Caveolae Restrict Tiger Frog Virus Release in HepG2 cells and Caveolae-Associated Proteins Incorporated into Virus Particles

Caveolae are flask-shaped invaginations of the plasma membrane. Caveolae play important roles in the process of viruses entry into host cells, but the roles of caveolae at the late stage of virus infection were not completely understood. Tiger frog virus (TFV) has been isolated from the diseased tadpoles of the frog, Rana tigrina rugulosa, and causes high mortality of tiger frog tadpoles cultured in Southern China. In the present study, the roles of caveolae at the late stage of TFV infection were investigated. We showed that TFV virions were localized with the caveolae at the late stage of infection in HepG2 cells. Disruption of caveolae by methyl-β-cyclodextrin/nystatin or knockdown of caveolin-1 significantly increase the release of TFV. Moreover, the interaction between caveolin-1 and TFV major capsid protein was detected by co-immunoprecipitation. Those results suggested that caveolae restricted TFV release from the HepG2 cells. Caveolae-associated proteins (caveolin-1, caveolin-2, cavin-1, and cavin-2) were selectively incorporated into TFV virions. Different combinations of proteolytic and/or detergent treatments with virions showed that caveolae-associated proteins were located in viral capsid of TFV virons. Taken together, caveolae might be a restriction factor that affects virus release and caveolae-associated proteins were incorporated in TFV virions.

Scientific RepoRts | 6:21663 | DOI: 10.1038/srep21663 morphology and cell type 15 . Cavin proteins function primarily as scaffolding and also regulate caveolin availability 16 . An increasing evidence shows that several pathogens, such as viruses, bacteria, and parasites, use caveolae for their own benefit 17 . On one hand, some viruses, such as HIV, SV40 (simian virus 40), RSV (respiratory syncytial virus), and echovirus 1, use caveolae to enter the host [18][19][20][21] . On the other hand, raft domains provide assembly and budding sites for viruses, such as influenza virus, measles virus, rotavirus, and herpes simplex virus 22-25 . Caveolae are a subset of lipid rafts, but the roles of caveolae at the late stage of virus infection not completely understood.
Iridoviruses are large, icosahedral cytoplasmic DNA viruses that contain circularly permutated, terminally redundant, double-stranded DNA genomes 26,27 . Iridoviruses cause systemic diseases in invertebrates, particularly insects and poikilothermic vertebrates (fish, amphibians and reptiles) 28 . To date, more than 100 iridovirus strains have been isolated, and entire genomes of more than 20 strains have been completely sequenced 28 . The Iridoviridae family is composed of five genera, as follows: Iridovirus, Chloriridovirus, Ranavirus, Lymphocystivirus, and Megalocytivirus 29 . The first reported complete genome sequence in the genus Ranavirus is tiger frog virus (TFV). TFV is isolated from the infected tadpoles of Rana tigrina rugulosa, causes high mortality rate among tiger frog tadpoles cultured in Southern China 30 . Recent reports showed that caveolae participate in the invasion of some iridoviruses. Infectious spleen and kidney necrosis virus (a megalocytivirus) enters mandarin fish fry (MFF-1) cells through a caveolae-dependent endocytic pathway and may colocalize with caveolin-1 31 . Research on the uptake of TFV into HepG2 cells at 27 °C show a pH-dependent, atypical caveolae-dependent endocytosis via the acidification of intracellular organelles 32 . In the present study, the roles of caveolae at the late stage of virus infection are explored, and the major structural proteins of caveolae are identified incorporated into TFV virions.

TFV virions localized with caveolae/caveolar endocytotic vesicle at the late stage of infection.
Dual-color fluorescence staining was used to prove the relationship between caveolae and TFV virions at 72 h postinfection. Caveolin-1 was used as the marker of caveolae or caveolar endocytotic vesicle. Caveolin-1 demonstrated punctuate staining pattern that is mainly localized in the plasma membrane of uninfected HepG2 cells (Fig.1A, Con, red fluorescence), which is consistent with its known localization in clustered caveolae microdomains 33 . After infecting with TFV, staining for TFV major capsid protein (MCP) showed that the protein was concentrated in patches in the cytoplasm of the virus-infected cells (Fig.1A, Inf, green fluorescence). The inset in Fig. 1A (yellow fluorescence, arrowed) shows that the punctate pattern caveolin-1 staining localized in the patches containing MCP stain, thereby suggesting that TFV MCP localizes with caveolin-1/caveolae/caveolar endocytotic vesicle at the late stage of infection.
Caveolae and raft membrane microdomains, enriched with sphingolipids and cholesterol can be isolated on low-density sucrose gradients by their insolubility in Triton X-100 at 4 °C 34,35 . The caveolae were isolated on low-density sucrose gradients 72 h postinfection, and equal volume fractions were separated using SDS-PAGE and immunoblotting. As shown in Fig. 1B, caveolin-1 and cavin-1 were highly enriched in fractions 5 and 6, which corresponded to the interphase between 5% and 30% sucrose, and those fractionations pattern are consistent with the known sedimentation properties of caveolae membranes 36,37 . In addition, TFV MCP floated into the caveolae-rich fractions (Fig. 1B), indicating that TFV MCP localized in caveolae at the late phase of infection. To further confirm the existence of TFV virions, viral genome DNA was extracted from each fraction and detected TFV orf031L gene and mcp gene (orf96R) using PCR and qPCR. TFV orf031L and mcp gene were determined in fractions 5 and 6 ( Fig. 1C). The highest expression level of TFV mcp gene was observed in fraction 5 (Fig.1D). Together, these results indicated that TFV virions localized in caveolae at the late phase of infection.
To investigate the profile of caveolae colocalization with TFV during virus infection, caveolae were isolated on sucrose gradients at different time points from 1 h to 72 h post-infection, and equal volume fractions were separated. The fractions 5 and 12 were detected by using SDS-PAGE and western blotting, since the fractions 5 were consistent with the known sedimentation properties of caveolae membranes while fraction 12 were with cytoplasm. As shown in Fig.1E, TFV MCP started to be detected at 36 h postinfection and the signal became stronger gradually at 48, 60 and 72 h in fractions 12, suggesting that TFV MCP begins to synthesis at 36 h postinfection in HepG2 cells. However, TFV MCP could not be detected in fraction 5 until 60 h postinfection, suggesting that TFV MCP colocalize with caveolae after 60 h postinfection. The results suggested that TFV colocalize with caveolae started at 60 h postinfection, indicating TFV detected in caveolae represent newly formed viruses but not the ones have entered inside the host cells.
Caveolae restricted the release of TFV virions. Depletion of cholesterol from membranes with Mβ CD or sequestration of cholesterol with nystatin impairs caveolae-mediated endocytosis 38 . To investigate the roles of caveolae at the late stage of TFV infection, HepG2 cells were treated with 5 mM Mβ CD or 200 μ g/ml nystatin at 60 h postinfection. After HepG2 cells were treated with Mβ CD or nystatin, caveolae were isolated on sucrose gradients using the above methods. As the results shown in Fig. 2G, caveolin-1 was not detected in the fractions 5 but in fractions 11 and 12 (cytoplasmic fragment), which suggested that caveolae were depolymerized after treated with Mβ CD or nystatin. TFV mcp gene from the supernatant of cells at 72 h postinfection was quantified using qPCR. As shown in Fig. 2A,D, the amount of TFV virions in the supernatant of treated-HepG2 cells increased. When the dose of Mβ CD or nystatin increased, the amount of TFV correspondingly increased in a dose-dependent manner (Fig. 2B,E). The absolute amount of TFV mcp gene from the infected cells had no significant change after treated with the different concentrations of Mβ CD or nystatin (Fig. 2C,F). This result showed that the concentrations of the chemicals used in this study did not affect TFV replication. The virus titers assay was used to measure the infectious virions that released from cell into the supernatant. HepG2 cells were treated with 5 mM Mβ CD or 200 μ g/ml nystatin at 60 h postinfection. The supernatant of cells at 72 h postinfection was collected and the TCID 50 was measured for each sample. As shown in Fig. 3A,B, the virus titer of TFV in the supernatant of treated-HepG2 cells increased. The single step growth curves (Fig. 3C) showed that infectious virions in the supernatant increased after cells treated with 5 mM Mβ CD. Caveolin-1 is the principal organizer of caveolae. Knockdown of caveolin-1 impairs the formation of caveolae 39 . In our study, si-caveolin-1 was used to knockdown the gene to detect the level of TFV release. The most effective si-caveolin-1 concentration is 50 nM (Fig. 2J), and TFV replication was not affected at this concentration (Fig. 2I). However, the amount of TFV in the supernatant was higher with the si-caveolin-1 transfection than with the si-NC control (Fig. 2H). Therefore, these data indicated that caveolae restricted TFV release from HepG2 cells.
To explore whether caveolae can restrict TFV release from more relevant cell lines. FHM cells were infected with TFV and were then treated with Mβ CD or nystatin at 60 h postinfection. Control cells were incubated in medium with the corresponding solvent. The TFV mcp gene from the supernatant of cells at 72 h postinfection was quantified using qPCR. As shown in Fig. 3D, the amount of TFV virions in the supernatant of treated-FHM cells increased. The results suggested that caveolae might also restrict TFV release from FHM cells. Caveolin-1 interacts with TFV MCP. To identify the caveolin-1 interaction with TFV protein, the interaction between human caveolin-1 and TFV MCP was observed using Co-IP experiments. HeLa cells were transiently transfected to produce Flag-tagged caveolin-1 protein and myc-tagged TFV MCP. Immunopecipitation of myc-tagged TFV MCP by using myc tag-specific monoclonal antibody led to coprecipitation of Flag-tagged caveolin-1 protein (Fig. 4A, lane 3). In a converse experiment, immunoprecipitation of Flag-tagged caveolin-1 protein using Flag-tag specific monoclonal resulted in coprecipitation of myc-tagged TFV MCP (Fig. 4A, lane 3). The results were further supported by colocalization of caveolin-1 with TFV MCP found in HeLa cells transiently expressing Flag-tagged caveolin-1 protein and myc-tagged TFV MCP (Fig. 4C). The results proved that caveolin-1 physically interacted with TFV MCP and suggested the mechanism underlying the restriction of TFV by caveolae might be via the interaction of caveolin-1 and TFV MCP.
To corroborate interaction between caveolin-1 and TFV MCP under non-over expression conditions. The immunoprecipitation experiments were carried out with the antibodies against caveolin-1 in the cells infected with virus. HepG2 cells were infected with TFV at an MOI of 10. At 72 h postinfection, we collected the cells to do the immunoprecipitation using anti-caveolin-1 antibody with uninfected HepG2 cells as control. As shown in Fig. 4B, TFV MCP can be immunoprecipitated by anti-caveolin-1 antibody, suggesting that TFV MCP interacts with caveolin-1 at the late stage of virus infection.

Caveolae-associated proteins were incorporated into TFV virions. HepG2 cells became round and
yielded TFV virions at 6 d postinfection with TFV at an MOI of 10. The supernatant was harvested to purify TFV by sedimentation in sucrose density gradients. Western blot analysis results showed that the viral envelope protein vp020R and the viral MCP were detected in our purified virus sample, but not CD63 (a marker of microvesicles; used as control), CD98, alpha 1 Sodium Potassium ATPase and Glut-1 (markers of plasma membrane; used as control) (Fig. 5A). Those results showed that the TFV used in the experiment was without microvesicles or cell debris. To explore whether caveolae-associated proteins were incorporated into TFV virions, the major structural proteins of caveolae and the cytoskeletal elements were detected by western blot. The major structural proteins (caveolin-1, caveolin-2, cavin-1, and cavin-2) were present in the TFV virions (Fig. 5B). To provide additional evidence for the incorporation of caveolae-associated proteins in TFV virions, immunogold labeling was performed. As shown in Fig. 5E, several gold particles were labeled on the virions using anti-caveolin-1, anti-caveolin-2 or anti-cavin-1 antibody as the primary antibody. Cytoskeletal elements, such as β -actin and filamin, were detected in the purified TFV virions (Fig. 5C). Interestingly, the phosphorylation form of caveolin-1 (p-caveolin-1-Y14) was also detected in TFV virions (Fig. 5B). Other lipid raft proteins, namely, clathrin and flotillin, were not detected in purified TFV virions (Fig. 5C). These observations suggested that caveolae-associated proteins were incorporated into TFV virions.
To test whether depolymerized caveolae affected the released virions in terms of incorporation of caveolar components. HepG2 cells were infected with TFV at an MOI of 10 and at 60 h postinfection were treated with 5 mM Mβ CD. The supernatant was harvested 12 h after treating with Mβ CD. TFV virions were purified by sedimentation in sucrose density gradients. Western blot analysis results showed that only caveolin-1 can be detected but not caveolin-2 and cavin-1 (Fig. 5D), suggesting that treating with Mβ CD changed the incorporation of caveolar components.

Caveolin-1 was selectively incorporated into virions.
To confirm that caveolin-1 was selectively incorporated into TFV virions, we tested whether the exogenous fusion caveolin-1-flag could also be incorporated into the TFV virions. HepG2 cells were transfected to express caveolin-1 with flag-tag, and the control cells were transfected to express green-fluorescent protein (GFP), Glut-1 (a membrane protein) or flotillin-1 (a lipid raft microdomain-associated protein) with flag-tag. The cells were then infected with TFV at 24 h post-transfection and the supernatant was harvested to purify TFV by sedimentation in sucrose density gradients at 6 d postinfection. As shown in Fig. 6C, caveolin-1 was detected by flag-tag specific monoclonal antibody. However, GFP (Fig. 6D), Glut-1 (Fig. 6E) and flotillin-1 (Fig. 6F) proteins were not detected in purified virions. Those results suggested that caveolin-1 was selectively incorporated into TFV virions.
Ranavirus particles consist of an inner DNA/protein core, an internal limiting membrane, a viral capsid, and an outer viral envelope 29 . To explore the location of caveolae-associated proteins within TFV virions, virions were subjected to different combinations of proteolytic and/or detergent treatments from the envelope to the capsids, after which gradient purification of the resulting particles and western blot analysis were performed. ORF020R is a TFV envelope protein 40 . As shown in Fig. 6A, after treating with 0.5% Triton X-100 for 10 min, purified TFV virions were centrifuged at 20000 rpm. ORF020R could be detected in supernatant (Fig. 6A, lane 1), but not in the precipitation (Fig. 6A, lane 2). The results proved that the outer viral envelope of TFV were removed by Triton X-100. Untreated purified TFV virions incorporated caveolin-1, p-caveolin-1-Y14, caveolin-2, cavin-1, and cavin-2 proteins (Fig. 6B, lane 1) and were used as positive controls. Proteins outside the envelope were removed (Fig. 6B, lane 2) by subtilisin digestion. To degrade the envelope, virions were treated with 0.5% Triton X-100 for 10 min (Fig. 6B, lane 3). The signal persisted until the naked virions were digested by subtilisin (Fig. 6B, lane 4). Taken together, these observations suggest that caveolae-associated proteins (caveolin-1, p-caveolin-1-Y14, caveolin-2, cavin-1, and cavin-2) were incorporated in the TFV virions and located in viral capsid of TFV virion.

Discussion
In the present study, we found that caveolae restrict TFV release and caveolae-associated proteins were incorporated into TFV particles in HepG2 cells. Virions localization with caveolae were observed at the last stages of infection. Disruption of caveolae by Mβ CD/nystatin or knockdown caveolin-1 increased the release of the virus, suggesting that caveolae play important roles at the late stages of viral life cycle and may restrict virus release. The mechanism underlying the restriction may be via the interaction of caveolin-1 and TFV MCP. Moreover, the major structural proteins of caveolae (caveolin-1, caveolin-2, cavin-1, and cavin-2) were selectively incorporated into the mature virions, and were present in the capsid of TFV virions.
Caveolae can be used by viruses for their own benefit. Caveola-dependent endocytosis was originally identified as an entry pathway for SV40 21 . Since then, the use of caveolae for entry into host cells has been demonstrated for other viruses, such as HIV, RSV, and echovirus 1 [18][19][20][21] . In this study, numerous TFV virions were localized in caveolae/caveolar endocytotic vesicle at 72 h postinfected (Fig. 1A-C). The time course experiment showed that TFV colocalized with caveolae started at 60 h postinfection (Fig. 1E), indicating that TFV detected in caveolae represent newly formed viruses but not the ones have entered inside the host cells. Those results suggested that caveolae may take part in the late stages of TFV life cycle.
To investigate the function of caveolae at the late phase of TFV life cycle, we disrupted the caveolae using MβCD and nystatin, and found that the amount of TFV virions in the supernatant increased (Figs 2 and 3). We used siRNA knockdown caveolin-1 to impair the formation of caveolae and acquired the same results (Fig. 2). These results show that caveolae restrict the release of TFV virions. As well known, antiviral activity is shown in human cells when the release of enveloped virus particles is inhibited. Up to now, BST-2 (bone marrow stromal cell antigen 2, CD317, Tetherin) is the only one host protein has been discovered to function as the factor to restrict the virus release 41 . BST-2, a membrane protein localized in lipid rafts, inhibits diverse families of enveloped viruses release 42,43 . In the case of HIV, the prevention of the release of HIV virions in the absence of Vpu is associated with nascent virions accumulation along the plasma membrane and within clathrin-coated endosomes 44,45 . HIV Vpu protein down-regulates BST-2 from the cell surface by interacting with it to antagonize the inhibition 46,47 . Our findings suggested that caveolae were possibly another restriction factor of virus release.
The mechanism of BST-2 restrict HIV release was suggested 46 . BST-2 is positioned to directly retain nascent HIV virions on the plasma membrane of infected cells and incorporated into virions. BST-2 restrict HIV release mediated directly by virion-associated and cell-surface-associated BST-2 molecules interact via their ectodomains 46 . In our study, caveolin-1 protein's incorporation into TFV virions (Fig. 5B), along with the other main structural proteins of caveolae (caveolin-2, cavin-1, and cavin-2) (Fig. 5B), was validated by western blot and immunoelectron microscopy (Fig. 5E). Caveolae-associated proteins (β -actin and filamin) were also detected in purified TFV virions (Fig. 5C). The caveolae-associated proteins were incorporated into TFV virions, but other lipid raft structural proteins, clathrin and flotillin, were not detected in TFV virions (Fig. 5C). We also found that depolymerized caveolae using Mβ CD affected the released virions in terms of incorporation of caveolar components. Caveolin-1 can be incorporated but no other caveolae associated proteins such as caveolin-2 and cavin-1 (Fig. 5D). Those results implied that the incorporations of caveolae associated proteins were related to the colocalization of TFV and caveolae. To explain how the caveolae-associated proteins were incorporated into the virions, the main structural protein of caveolae, caveolin-1, was investigated. We determined whether the exogenous fusion caveolin-1 protein could be incorporated into the TFV mature virion. The results showed that caveolin-1-flag can incorporated in the virion, but GFP-flag or Glut-1-flag or flotillin-1-flag protein wasn't, thereby suggesting that caveolin-1 is not just attached non-specifically to the external or that this protein is perhaps derived from microvesicle or cell debris that was co-purified with the virus (Fig. 6). Furthermore, the interaction of caveolin-1 and TFV MCP were detected using Co-IP/IP and IFA (Fig. 4). Caveolin-1 is the major structure protein of caveolae, like a Scaffold for caveolae to recruit other proteins to this plasma membrane domain. Caveolin-1 has been shown to interact directly with caveolin-2, cavin-1, and cavin-2 [48][49][50] . Thus, we speculate that the caveolae-associated proteins incorporated into TFV virion might be obligated to participate in the interaction between caveolin-1 and TFV MCP at the late stage of infection. To investigate the location of the caveolae-associated proteins within the virion, the virions were subjected to different combinations of proteolytic and/or detergent treatments from the envelope to the capsids. From the inside out, Ranavirus consists of an inner DNA/protein core, an internal limiting membrane, a viral capsid, and an outer viral envelope 29 . Caveolae-associated proteins, caveolin-1, caveolin-2, cavin-1, and cavin-2, were present in the envelope and on the capsid of TFV virions (Fig. 3). Thus, the location of caveolae-associated proteins the further proved these proteins were selectively incorporated.
Our results showed that caveolae restrict the TFV virion release. The potential mechanism of the restriction may be through the interaction of caveolin-1 incorporate in the TFV nascent virion and the proteins in the caveolae (Fig. 7). We found that caveolae-associated proteins can be incorporated in the TFV. However the function of those proteins and detail mechanism of caveolae restrict virus release would need further research.

Virus purification and Western blot analysis. Confluent monolayers of HepG2 cells were infected with
TFV with a multiplicity of infection (MOI) of 10 and incubated at 27 °C. At 6 d postinfection, the supernatant was harvested before the cells detached, and cell debris was removed by low-speed centrifugation. Virus purification by sedimentation in sucrose density gradients was done as described previously 40 . Protein concentrations of the purified virus stocks were determined using the DC protein assay kit from Bio-Rad (California USA). The purified sample (20 μ g) was boiled in sodium dodecyl sulfate (SDS) loading buffer and analyzed using SDS-PAGE on 12% or 15% polyacrylamide gel. Western blot analysis was performed as described previously 52   Co-immunopreciptitaion (Co-IP). HeLa cells were grown in 6 cm dishes and transfected with plasmids. At 24 h post-transfection, cells were lysed with ice-cold lysis buffer containing 10 mM Tris-HCl pH 7.5, 0.4 M NaCl, 1% NP-40, 0.4% Triton X-100, 0.2% sodium deoxycholate, 1 mM EDTA, and protease inhibitors (Calbiochem, USA) for 30 min. Cellular debris were removed by centrifugation at 12,000 g for 15 min at 4 °C. The lysates were immunoprecipitated with antibodies and subsequently adsorbed onto Pierce ™ Protein A/G Magnetic Beads (Rockford, USA). Lysates were then collected by centrifugation and washed extensively with 1 ml of washing buffer (10 mM Tris-HCl, pH 7.5, 0.2 M NaCl, and 1 mM EDTA). Immunoprecipitated proteins were solubilized by boiling in alkaline SDS loading buffer and were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) before analysis by immunoblotting as described previously 52 .
Isolation of caveolae-rich membrane fractions from HepG2 cells. Isolation of caveolae-rich membrane fractions from TFV-infected HepG2 cells in sucrose density gradients was performed. TFV-infected HepG2 cells were washed twice with Ca 2+ -and Mg 2+ -deficient phosphate-buffered saline and scraped in 1 ml of ice-cold lysis buffer containing 1% Triton X-100, 25 mM morpholineethanesulfonic acid (pH 6.5), 150 mM NaCl, 5 mM EDTA, and protease inhibitors as described previously 36,37 . After homogenization, cell extracts were adjusted to 40% sucrose by mixing with an equal volume of the lysis buffer containing 90% (w/v) sucrose (without Triton X-100 and protease inhibitors). Subsequently, cell extracts were placed at the bottom of a 12 ml ultracentrifuge tube. A discontinuous gradient was formed above the lysate by adding 6 ml of 30% sucrose solution and 4 ml of 5% sucrose solution. Samples were centrifuged at 190,000 g for 16 to 20 h at 4 °C. Fractions (1 ml) were collected from the top of the gradient. Aliquots of each sucrose density gradient fraction were resolved by SDS-PAGE and analyzed by immunoblotting.
Immunofluorescence assay (IFA). HepG2 cells were grown at low density on glass coverslips. Cells were infected with TFV at an MOI of 10. At 72 h postinfection, cells were fixed for 15 min with 4% formaldehyde in PBS at room temperature. Nonspecific binding was blocked by 30 min incubation in 5% normal goat serum. TFV MCP and caveolin-1 were detected by simultaneously staining cells with specific antibodies diluted in PBS. Cells were then washed, and antibody binding was detected using host-specific Alex Fluor-conjugated secondary IgGs (Invitrogen, Carlsbad, CA, USA) as described previously. The coverslips were then washed several times with phosphate-buffered saline with Tween 20 and incubated with Hochest 33342 (Invitrogen, Carlsbad, CA, USA). The samples were examined under a confocal microscope (Zeiss LSM510, Germany). Mock-infected cells were similarly stained as controls.
Drug treatments. Drug treatments of HepG2 cells at late phase postinfection were performed. HepG2 cells were infected with TFV at an MOI of 10 and were treated with Mβ CD or nystatin at 60 h postinfection, control cells were incubated in medium with the corresponding solvent. PCR analysis TFV genome. TFV genome was determined using two TFV genes, TFV mcp gene orf096R and TFV gene orf031L, located at different region of TFV genome. The primer of TFV mcp gene orf096R (forward primer 5′ -ATGTCTTCTGTAACTGGTTC-3′ ) and (reverse primer 5′ -TTACAAGATTGGGAATCCCATC-3′ ) were used to amplify a region from 95938 to 97329 of the TFV genome (GenBank Accession No. AF389451.1). the primer of TFV orf031L, forward (forward primer 5′ -ATGCGTTGTATGCGCTCCGG -3′ ) and reverse (reverse primer 5′ -TCAAGTCCTCGGAGACGTGA-3′ ) were used to amplify a region from 3582 to 36122 of the TFV genome (GenBank Accession No. AF389451.1).
Absolute quantitative real-time PCR analysis. TFV genome was determined using a LightCycler 480 instrument (Roche, Germany). Absolute qPCR was performed using TFV mcp gene orf096R forward (forward primer 5′ -TCGCTGGTGGAGCCCTGGTA-3′ ) and reverse (reverse primer 5′ -GGCGTTGGTCAGTCTGCCGTA -3′ ) primers. The primers were used to amplify a region from 97034 to 97163 of the TFV genome (GenBank Accession No. AF389451.1). Plasmid pCMV-myc, which contains TFV orf096R, was used as the internal standard. The internal standard plasmid was serially diluted by 10-folds to generate a standard curve of absolute qPCR. DNA from the supernatant of infected cells was extracted using PureLink ™ Viral RNA/DNA Mini Kit (Invitrogen, Carlsbad, CA, USA). The extracted DNA template and the internal standard plasmid were subjected to absolute qPCR. The PCR reaction mixture (10 μ l) contained 5 μ l 2 × SYBR Premix Ex Taq (TaKaRa, China), 1 μ l DNA template, 0.2 μ l of 10 μ M primers, and 3.6 μ l H 2 O. The absolute qPCR conditions were as follows: one cycle at 95 °C for 10 s, 40 cycles of 5 s at 95 °C, 40 s at 60 °C, and 1 s at 72 °C. Absolute qPCR was performed at three replicates per sample. Immunogold labeling. The purified viral particles were absorbed onto Formvar-coated, carbon-stabilized 200-mesh nickel grids. After semi-drying for 15 min, the grids were blocked with 3% bovine serum albumin for 30 min at 37 °C. The grids were rinsed with PBS and then incubated in antiserum against caveolin-1 or the thioredoxin control (1:100 dilution) for 1 h at 37 °C. After washing with PBS, the grids were incubated with goat anti-rabbit IgG conjugated with 10 nm colloid gold (Sigma, USA) for 1 h at 37 °C. The grids were then washed with distilled water and negatively stained with 2% phosphotungstic acid. The viral envelope was removed with 1% (v/v) alkyl phenoxy polythoxy enthanol (Triton X-100) as described previously. The specimens were examined under a transmission electron microscope (JEM-100CX II, Japan).