneumovirus (hMPV) was first identified in Holland [1], many research groups have found evidence of hMPV infection worldwide [2e5]. Indeed, hMPV is considered a major pathogen responsible for lower respiratory tract disease in infants and children [6,7]. It also causes acute wheezing in young children [8] and can exacerbate asthma [9]. The importance of hMPV is not related to its prevalence, but rather to the severity of resultant disease. However, how this virus infects cells is still entirely unclear. There are two primary routes by which enveloped viruses can enter host cells: membrane fusion and endocytosis. The endocytosis can occur via four major kinds of pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, or clathrin/caveolae-independent endocytosis. Most enveloped viruses are believed to fuse their viral membranes and cells membranes to achieve effective infection [10]. Srinivasakumar et al. [11]
* Corresponding author. Present Address. No. 136, Zhongshan 2nd Road, Yuzhong District, Chongqing, 400014, China. ** Corresponding author. Present Address. No. 136, Zhongshan 2nd Road, Yuzhong District, Chongqing, 400014, China. E-mail addresses: [email protected] (X. Zhao), [email protected] (Y. Zhao).
showed that ~ 35% of Respiratory Syncytial Virus (RSV) viral particles had fused to the plasma membrane 1 h following infection. Although the fusion mechanism is recognised by many researchers, various endocytic pathways have now been identified. For example, one study showed that RSV enters HeLa cells via macropinocytosis [12], whereas other studies showed that it infects HeLa cells via clathrin-mediated endocytosis [13]. Moreover, another investigation showed that dendritic cells present RSV antigen by caveolaedependent endocytosis [14]. It seems that RSV mediates different entry pathways at different infection times to enter the same host cells, or RSV via different ways to infect different cells. hMPV, like RSV, represents the subfamily Pneumovirinae within the family Paramyxoviridae and these viruses cause similar clinical symptoms. So, we speculated that hMPV could choose different avenues to enter different cells, depending on infection conditions, although until now, membrane fusion was regarded to be the major pathway of hMPV cell entry [15,16]. Recently, Cox et al. [17] found that hMPV enters human bronchial epithelial cells via clathrin-mediated endocytosis in a dynamin-dependent manner. This information suggests that hMPV might exploit an endocytosis pathway in different host cells. Here, we selected Vero E6 and LLC-MK2 as target cells, not only because they are the most common used cells for propagating hMPV, but also owing to their higher efficiency of hMPV infection than other airway epithelial cells [18,19], and used a series of
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fluorescent dyes to label hMPV particles. We have followed the hMPV cell entry process with a confocal laser-scanning microscope. The results indicated that hMPV entered cells primarily via an endocytic pathway. Moreover, a low intracellular pH played a key role in hMPV entry. 2. Materials and methods 2.1. Cell lines Vero E6 and LLC-MK2 cells were purchased from the China Center for Type Culture Collection and maintained in a humid 5% CO2 incubator at 37 C in Dulbecco’s Modified Eagle Medium (DMEM), (Gibco, NY, USA) or Minimum Essential Medium (MEM), (Gibco, NY, USA), respectively, supplemented with 10% fetal bovine serum (FBS), (Gibco, NY, USA) and antibiotics (penicillin 100 IU/ml; streptomycin 100 mg/ml); (Hyclone, UT, USA). 2.2. Virus stocks hMPV was successfully recovered from full-length cDNA clones of hMPV NL/1/00 by reverse genetics as described previously [20]; hMPV was propagated in Vero E6 cells, and hMPV virions were purified by ultracentrifugation on a modified 35% glucose gradient. The viral supernatant was harvested and gently layered on to the surface of a 35% glucose-HBSS solution (Hyclone, UT, USA). Then, they were centrifuged at 51,500 g for 16 h at 4 C (Beckman Coulter, FL, USA). Finally, the supernatant was discarded and the virus particles were suspended in medium and maintained as stocks at 80 C. 2.3. Dyes and antibodies Octadecyl Rhodamine B Chloride (R18) was obtained from Biotium Inc, USA. DiOC was obtained from Beyotime Biotechnology, China. Cy3 and CypHer5 NHS ester were obtained from GE Healthcare, UK. The monoclonal mouse anti-hMPV fusion protein (F) and monoclonal mouse anti-hMPV nuclear protein (N) antibodies were purchased from Merck Millipore, Germany. The Alexa Fluor® 488/546 donkey anti-mouse IgG antibodies were purchased from Life Technologies, USA. 2.4. Indirect fluorescence assay hMPV was inoculated on to the cells (MOI ¼ 1). First, the cells were placed at 4 C for 30 min to synchronize host cell entry. Unbound virus was washed away and the temperature raised to 37 C to initialize the entry process. After incubation at 37 C for different times, cells were fixed and permeated by exposure to 0.1% Triton X100 (Sigma-Aldrich). Albumin bovine V (1%; BSA, Sigma-Aldrich) was used as a blocking agent. The cells were then incubated with anti-hMPV F and N antibodies, followed by fluorescently conjugated anti-IgG antibodies. F-actin was stained by incubating cells with phalloidin. Finally, cell nuclei were labeled by DAPI (Beyotime). Fluorescence was observed under a confocal microscope (Nikon, Tokyo, Japan). 2.5. R18 fusion assay R18 (1 ml) was added into 1 ml of hMPV (pfu ¼ 106/ml) to yield a final R18 concentration of 10 mM. The mixture was shaken gently for 1 h to allow R18 binding. Impurities were removed using a 0.22 mm filter, and unbound dye was removed using CaptoCore 700 (GE Healthcare). R18 alone was also added to medium as a negative control. Both R18-hMPV and R18-control were inoculated on to
Vero E6 and LLC-MK2 cells (MOI ¼ 1) for 30 min at 4 C, and then replaced with fresh medium. Once the cells were placed under the confocal microscope, the temperature was raised to 37 C, and the fluorescence change was recorded for 4 h. Variations in fluorescence intensity were also measured every 2 min in a microplate reader. The amount of R18 fluorescence dequenching was expressed as the DQ%, calculated using the formula: DQ%¼(Fd-F0)/ Ft*100%, where F0 is the fluorescence intensity at the beginning, Fd is the fluorescence intensity at each detection time, and Ft is the maximum fluorescence intensity when R18 was infinitely diluted with 1% Triton X-100 [21]. 2.6. DiOC/R18 FRET A mixture of DiOC/R18 was added to 1 ml of hMPV. The final concentration of DiOC was 3.3 mM and that of R18 was 6.7 mM. Gentle shaking was performed to facilitate virus staining and unincorporated dyes were removed in the same way as for the R18 fusion assay. The negative control was treated in the same way. FRET was observed under a confocal microscope. DiOC/R18 labeled hMPV was excited with a laser (488 nm), and fluorescence was detected simultaneously at 500 nm and 575 nm. 2.7. Cy3/CypHer5 dual labeling Before labeling, hMPV was treated with 0.5 M Na2CO3 to increase the pH of the solution to 9.3. Cy3 and CypHer5 were then added to 1 ml hMPV; the final concentration of each was 10 mg/ml. The mixture was then shaken and filtered as described for previous experiments. Dual-labeled virus was then inoculated on to cells at 37 C and excited with a laser at 550 nm. The fluorescence emitted by the two dyes was detected at 575 nm and 660 nm. 3. Results 3.1. Fusion protein (F) and nucleoprotein (N) co-localize during hMPV entry Cells were infected with hMPV for different times and then stained with anti-hMPV F and anti-hMPV N antibodies to identify the viral membrane and the nuclear capsid, respectively. At the same time, F-actin was labeled with Alexa Fluor® 647 phalloidin to reveal the cytoskeleton. Since F and N are located on the virus membrane and capsid, respectively, staining by both F and N antibodies indicated intact particles within infected cells. Serial confocal Z-stack images revealed that fluorescent spots of F and N co-localized on the cell surface and in the cytoplasm at 2 h posthMPV infection (Fig. 1A and B). Vero E6 cells without infection were used as a negative control (Fig. 1C). Krzyzaniak et al. [12] defined capsid-free envelopes (or virus-like particles; VLPs) as particles with F spots but without N spots. Here, we found that the number of VLPs (F only) (Fig. 1D) and intact viral particles (FN both) increased 2 h after infection. We believe that the N spots represent the total number of viral particles (N only), whereas the FN spot represents the number of endocytosed viral particles. Thus, 2 h after infection, both the total number of particles and the number of endocytosed particles increased (Fig. 1E, F). The ratio of FN to N spots in LLC-MK2 cells increased from 0.58 to 0.84 (Fig. 1G, right panel), and that in Vero E6 cells varied from 0.65 to 0.70 (Fig. 1G, left panel) during 2 h of incubation. A possible explanation for this finding is that more hMPV particles were endocytosed by LLC-MK2 during the 37 C incubation period. In other words, whether incubation at 37 C is optimal for endocytosis depends on the host cell type. However, in both groups, about two-thirds of hMPV particles entered via the endocytosis pathway.
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Fig. 1. The F and N proteins colocalize in host cells. Vero E6 (A) and LLC-MK2 cells (B) were incubated with hMPV and fixed. The control group (C) (Vero E6) was incubated with medium instead. Cells were stained with both anti-F-AF488 (green) and anti-N-AF546 (red) antibodies, and phalloidin-AF647 (purple) and DAPI (blue). Z-stack images were taken (bottom and right of A and B). The fluorescence spots of anti-F only (D), anti-N only (E) and both anti-F and anti-N colocalization (F) were analyzed using NIS elements software Ver. 4.00 (Nikon, Tokyo, Japan). (G) Fluorescence spots of FN colocalization and N spots ratio was calculated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.2. Membrane fusion occurs during hMPV entry A lipophilic dye (octadecyl rhodamine B; R18) was used to confirm whether membrane fusion occurred during hMPV entry. R18 has a specific property in that its fluorescence is quenched after incorporation into the viral membrane. Once the R18-labeled virus
fuses with the unlabeled membrane, the dye is diluted and the fluorescence dequenches [21]. This feature makes R18 a useful fusion probe. In this assay, the fluorescence counts and intensity of R18 (red) increased over time in both cell lines (Fig. 2A and Movies S1 and S2). These findings indicate that membrane fusion events occur during the hMPV cell entry process. Measuring fluorescence
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Fig. 2. Membrane fusion events occur during infection. (A): Vero E6 (middle) and LLC-MK2 (bottom) cells were incubated with R18-hMPV and images were captured in real time. R18-control was added to Vero E6 cells as a control. (B): Fluorescence intensity of R18 at different times was analyzed. (C): Cells were cultured with R18-hMPV in a 96-well plate at 37 C, and the fluorescence intensity of R18 was measured with a microplate reader every 2 min. After 4 h, an equal volume of 2% Triton-X 100 was added to achieve an infinite dilution of the R18 dye. DQ% of R18 was calculated.
over time in a microplate reader yielded the same results. The fluorescence intensity increased to a maximum of 27.34% and 88.24% in Vero E6 and LLC-MK2 cells, respectively. However, there was a small difference between the two cell lines. In Vero E6 cells, the intensity of R18 increased, peaking around 60 min after infection, before declining again. However, in LLC-MK2 cells, the intensity of R18 fluorescence continued to increase and reached a plateau at 140 min during the 4 h incubation period (Fig. 2B). In both cell lines, the DQ% increased rapidly at first and then decreased slowly. The maximum DQ% for Vero E6 and LLC-MK2 cells was 30.1% and 32.0%, respectively. These peak values were reached at 90 min and 110 min after infection, respectively (Fig. 2C). These results showed that about one-third of hMPV particles fused with other biomembrane during infection. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.mcp.2016.06.003. 3.3. Intracellular fusion occurs after endocytosis In this experiment, two fluorescent dyes (R18 and DiOC) were introduced into cells. R18 and DiOC are used to examine intracellular membrane fusion [22]. When hMPV membrane was labeled with the two lipophilic dyes, both were located in close proximity on the viral membrane; therefore, fluorescent resonance energy transfer (FRET) occurred. This means that, when the labeled virus was excited by a laser, the signal generated by DiOC was quenched by R18. Thus, only red fluorescence emitted by R18 was observed. However, when the labeled virus fused with unlabeled biological membranes, the two dyes separated. FRET reduced, and DiOC (green fluorescence) was detected, which indicated that the fusion occured inside the cell. The results showed that, at the beginning of the incubation period, only a few red spots were detected in both Vero E6 and LLC-MK2. In addition, the numbers of intracellular green or yellow spots increased over time (Fig. 3A and Movies S3 and S4). As shown for the single laser channel, no green spots were visible until 30 min in LLC-MK2; thereafter, green spots gradually began to appear, but green spots were not visible in Vero E6 cells until 1 h after infection (Fig. 3A, middle and bottom panels). This
information implies that intracellular fusion occurred (i.e., DiOC was no longer quenched by R18), and that these fusion events occurred earlier in LLC-MK2 than in Vero E6. Analysis with NIS Elements software revealed that the intensity of R18 and DiOC in Vero E6 cells increased by ~141.1% and 141.7% respectively, after 2 h of incubation, and by ~292.7% and 395.1% respectively, in LLC-MK2 cells (Fig. 3B and C). (Fig. 3B and C). Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.mcp.2016.06.003. 3.4. hMPV entry is associated with a decrease in intracellular pH Since extracellular pH plays an important role in paramyxovirus infection, we attempted to establish whether the pH in endocytic vacuoles changed after hMPV infection. To do this, we labeled hMPV with two dyes: pH-insensitive Cy3 and pH-sensitive CypHer5. The ratio of the fluorescence emissions of them is an indicator of pH [23]. In LLC-MK2 cells, green fluorescence increased during 2 h following infection (Fig. 4A, Movie S5). The fluorescence intensity of Cy3 increased slowly throughout the 2 h period, whereas that of CypHer5 increased, peaking at ~80 min, before declining sharply. The increase level in CypHer5 fluorescence was much greater than that of Cy3. The fluorescence intensity of CypHer5 increased by 60.00% at 80 min; the increase for Cy3 at the same time point was 12.9% (Fig. 4B). Since CypHer5 is pH-sensitive, the ratio of the two dyes was expressed as the G/R ratio. The G/R ratio increased from 0.38 to 0.56, before declining to 0.31 (Fig. 4C). Changes in the G/R ratio mirrored changes in CypHer5 fluorescence. These results suggest that hMPV requires a low pH environment in the cytoplasm. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.mcp.2016.06.003. 4. Discussion Productive viral infection occurs only after infectious virus particles enter susceptible cells. At present, there are two major pathways by which viruses enter host cells, i.e. via membrane
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Fig. 3. hMPV undergoes intracellular fusion. (A): Target host cells were inoculated with the DiOC/R18 dual-labeled hMPV. Real-time images were captured. Vero E6 without hMPV were exposed to DIOC/R18 mixture as a control (top). Pictures of individual fluorescence channels of Vero E6 (middle) and LLC-MK2 cells (bottom) are shown. (B), (C): Changes in the fluorescence intensity of R18 (red) and DIOC (green) were measured in the two cell lines at different time points. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
fusion and endocytosis. Research results achieved using modern techniques reveal that a single virus can utilize both mechanisms simultaneously [12e14]. We speculate that hMPV mediates primarily endocytosis to achieve maximal infection, but which pathway that hMPV uses might depend on the host cell type. Classical virology suggests that paramyxovirus enters cells via membrane fusion. Although hMPV is very similar to RSV, they also have different characteristics; hMPV replicates more slowly than RSV [18]. Unlike syncytium formation by RSV, CPE caused by hMPV are atypical, including formation of granular, small, rounded and refringent cells [24]. The particularities associated with hMPV suggest that it may enter host cells via a specific mechanism. The present findings suggest the endocytosis pathway plays a major role in hMPV infection. The enveloped virus comprises lipid membrane and nucleocapsid. Where the viral envelope fuses is the key factor to differentiate membrane and endocytosis. During membrane fusion, the viral membrane fuses with the plasma membrane before releasing its nucleocapsid into the cytoplasm [10]. Therefore, the viral membrane components remain at the cell surface after entry. When a virus enters via endocytic pathway, the entire intact particle enters the host cell; thus, the components of the viral membrane are present within the cell. We found that the hMPV viral membrane was still detectable in cells after the virus particle was endocytosed, since the F protein was observed intracellularly after entry. In the Z-stack images, both the anti-hMPV F and anti-hMPV N
antibodies co-localized deep inside the same cell (Fig. 1A, B), confirming that the viral membrane and its nuclear material were within the cell. According to the IFA, 70.1% and 83.9% of hMPV particles were present in Vero E6 and LLC-MK2, respectively, as intact virions at 2 h after infection (Fig. 1G). However, about 20%e 30% of hMPV was present in the form of N protein only, suggesting that some virions entered the cell via membrane fusion. In order to establish which pathway is the major one for hMPV entry, we used R18 and DiOC to detect the key events. We found that upon incubation of R18-hMPV with both Vero E6 and LLCMK2, the fluorescence intensity of R18 increased during the first hour (Fig. 2B). After 1 h, the fluorescence intensity declined in Vero E6 cells, but still increased and reached a plateau in LLC-MK2. A possible interpretation here is that the virus does not enter these cell lines by exactly the same mechanism. The data suggest that fusion events occur much more during infection of LLC-MK2 cells. Dequenching also increased in both cell lines; indeed, the DQ% was similar in both (Fig. 2C). This information suggests that approximately one-third of the virus particles fused during infection. The pathway by which hMPV enters depends on where the fusion occurs. If fusion is within a cell, then hMPV is endocytosed. Results (Fig. 2A and Movie S1, S2) indicated that most hMPVs were endocytosed in Vero E6 and LLC-MK2. DiOC/R18 duallabelling [22] revealed that the intensity of R18 and DiOC in both Vero E6 and LLC-MK2 (Fig. 3B and C). Variation in the fluorescence intensity of individual spots suggested that intracellular fusion occurred
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Fig. 4. hMPV entry is associated with a decrease in intracellular pH. (A): Cy3/CypHer5-hMPV was added to LLC-MK2 cells. Images were captured in real time. Photos of per channel are shown. (B): Fluorescence intensities of Cy3 (red) and CypHer5 (green) were measured at different time points following infection. (C): The ratio of the two fluorescence intensities was calculated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
intracellularly, i.e., virus-endosome fusion, endosome-endosome fusion, or endosome-lysosome fusion. At the same time, these results indicated that LLC-MK2 was more suitable for hMPV infection compared with Vero E6. Since viruses are parasitic microorganisms, the pH of the environment plays an important role in determining the route of infection. For example, Newcastle Disease Virus enters HeLa and ELL-0 cells via low pH-dependent endocytosis [25]. The role of pH in hMPV entry is controversial. Schowalter et al. [26] found that trypsin treatment combined with low pH promoted syncytial formation by the hMPV F protein in BHK cells; however, Herfst et al. [27] suggested that low-pH-induced fusion is a rare phenomenon, since only a few hMPV strains harbouring a specific amino acid residue in the F protein require low pH for efficient membrane fusion. Some researchers report intracellular pH changes during viral entry and transport. Lakadamyali et al. [23] revealed that influenza virus showed a three-stage transport pattern when infecting CHO cells. The present study also showed that low pH is beneficial for hMPV infection. According to the present data, the fluorescence counts and the intensity of the two tracker dyes increased during the 2 h observation period (Fig. 4B). The fluorescence counts for CypHer5 and Cy3 were 48-fold and 3.5-fold higher, respectively, than at the beginning of the experiment (data not shown). Despite differences between the properties of the two dyes, the ratio of the fluorescence (G/R ratio) that they emitted also increased after viral infection (Fig. 4C). In accordance with the fluorescence intensity, the G/R ratio peaked at around 80 min. These findings suggested that intracellular pH declined first and
then recovered. At the same time, it meant that hMPV was exposed to acidic pH at least during 80 min. These results are approximately concordant with those of the DiOC/R18 fusion assay, because DiOC/ R18 FRET significantly increased during this period, and suggested that acidification of endocytic vesicles occurred during intracellular fusion. Therefore, we conclude that decreases in intracellular pH trigger fusion between the viral and endosomal membranes. A low pH may help to release the viral nuclear material. It is notable that changes in Cy3/CypHer5 fluorescence were not so clear in Vero E6 cells, which might relate to different cell types showing different sensitivities to pH. Therefore, pH-dependency not only relates to virus strains, but also to the cell type that they infect. In conclusion, we established that hMPV can use an endocytosis pathway to enter host cells (Vero E6 and LLC-MK2). We showed that a low pH is required for hMPV/endosome fusion in one of the two host cells. The present findings reveal that Paramyxoviridae (such as hMPV) may require acidification of the intracellular milieu to facilitate viral/membrane fusion with other organelles. The next plan is to investigate precisely which endocytosis pathway hMPV uses for cell entry. Acknowledgements This research was supported by Natural Science Foundation Project of CQ CSTC (NO. cstc2012jjA10091, cstc2013jjB10029), National Natural Science Foundation of China (NO. 30800972, NO. 81371876), Research Fund for the Doctoral Program of Higher Education of China (NO. 20125503110002), Outstanding Youth Fund
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of Children’s Hospital of Chongqing Medical University and Chongqing Municipal Colleges and Universities Outstanding Talent Support Program (2014-47-1-11). We are grateful to Professor Wenxia Song (University of Maryland, USA) and Chaohong Liu for their helpful advice. References [1] B.G. van den Hoogen, J.C. de Jong, J. Groen, T. Kuiken, R. de Groot, R.A. Fouchier, A.D. Osterhaus, A newly discovered human pneumovirus isolated from young children with respiratory tract disease, Nat. Med. 7 (6) (2001) 719e724. [2] T. Ebihara, R. Endo, H. Kikuta, N. Ishiguro, H. Ishiko, M. Hara, Y. Takahashi, K. Kobayashi, Human metapneumovirus infection in Japanese children, J. Clin. Microbiol. 42 (1) (2004) 126e132. [3] N. Bastien, D. Ward, P. Van Caeseele, K. Brandt, S.H. Lee, G. McNabb, B. Klisko, E. Chan, Y. Li, Human metapneumovirus infection in the Canadian population, J. Clin. Microbiol. 41 (10) (2003) 4642e4646. [4] H. Dollner, K. Risnes, A. Radtke, S.A. Nordbo, Outbreak of human metapneumovirus infection in norwegian children, Pediatr. Infect. Dis. J. 23 (5) (2004) 436e440. [5] M. Galiano, C. Videla, S.S. Puch, A. Martinez, M. Echavarria, G. Carballal, Evidence of human metapneumovirus in children in Argentina, J. Med. Virol. 72 (2) (2004) 299e303. [6] A. Mejias, S. Chavez-Bueno, O. Ramilo, Human metapneumovirus: a not so new virus, Pediatr. Infect. Dis. J. 23 (1) (2004), 1-7; quiz 8e10. [7] P. Xepapadaki, S. Psarras, A. Bossios, M. Tsolia, D. Gourgiotis, G. Liapi-Adamidou, A.G. Constantopoulos, D. Kafetzis, N.G. Papadopoulos, Human Metapneumovirus as a causative agent of acute bronchiolitis in infants, J. Clin. Virol. 30 (3) (2004) 267e270. [8] T. Jartti, B. van den Hoogen, R.P. Garofalo, A.D. Osterhaus, O. Ruuskanen, Metapneumovirus and acute wheezing in children, Lancet 360 (9343) (2002) 1393e1394. [9] J.S. Peiris, W.H. Tang, K.H. Chan, P.L. Khong, Y. Guan, Y.L. Lau, S.S. Chiu, Children with respiratory disease associated with metapneumovirus in Hong Kong, Emerg. Infect. Dis. 9 (6) (2003) 628e633. [10] T.S. Jardetzky, R.A. Lamb, Activation of paramyxovirus membrane fusion and virus entry, Curr. Opin. Virol. 5 (2014) 24e33. [11] N. Srinivasakumar, P.L. Ogra, T.D. Flanagan, Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay, J. Virol. 65 (8) (1991) 4063e4069. [12] M.A. Krzyzaniak, M.T. Zumstein, J.A. Gerez, P. Picotti, A. Helenius, Host cell entry of respiratory syncytial virus involves macropinocytosis followed by proteolytic activation of the F protein, PLoS Pathog. 9 (4) (2013) e1003309. [13] A.A. Kolokoltsov, D. Deniger, E.H. Fleming, N.J. Roberts Jr., J.M. Karpilow, R.A. Davey, Small interfering RNA profiling reveals key role of clathrinmediated endocytosis and early endosome formation for infection by
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respiratory syncytial virus, J. Virol. 81 (14) (2007) 7786e7800. [14] D. Werling, J.C. Hope, P. Chaplin, R.A. Collins, G. Taylor, C.J. Howard, Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells, J. Leukoc. Biol. 66 (1) (1999) 50e58. [15] R.G. Cox, S.B. Livesay, M. Johnson, M.D. Ohi, J.V. Williams, The human metapneumovirus fusion protein mediates entry via an interaction with RGDbinding integrins, J. Virol. 86 (22) (2012) 12148e12160. [16] Y. Wei, Y. Zhang, H. Cai, A.M. Mirza, R.M. Iorio, M.E. Peeples, S. Niewiesk, J. Li, Roles of the putative integrin-binding motif of the human metapneumovirus fusion (f) protein in cell-cell fusion, viral infectivity, and pathogenesis, J. Virol. 88 (8) (2014) 4338e4352. [17] R.G. Cox, B.A. Mainou, M. Johnson, A.K. Hastings, J.E. Schuster, T.S. Dermody, J.V. Williams, Human metapneumovirus is capable of entering cells by fusion with endosomal membranes, PLoS Pathog. 11 (12) (2015) e1005303. [18] C. Deffrasnes, S. Cote, G. Boivin, Analysis of replication kinetics of the human metapneumovirus in different cell lines by real-time PCR, J. Clin. Microbiol. 43 (1) (2005) 488e490. [19] X. Bao, T. Liu, L. Spetch, D. Kolli, R.P. Garofalo, A. Casola, Airway epithelial cell response to human metapneumovirus infection, Virology 368 (1) (2007) 91e101. [20] J. Zhang, Y. Dou, J. Wu, W. She, L. Luo, Y. Zhao, P. Liu, X. Zhao, Effects of Nlinked glycosylation of the fusion protein on replication of human metapneumovirus in vitro and in mouse lungs, J. Gen. Virol. 92 (Pt 7) (2011) 1666e1675. [21] D. Hoekstra, T. de Boer, K. Klappe, J. Wilschut, Fluorescence method for measuring the kinetics of fusion between biological membranes, Biochemistry 23 (24) (1984) 5675e5681. [22] T. Sakai, M. Ohuchi, M. Imai, T. Mizuno, K. Kawasaki, K. Kuroda, S. Yamashina, Dual wavelength imaging allows analysis of membrane fusion of influenza virus inside cells, J. Virol. 80 (4) (2006) 2013e2018. [23] M. Lakadamyali, M.J. Rust, H.P. Babcock, X. Zhuang, Visualizing infection of individual influenza viruses, Proc. Natl. Acad. Sci. U. S. A. 100 (16) (2003) 9280e9285. [24] C. Abiko, K. Mizuta, T. Itagaki, N. Katsushima, S. Ito, Y. Matsuzaki, M. Okamoto, H. Nishimura, Y. Aoki, T. Murata, H. Hoshina, S. Hongo, K. Ootani, Outbreak of human metapneumovirus detected by use of the Vero E6 cell line in isolates collected in Yamagata, Japan, in 2004 and 2005, J. Clin. Microbiol. 45 (6) (2007) 1912e1919. [25] L. Sanchez-Felipe, E. Villar, I. Munoz-Barroso, Entry of Newcastle Disease Virus into the host cell: role of acidic pH and endocytosis, Biochim. Biophys. Acta 1838 (1 Pt B) (2014) 300e309. [26] R.M. Schowalter, S.E. Smith, R.E. Dutch, Characterization of human metapneumovirus F protein-promoted membrane fusion: critical roles for proteolytic processing and low pH, J. Virol. 80 (22) (2006) 10931e10941. [27] S. Herfst, V. Mas, L.S. Ver, R.J. Wierda, A.D. Osterhaus, R.A. Fouchier, J.A. Melero, Low-pH-induced membrane fusion mediated by human metapneumovirus F protein is a rare, strain-dependent phenomenon, J. Virol. 82 (17) (2008) 8891e8895.
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Original research article
Human metapneumovirus uses endocytosis pathway for host cell entry Hui Yang, Hao He, Bin Tan, Enmei Liu, Xiaodong Zhao*, Yao Zhao** Chongqing Key Laboratory of Child Infection and Immunity, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children’s Hospital of Chongqing Medical University, Chongqing, 400014, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 March 2016 Received in revised form 15 June 2016 Accepted 15 June 2016 Available online xxx
Human metapneumovirus (hMPV) is a prevalent pathogen worldwide and causes various respiratory infections. Although it is a critical pathogen in pediatric patients, it is unclear how it enters host cells. In this study, we focused on hMPV cell entry using two kinds of cell lines (Vero E6 and LLC-MK2), which are most commonly used for isolating and propagating for hMPV, and we used fluorescent dyes to label the virus particles and monitored how they enter the host cell in real time. We found that endocytosis was the predominant pathway by which hMPV entered host cells. When the virus particles were traced inside host cells, we found that a low intracellular pH was needed for intracellular fusion in LLC-MK2 cells. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Human metapneumovirus Cell entry Endocytosis Low pH
1. Introduction Since 2001, when human metapneumovirus (hMPV) was first identified in Holland [1], many research groups have found evidence of hMPV infection worldwide [2e5]. Indeed, hMPV is considered a major pathogen responsible for lower respiratory tract disease in infants and children [6,7]. It also causes acute wheezing in young children [8] and can exacerbate asthma [9]. The importance of hMPV is not related to its prevalence, but rather to the severity of resultant disease. However, how this virus infects cells is still entirely unclear. There are two primary routes by which enveloped viruses can enter host cells: membrane fusion and endocytosis. The endocytosis can occur via four major kinds of pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, or clathrin/caveolae-independent endocytosis. Most enveloped viruses are believed to fuse their viral membranes and cells membranes to achieve effective infection [10]. Srinivasakumar et al. [11]
* Corresponding author. Present Address. No. 136, Zhongshan 2nd Road, Yuzhong District, Chongqing, 400014, China. ** Corresponding author. Present Address. No. 136, Zhongshan 2nd Road, Yuzhong District, Chongqing, 400014, China. E-mail addresses: [email protected] (X. Zhao), [email protected] (Y. Zhao).
showed that ~ 35% of Respiratory Syncytial Virus (RSV) viral particles had fused to the plasma membrane 1 h following infection. Although the fusion mechanism is recognised by many researchers, various endocytic pathways have now been identified. For example, one study showed that RSV enters HeLa cells via macropinocytosis [12], whereas other studies showed that it infects HeLa cells via clathrin-mediated endocytosis [13]. Moreover, another investigation showed that dendritic cells present RSV antigen by caveolaedependent endocytosis [14]. It seems that RSV mediates different entry pathways at different infection times to enter the same host cells, or RSV via different ways to infect different cells. hMPV, like RSV, represents the subfamily Pneumovirinae within the family Paramyxoviridae and these viruses cause similar clinical symptoms. So, we speculated that hMPV could choose different avenues to enter different cells, depending on infection conditions, although until now, membrane fusion was regarded to be the major pathway of hMPV cell entry [15,16]. Recently, Cox et al. [17] found that hMPV enters human bronchial epithelial cells via clathrin-mediated endocytosis in a dynamin-dependent manner. This information suggests that hMPV might exploit an endocytosis pathway in different host cells. Here, we selected Vero E6 and LLC-MK2 as target cells, not only because they are the most common used cells for propagating hMPV, but also owing to their higher efficiency of hMPV infection than other airway epithelial cells [18,19], and used a series of
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fluorescent dyes to label hMPV particles. We have followed the hMPV cell entry process with a confocal laser-scanning microscope. The results indicated that hMPV entered cells primarily via an endocytic pathway. Moreover, a low intracellular pH played a key role in hMPV entry. 2. Materials and methods 2.1. Cell lines Vero E6 and LLC-MK2 cells were purchased from the China Center for Type Culture Collection and maintained in a humid 5% CO2 incubator at 37 C in Dulbecco’s Modified Eagle Medium (DMEM), (Gibco, NY, USA) or Minimum Essential Medium (MEM), (Gibco, NY, USA), respectively, supplemented with 10% fetal bovine serum (FBS), (Gibco, NY, USA) and antibiotics (penicillin 100 IU/ml; streptomycin 100 mg/ml); (Hyclone, UT, USA). 2.2. Virus stocks hMPV was successfully recovered from full-length cDNA clones of hMPV NL/1/00 by reverse genetics as described previously [20]; hMPV was propagated in Vero E6 cells, and hMPV virions were purified by ultracentrifugation on a modified 35% glucose gradient. The viral supernatant was harvested and gently layered on to the surface of a 35% glucose-HBSS solution (Hyclone, UT, USA). Then, they were centrifuged at 51,500 g for 16 h at 4 C (Beckman Coulter, FL, USA). Finally, the supernatant was discarded and the virus particles were suspended in medium and maintained as stocks at 80 C. 2.3. Dyes and antibodies Octadecyl Rhodamine B Chloride (R18) was obtained from Biotium Inc, USA. DiOC was obtained from Beyotime Biotechnology, China. Cy3 and CypHer5 NHS ester were obtained from GE Healthcare, UK. The monoclonal mouse anti-hMPV fusion protein (F) and monoclonal mouse anti-hMPV nuclear protein (N) antibodies were purchased from Merck Millipore, Germany. The Alexa Fluor® 488/546 donkey anti-mouse IgG antibodies were purchased from Life Technologies, USA. 2.4. Indirect fluorescence assay hMPV was inoculated on to the cells (MOI ¼ 1). First, the cells were placed at 4 C for 30 min to synchronize host cell entry. Unbound virus was washed away and the temperature raised to 37 C to initialize the entry process. After incubation at 37 C for different times, cells were fixed and permeated by exposure to 0.1% Triton X100 (Sigma-Aldrich). Albumin bovine V (1%; BSA, Sigma-Aldrich) was used as a blocking agent. The cells were then incubated with anti-hMPV F and N antibodies, followed by fluorescently conjugated anti-IgG antibodies. F-actin was stained by incubating cells with phalloidin. Finally, cell nuclei were labeled by DAPI (Beyotime). Fluorescence was observed under a confocal microscope (Nikon, Tokyo, Japan). 2.5. R18 fusion assay R18 (1 ml) was added into 1 ml of hMPV (pfu ¼ 106/ml) to yield a final R18 concentration of 10 mM. The mixture was shaken gently for 1 h to allow R18 binding. Impurities were removed using a 0.22 mm filter, and unbound dye was removed using CaptoCore 700 (GE Healthcare). R18 alone was also added to medium as a negative control. Both R18-hMPV and R18-control were inoculated on to
Vero E6 and LLC-MK2 cells (MOI ¼ 1) for 30 min at 4 C, and then replaced with fresh medium. Once the cells were placed under the confocal microscope, the temperature was raised to 37 C, and the fluorescence change was recorded for 4 h. Variations in fluorescence intensity were also measured every 2 min in a microplate reader. The amount of R18 fluorescence dequenching was expressed as the DQ%, calculated using the formula: DQ%¼(Fd-F0)/ Ft*100%, where F0 is the fluorescence intensity at the beginning, Fd is the fluorescence intensity at each detection time, and Ft is the maximum fluorescence intensity when R18 was infinitely diluted with 1% Triton X-100 [21]. 2.6. DiOC/R18 FRET A mixture of DiOC/R18 was added to 1 ml of hMPV. The final concentration of DiOC was 3.3 mM and that of R18 was 6.7 mM. Gentle shaking was performed to facilitate virus staining and unincorporated dyes were removed in the same way as for the R18 fusion assay. The negative control was treated in the same way. FRET was observed under a confocal microscope. DiOC/R18 labeled hMPV was excited with a laser (488 nm), and fluorescence was detected simultaneously at 500 nm and 575 nm. 2.7. Cy3/CypHer5 dual labeling Before labeling, hMPV was treated with 0.5 M Na2CO3 to increase the pH of the solution to 9.3. Cy3 and CypHer5 were then added to 1 ml hMPV; the final concentration of each was 10 mg/ml. The mixture was then shaken and filtered as described for previous experiments. Dual-labeled virus was then inoculated on to cells at 37 C and excited with a laser at 550 nm. The fluorescence emitted by the two dyes was detected at 575 nm and 660 nm. 3. Results 3.1. Fusion protein (F) and nucleoprotein (N) co-localize during hMPV entry Cells were infected with hMPV for different times and then stained with anti-hMPV F and anti-hMPV N antibodies to identify the viral membrane and the nuclear capsid, respectively. At the same time, F-actin was labeled with Alexa Fluor® 647 phalloidin to reveal the cytoskeleton. Since F and N are located on the virus membrane and capsid, respectively, staining by both F and N antibodies indicated intact particles within infected cells. Serial confocal Z-stack images revealed that fluorescent spots of F and N co-localized on the cell surface and in the cytoplasm at 2 h posthMPV infection (Fig. 1A and B). Vero E6 cells without infection were used as a negative control (Fig. 1C). Krzyzaniak et al. [12] defined capsid-free envelopes (or virus-like particles; VLPs) as particles with F spots but without N spots. Here, we found that the number of VLPs (F only) (Fig. 1D) and intact viral particles (FN both) increased 2 h after infection. We believe that the N spots represent the total number of viral particles (N only), whereas the FN spot represents the number of endocytosed viral particles. Thus, 2 h after infection, both the total number of particles and the number of endocytosed particles increased (Fig. 1E, F). The ratio of FN to N spots in LLC-MK2 cells increased from 0.58 to 0.84 (Fig. 1G, right panel), and that in Vero E6 cells varied from 0.65 to 0.70 (Fig. 1G, left panel) during 2 h of incubation. A possible explanation for this finding is that more hMPV particles were endocytosed by LLC-MK2 during the 37 C incubation period. In other words, whether incubation at 37 C is optimal for endocytosis depends on the host cell type. However, in both groups, about two-thirds of hMPV particles entered via the endocytosis pathway.
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Fig. 1. The F and N proteins colocalize in host cells. Vero E6 (A) and LLC-MK2 cells (B) were incubated with hMPV and fixed. The control group (C) (Vero E6) was incubated with medium instead. Cells were stained with both anti-F-AF488 (green) and anti-N-AF546 (red) antibodies, and phalloidin-AF647 (purple) and DAPI (blue). Z-stack images were taken (bottom and right of A and B). The fluorescence spots of anti-F only (D), anti-N only (E) and both anti-F and anti-N colocalization (F) were analyzed using NIS elements software Ver. 4.00 (Nikon, Tokyo, Japan). (G) Fluorescence spots of FN colocalization and N spots ratio was calculated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.2. Membrane fusion occurs during hMPV entry A lipophilic dye (octadecyl rhodamine B; R18) was used to confirm whether membrane fusion occurred during hMPV entry. R18 has a specific property in that its fluorescence is quenched after incorporation into the viral membrane. Once the R18-labeled virus
fuses with the unlabeled membrane, the dye is diluted and the fluorescence dequenches [21]. This feature makes R18 a useful fusion probe. In this assay, the fluorescence counts and intensity of R18 (red) increased over time in both cell lines (Fig. 2A and Movies S1 and S2). These findings indicate that membrane fusion events occur during the hMPV cell entry process. Measuring fluorescence
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Fig. 2. Membrane fusion events occur during infection. (A): Vero E6 (middle) and LLC-MK2 (bottom) cells were incubated with R18-hMPV and images were captured in real time. R18-control was added to Vero E6 cells as a control. (B): Fluorescence intensity of R18 at different times was analyzed. (C): Cells were cultured with R18-hMPV in a 96-well plate at 37 C, and the fluorescence intensity of R18 was measured with a microplate reader every 2 min. After 4 h, an equal volume of 2% Triton-X 100 was added to achieve an infinite dilution of the R18 dye. DQ% of R18 was calculated.
over time in a microplate reader yielded the same results. The fluorescence intensity increased to a maximum of 27.34% and 88.24% in Vero E6 and LLC-MK2 cells, respectively. However, there was a small difference between the two cell lines. In Vero E6 cells, the intensity of R18 increased, peaking around 60 min after infection, before declining again. However, in LLC-MK2 cells, the intensity of R18 fluorescence continued to increase and reached a plateau at 140 min during the 4 h incubation period (Fig. 2B). In both cell lines, the DQ% increased rapidly at first and then decreased slowly. The maximum DQ% for Vero E6 and LLC-MK2 cells was 30.1% and 32.0%, respectively. These peak values were reached at 90 min and 110 min after infection, respectively (Fig. 2C). These results showed that about one-third of hMPV particles fused with other biomembrane during infection. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.mcp.2016.06.003. 3.3. Intracellular fusion occurs after endocytosis In this experiment, two fluorescent dyes (R18 and DiOC) were introduced into cells. R18 and DiOC are used to examine intracellular membrane fusion [22]. When hMPV membrane was labeled with the two lipophilic dyes, both were located in close proximity on the viral membrane; therefore, fluorescent resonance energy transfer (FRET) occurred. This means that, when the labeled virus was excited by a laser, the signal generated by DiOC was quenched by R18. Thus, only red fluorescence emitted by R18 was observed. However, when the labeled virus fused with unlabeled biological membranes, the two dyes separated. FRET reduced, and DiOC (green fluorescence) was detected, which indicated that the fusion occured inside the cell. The results showed that, at the beginning of the incubation period, only a few red spots were detected in both Vero E6 and LLC-MK2. In addition, the numbers of intracellular green or yellow spots increased over time (Fig. 3A and Movies S3 and S4). As shown for the single laser channel, no green spots were visible until 30 min in LLC-MK2; thereafter, green spots gradually began to appear, but green spots were not visible in Vero E6 cells until 1 h after infection (Fig. 3A, middle and bottom panels). This
information implies that intracellular fusion occurred (i.e., DiOC was no longer quenched by R18), and that these fusion events occurred earlier in LLC-MK2 than in Vero E6. Analysis with NIS Elements software revealed that the intensity of R18 and DiOC in Vero E6 cells increased by ~141.1% and 141.7% respectively, after 2 h of incubation, and by ~292.7% and 395.1% respectively, in LLC-MK2 cells (Fig. 3B and C). (Fig. 3B and C). Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.mcp.2016.06.003. 3.4. hMPV entry is associated with a decrease in intracellular pH Since extracellular pH plays an important role in paramyxovirus infection, we attempted to establish whether the pH in endocytic vacuoles changed after hMPV infection. To do this, we labeled hMPV with two dyes: pH-insensitive Cy3 and pH-sensitive CypHer5. The ratio of the fluorescence emissions of them is an indicator of pH [23]. In LLC-MK2 cells, green fluorescence increased during 2 h following infection (Fig. 4A, Movie S5). The fluorescence intensity of Cy3 increased slowly throughout the 2 h period, whereas that of CypHer5 increased, peaking at ~80 min, before declining sharply. The increase level in CypHer5 fluorescence was much greater than that of Cy3. The fluorescence intensity of CypHer5 increased by 60.00% at 80 min; the increase for Cy3 at the same time point was 12.9% (Fig. 4B). Since CypHer5 is pH-sensitive, the ratio of the two dyes was expressed as the G/R ratio. The G/R ratio increased from 0.38 to 0.56, before declining to 0.31 (Fig. 4C). Changes in the G/R ratio mirrored changes in CypHer5 fluorescence. These results suggest that hMPV requires a low pH environment in the cytoplasm. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.mcp.2016.06.003. 4. Discussion Productive viral infection occurs only after infectious virus particles enter susceptible cells. At present, there are two major pathways by which viruses enter host cells, i.e. via membrane
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Fig. 3. hMPV undergoes intracellular fusion. (A): Target host cells were inoculated with the DiOC/R18 dual-labeled hMPV. Real-time images were captured. Vero E6 without hMPV were exposed to DIOC/R18 mixture as a control (top). Pictures of individual fluorescence channels of Vero E6 (middle) and LLC-MK2 cells (bottom) are shown. (B), (C): Changes in the fluorescence intensity of R18 (red) and DIOC (green) were measured in the two cell lines at different time points. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
fusion and endocytosis. Research results achieved using modern techniques reveal that a single virus can utilize both mechanisms simultaneously [12e14]. We speculate that hMPV mediates primarily endocytosis to achieve maximal infection, but which pathway that hMPV uses might depend on the host cell type. Classical virology suggests that paramyxovirus enters cells via membrane fusion. Although hMPV is very similar to RSV, they also have different characteristics; hMPV replicates more slowly than RSV [18]. Unlike syncytium formation by RSV, CPE caused by hMPV are atypical, including formation of granular, small, rounded and refringent cells [24]. The particularities associated with hMPV suggest that it may enter host cells via a specific mechanism. The present findings suggest the endocytosis pathway plays a major role in hMPV infection. The enveloped virus comprises lipid membrane and nucleocapsid. Where the viral envelope fuses is the key factor to differentiate membrane and endocytosis. During membrane fusion, the viral membrane fuses with the plasma membrane before releasing its nucleocapsid into the cytoplasm [10]. Therefore, the viral membrane components remain at the cell surface after entry. When a virus enters via endocytic pathway, the entire intact particle enters the host cell; thus, the components of the viral membrane are present within the cell. We found that the hMPV viral membrane was still detectable in cells after the virus particle was endocytosed, since the F protein was observed intracellularly after entry. In the Z-stack images, both the anti-hMPV F and anti-hMPV N
antibodies co-localized deep inside the same cell (Fig. 1A, B), confirming that the viral membrane and its nuclear material were within the cell. According to the IFA, 70.1% and 83.9% of hMPV particles were present in Vero E6 and LLC-MK2, respectively, as intact virions at 2 h after infection (Fig. 1G). However, about 20%e 30% of hMPV was present in the form of N protein only, suggesting that some virions entered the cell via membrane fusion. In order to establish which pathway is the major one for hMPV entry, we used R18 and DiOC to detect the key events. We found that upon incubation of R18-hMPV with both Vero E6 and LLCMK2, the fluorescence intensity of R18 increased during the first hour (Fig. 2B). After 1 h, the fluorescence intensity declined in Vero E6 cells, but still increased and reached a plateau in LLC-MK2. A possible interpretation here is that the virus does not enter these cell lines by exactly the same mechanism. The data suggest that fusion events occur much more during infection of LLC-MK2 cells. Dequenching also increased in both cell lines; indeed, the DQ% was similar in both (Fig. 2C). This information suggests that approximately one-third of the virus particles fused during infection. The pathway by which hMPV enters depends on where the fusion occurs. If fusion is within a cell, then hMPV is endocytosed. Results (Fig. 2A and Movie S1, S2) indicated that most hMPVs were endocytosed in Vero E6 and LLC-MK2. DiOC/R18 duallabelling [22] revealed that the intensity of R18 and DiOC in both Vero E6 and LLC-MK2 (Fig. 3B and C). Variation in the fluorescence intensity of individual spots suggested that intracellular fusion occurred
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Fig. 4. hMPV entry is associated with a decrease in intracellular pH. (A): Cy3/CypHer5-hMPV was added to LLC-MK2 cells. Images were captured in real time. Photos of per channel are shown. (B): Fluorescence intensities of Cy3 (red) and CypHer5 (green) were measured at different time points following infection. (C): The ratio of the two fluorescence intensities was calculated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
intracellularly, i.e., virus-endosome fusion, endosome-endosome fusion, or endosome-lysosome fusion. At the same time, these results indicated that LLC-MK2 was more suitable for hMPV infection compared with Vero E6. Since viruses are parasitic microorganisms, the pH of the environment plays an important role in determining the route of infection. For example, Newcastle Disease Virus enters HeLa and ELL-0 cells via low pH-dependent endocytosis [25]. The role of pH in hMPV entry is controversial. Schowalter et al. [26] found that trypsin treatment combined with low pH promoted syncytial formation by the hMPV F protein in BHK cells; however, Herfst et al. [27] suggested that low-pH-induced fusion is a rare phenomenon, since only a few hMPV strains harbouring a specific amino acid residue in the F protein require low pH for efficient membrane fusion. Some researchers report intracellular pH changes during viral entry and transport. Lakadamyali et al. [23] revealed that influenza virus showed a three-stage transport pattern when infecting CHO cells. The present study also showed that low pH is beneficial for hMPV infection. According to the present data, the fluorescence counts and the intensity of the two tracker dyes increased during the 2 h observation period (Fig. 4B). The fluorescence counts for CypHer5 and Cy3 were 48-fold and 3.5-fold higher, respectively, than at the beginning of the experiment (data not shown). Despite differences between the properties of the two dyes, the ratio of the fluorescence (G/R ratio) that they emitted also increased after viral infection (Fig. 4C). In accordance with the fluorescence intensity, the G/R ratio peaked at around 80 min. These findings suggested that intracellular pH declined first and
then recovered. At the same time, it meant that hMPV was exposed to acidic pH at least during 80 min. These results are approximately concordant with those of the DiOC/R18 fusion assay, because DiOC/ R18 FRET significantly increased during this period, and suggested that acidification of endocytic vesicles occurred during intracellular fusion. Therefore, we conclude that decreases in intracellular pH trigger fusion between the viral and endosomal membranes. A low pH may help to release the viral nuclear material. It is notable that changes in Cy3/CypHer5 fluorescence were not so clear in Vero E6 cells, which might relate to different cell types showing different sensitivities to pH. Therefore, pH-dependency not only relates to virus strains, but also to the cell type that they infect. In conclusion, we established that hMPV can use an endocytosis pathway to enter host cells (Vero E6 and LLC-MK2). We showed that a low pH is required for hMPV/endosome fusion in one of the two host cells. The present findings reveal that Paramyxoviridae (such as hMPV) may require acidification of the intracellular milieu to facilitate viral/membrane fusion with other organelles. The next plan is to investigate precisely which endocytosis pathway hMPV uses for cell entry. Acknowledgements This research was supported by Natural Science Foundation Project of CQ CSTC (NO. cstc2012jjA10091, cstc2013jjB10029), National Natural Science Foundation of China (NO. 30800972, NO. 81371876), Research Fund for the Doctoral Program of Higher Education of China (NO. 20125503110002), Outstanding Youth Fund
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