Impact of temperature on DENV RVP production kinetics and decay

Because protein stability and folding efficiency may be greater at lower temperatures, we investigated whether more efficient DENV RVPs production could be achieved at reduced temperatures. T-REx-293-DGZ-WP cells were induced with tetracycline, incubated at the indicated temperatures and sampled for the release of infectious RVPs over the course of several days (Fig. 2A). Cells incubated at lower temperatures produced infectious RVPs more slowly than those incubated at 37 °C, consistent with the slower kinetics of structural gene expression in cells at lower temperatures (data not shown). In contrast, production of DENV RVPs at 37 °C was relatively transient and achieved modest titers relative to the peak RVP titers achieved using lower temperatures (Fig. 2A). The titer of DENV RVPs in supernatants of cells at 37 °C decreased significantly (46.6±0.1 fold, n=6, p<0.0001) between 24 and 48 h (Fig. 2D), whereas titers at 28 °C and 30 °C increased during the same time frame ((16.4±0.4 fold, p=0.0004) and (3.4±0.3 fold, p=0.0012) for 28 °C and 30 °C, respectively). By comparison, while the kinetics of WNV RVP production were also affected by temperature in a similar fashion, WNV RVP infectious titers at both 24 and 48 h were similar (Figs. 2B and 2E). Furthermore, that the production of DENV RVPs by HEK-293T cells transfected with DENV structural genes and a WNV replicon was also transient at 37 °C (Figs. 2C and 2F) suggests the rapid decrease in DENV RVP titers released from T-REx-293-DGZ-WP cells does not reflect unique properties of the DENV replicon packaged by these particles nor the inducible cell line used to produce them. We noted that the kinetics of RVPs released from transfected cells are slightly delayed relative to the inducible cell line. This may reflect the time required for transcription of the WNV DNA “launched” replicon RNA and the initiation of replicon RNA replication. This was not due to an intrinsic difference in the replication rate between WNV and DENV replicons, as the former replicates faster and to higher levels than the DENV replicon (Fig. S2).

Open in a separate window

Fig. 2

Temperature-dependent production of RVPs. (A) DENV1 RVPs were produced by induction of T-REx-293-DGZ-WP cells with tetracycline, followed by culture at the indicated temperatures. Culture supernatants were collected at the indicated times post-induction and titered using Raji-DCSIGNR cells. Infection was measured as a function of the percentage of GFP expressing cells at 72 h post-infection by flow-cytometry. (B) WNV RVPs were produced by transfection of HEK-293T cells with plasmids encoding a sub-genomic WNV replicon and WNV C-prM-E. Supernatants were collected at the indicated times post-transfection and titered as described above. (C) DENV1 RVPs that encapsidate a WNV genome were produced by transfection of HEK-293T cells with plasmids encoding a sub-genomic WNV replicon and DENV1 C-prM-E (Western Pacific strain). DENV RVPs were harvested at the indicated times and titered as described above. (D–F) To obtain a more quantitatively rigorous understanding of the temperature-dependent differences in DENV and WNV RVP production, RVPs were produced in replicate wells of a 6-well plate as described for A–C. RVP-containing supernatants were collected at 24 h and 48 h after induction and titered using Raji-DCSIGNR cells in triplicate. Infection is expressed relative to the titer achieved at 48 h post-infection at 37 °C. Error bars reflect the standard error of two independent production experiments.

To investigate the mechanisms that limit the duration and overall titer of DENV RVP production at 37 °C, we next measured the rate of RVP release from producer cells, and their decay in culture supernatants. The rate of virion release was estimated by measuring the titer of RVPs produced from cells during a four-hour window at 24 or 48 h post-induction or transfection (DENV and WNV, respectively). As suggested by our longitudinal and cross-sectional studies above (Figs. 2A and 2B), the rate of infectious DENV RVP production decreased significantly between days one and two for cells cultured at 37 °C (99.5± 0.1 fold, p=0.0032)(Fig. 3A), corresponding to the dramatic decrease in infectious DENV RVP titer present in culture supernatants by day two observed in longitudinal and cross-sectional sampling (Fig. 2A). By comparison, the rate of infectious WNV RVP release was only modestly reduced during the same interval. Infectious DENV RVP release from T-REx-293-DGZ-WP cells incubated at 28 °C and 30 °C increased significantly between day one and two (5.74±0.04 fold, p=0.0022, and 1.94±0.04 fold, p=0.0057, respectively). Analysis of the decay of the infectivity of DENV RVPs at each production temperature indicates that they are significantly less stable at 37 °C (T_1/2=8.3 h, n=7) as compared to 28 °C (T_1/2=34.2 h, n=6)(p<0.0001)(Fig. 4). WNV RVPs are significantly more stable than DENV RVPs at both 28 °C (T_1/2=65.7 h, n=4) (p=0.0025) and 37 °C (T_1/2=15.1 h, n=4) (p=0.0294).

Open in a separate window

Fig. 3

Temperature-dependent changes in the rate of DENV RVP production. The rate of infectious DENV and WNV RVP production at 24 and 48 h post-induction/transfection was investigated. DENV RVPs were produced by induction of T-REx-293-DGZ-WP cells (A) and WNV RVPs were produced by transfection of HEK-293T cells (B) as described in Fig. 2 and the Materials and methods. Culture supernatants were removed from duplicate wells at 22 h (d1) or 46 h (d2) post-induction and immediately replaced with conditioned media pre-equilibrated at the indicated temperature. Supernatant was collected 4 h later and titered in duplicate using Raji-DCSIGNR cells. Infection was measured as a function of the percent of GFP expressing cells at 72 h (A) or 48 h (B) post-infection by flow-cytometry. The data presented is representative of three independent experiments.

Open in a separate window

Fig. 4

Stability of WNV and DENV RVPs. (A) DENV RVPs were produced using T-REx-293-DGZ-WP cells and aliquoted (500 μl) into wells of a 48-well plate. Plates were incubated at the indicated temperatures. At the indicated times, RVPs were removed from duplicate wells and frozen at −80 °C. The titer of RVPs from each time point was determined simultaneously by infecting Raji-DCSIGNR cells with serial two-fold dilutions of RVPs in complete RPMI-1640 medium. Infection was measured as a function of GFP expression at 72 h post-infection by flow-cytometry. Non-linear regression analysis was performed to generate one-phase exponential decay curves using GraphPad Prism version 4.0b. Dotted lines indicate the 95% confidence interval for the regression analysis. (B) The average half-life of WNV and DENV RVPs at 28 °C or 37 °C is displayed. Error bars indicate the standard error of multiple independent measurements of the stability of DENV and WNV at each temperature (n=6 and n=7 for DENV and WNV at 28 °C; n=7 and n=4 for DENV and WNV at 37 °C). Statistical comparisons were made using GraphPad Prism version 4.0b (unpaired t test).

Together, our data indicates that differences in the efficiency of WNV and DENV RVP production reflect both the dynamics of RVP production and differences in the stability of infectious RVPs at 37 °C. The titer of DENV RVPs produced at 37 °C is limited due to the transient release of virions by cells as measured functionally (Fig. 3A) and by the presence of RVP-associated replicon RNA in the supernatant (Fig. S3). Why DENV RVP producing cells stop releasing infectious virus particles is presently unclear, but does not appear to reflect overt cytotoxicity conferred by DENV protein expression. LDH-release from RVP producing cells increased only modestly with time at 37 °C, and was not dependent upon DENV structural gene expression. The level of replicon replication in 293T producer cells also decreased with time in culture at this temperature (~2-fold) (Fig. S4). As neither change in producer cell phenotype occurred with kinetics that parallel the rapid reduction of DENV RVP production observed above, it remains unclear whether these factors govern RVP production efficiency, or reflect changes that occur during prolonged culture of 293T cells at high density. Instead, the transient production of DENV RVPs at 37 °C revealed by longitudinal sampling (Fig. 2A) can be explained by the termination of infectious RVP release shortly after day one post-induction/transfection, followed by the decay of virions already released into the media with a half-life of approximately 8 h. Production at lower temperatures is more efficient because it increases the time during which producer cells release DENV RVPs, as well as decreases the rate of virion decay in the culture supernatant.

Infection of multiple cell lines with DENV RVPs

The expression of DCSIGN (CD209) and DCSIGNR (CD209L) on cell lines has been shown to significantly increase the efficiency of DENV infection, reflecting the role of these lectins in promoting more efficient and durable attachment of the virus to target cells (Navarro-Sanchez et al., 2003; Tassaneetrithep et al., 2003). Thus, to maximize the sensitivity of our RVP production optimization experiments we utilized a cell line that expresses DCSIGNR. We next explored whether DENV RVPs produced using the plasmid complementation approach described above were capable of infecting cell lines that do not express known attachment factors, including those commonly employed in studies of flaviviruses (BHK21 and Vero). DENV and WNV RVPs were produced by transfection of HEK-293Ts with plasmids encoding the structural proteins of either virus and a WNV replicon encoding the Renilla luciferase gene. The rationale for the selection of a WNV replicon was: i.) our studies described above did not suggest that the use of a heterologous replicon was associated with a significant reduction in RVP titer (compare Figs. 2B and C), ii.) to control for post-entry differences in replication between DENV and WNV, and iii.) the kinetics of reporter gene expression by WNV replicons is more rapid than that observed with DENV replicons of similar design (25 h vs 98 h for WNV and DENV replicons, respectively). Serial four-fold dilutions of RVPs were used to infect eleven cell lines of varying origin. Analysis of the signal obtained at the highest RVP dilution tested indicates varying degrees of permissiveness, as expected from published studies (Fig. 5) (Anderson, 2003; Clyde et al., 2006; Diamond et al., 2000). Signals greater than 1000-fold above background were obtained for both DENV and WNV RVPs on the most permissive lines, whereas infection of the Jurkat T cell line was not observed with either WNV or DENV RVPs. To get a more precise estimate of the relative infectivity of DENV and WNV RVPs on these lines, we next measured the genome content of each preparation and used the resulting data to normalize the level of infectivity between the two types of virus particles. To our surprise, we found that the ratio between infectivity and the relative numbers of replicon genomes in stocks of DENV RVPs was relatively low as compared to WNV RVPs, despite the similar infectious titers observed using several different cell lines (Fig. 5B). Interestingly, as a function of the relative number of genome copies, DENV RVPs were more infectious than WNV in certain cell lines (e.g. BHK) that do not express any known flavivirus attachment factor, suggesting that these particular strains of DENV (16681) and WNV (NY99) might utilize distinct attachment factors to infect certain cell lines (Fig. 5B).

Open in a separate window

Fig. 5

Infection of a panel of cell lines with DENV RVPs. (A) WNV and DENV RVPs were produced in HEK-293T cells transfected with a WNV sub-genomic replicon and plasmids encoding either WNV (blue) or DENV2 16881 (red) structural genes. Serial four-fold dilutions of RVPs were used to infect each cell line in quadruplicate as described in the Materials and methods section. Infection was scored as a function of Renilla luciferase activity 48 h post-infection. The signal obtained with undiluted RVPs is shown, with error bars indicating the standard error. (B) The infectivity of each population of RVPs was normalized as a function of the relative genomic RNA content of each preparation. Genomic RNA was measured using real-time qRT-PCR as described (Geiss et al., 2005; Houng et al., 2001).

Plasmids

The WNV sub-genomic replicon and C-prM-E expression plasmid used throughout the study were described previously (Pierson et al., 2006). The DENV2 replicon was constructed as described below. DENV C-prM-E expression constructs (Western Pacific and 16681 strains) were constructed by topoisomerase mediated cloning into the pENTR/ D Gateway cloning entry vector. Inserts were sequenced and then transferred into pcDNA 6.2 and pT-REx-DEST30 destination vectors. The latter vector was used to construct the tetracycline-inducible cell lines described above.

Construction of a DNA-launched DENV2 replicon

A sub-genomic DENV2 replicon was constructed by modification of a molecular clone of the DENV2 strain 16681 using an approach described initially by Varnavski et al. (2000). Starting material for the work was the pD2/IC-30P molecular clone of the 16681 strain of DENV (Kinney et al., 1997). First, the majority of sequence encoding the structural genes of 16681 was deleted, and the T7 promoter of the pD2/IC-30P construct was replaced with the CMV immediate-early promoter/enhancer. To do this in the smallest number of steps, we performed overlapping PCR to fuse a fragment of a previously described sub-genomic WNV replicon that encodes the CMV promoter, WNV 5′ untranslated region, a small portion of the WNV capsid, and FMDV 2a autoprotease, and a MluI site with the DEN ORF (Pierson et al., 2006). The resulting PCR fragment was cleaved at BstX1 and MluI sites engineered into the primers and inserted into the pD2/IC-30P vector using unique SacI and KpnI sites. Importantly, the SacI site present in pD2/IC-30P was destroyed during this step, allowing the replacement of the WNV sequence in this cloning intermediate with sequence encoding the DEN 5′ UTR and 24 residues of DENV capsid. This was accomplished using a single PCR fragment and novel SacI and MluI sites engineered into the primers. The ribozyme of hepatitis delta virus (HDV) was then introduced into the 3′ end of the replicon using overlap extension PCR and introduced into the replicon via unique AvrII and ClaI sites present in the vector backbone. Subsequently, a GFP/Zeocin resistance reporter gene was inserted into the unique MluI site located just downstream of the FMDV2a autoprotease.

Production of reporter virus particles

RVPs were generated using two different approaches: i.) by transfection with plasmids encoding a sub-genomic replicon and structural genes (C-prM-E) as described (Pierson et al., 2006), or ii.) by induction of C-prM-E expression in 293T-Rex cell lines that stably propagate a DENV2 replicon. Production of RVPs by transfection was accomplished by introducing plasmids encoding a replicon and structural proteins (in a 1:2 ratio, by mass) into pre-plated HEK-293T cells using Lipofectamine 2000. RVPs were also produced by transfection of T-REx-293-DGZ cells with plasmids encoding DENV structural proteins. Four hours after transfection, culture media was removed and replaced with a low glucose formulation of DMEM containing 10% FBS and 100 U/ml PS and incubated at the indicated temperatures. RVP production using T-REx-293-DGZ-WP or T-REx-293-DGZ-16681 inducible cells lines was achieved following the addition of 1 μg/ml tetracycline. Using optimal production conditions for DENV RVPs, transfected cellswere incubated at 30 °C for approximately 72 h. RVP-containing supernatants were harvested, filtered using a 0.22 μm syringe filter and stored at −80 °C in complete low glucose DMEM.

RVP infections

The infectious titer of RVPs was typically determined by using Raji-DCSIGNR cells. Serial two-fold dilutions of RVPs were prepared using complete RPMI, and then added to Raji-DCSIGNR cells plated in 96-well plates at 5×10⁴ cells per well on the day of the assay. Infections were performed in a total volume of 200 μl. The percentage of cells infected at each dilution was scored as a function of GFP expression using flow-cytometry two days (when RVPs package a WNV replicon) or three days (when RVPs package a DENV replicon) post-infection. The rationale for performing serial dilutions of virus in each experiment reflects the lack of a linear relationship between virus dilution and the percentage of infected cells at all dilutions. In all cases, comparisons between RVP preparations were made at dilutions on the linear parts of the infection–dilution curve.

To compare the relative capacity of different cell lines to support E protein-mediated WNV or DENV entry, adherent cells were plated the day before the assay in 48-well plates ((BHK21, H1080, Huh-7 (20 k cells/well); Vero (30 k cells/well); HEK293T (40 k cells/well)). On the next day, media was removed and cells were infected with 100 μl of serial four-fold dilutions of RVPs. Cells were cultured at 37 °C overnight, and then media was replaced with 200 μl of a low glucose formulation of DMEM. In contrast, suspension cell lines (Raji, K562, and Jurkat) were plated in 96-well plates on the day of the assay (50 k cells/well) and infected with serial dilutions of RVPs (200 μl total volume). The relative infectivity was measured as a function of Renilla luciferase reporter gene expression at two days post-infection according to the manufacturers instructions.

Western blotting

Envelope protein expression in RVP-producing cells was detected by Western blot using the flavivirus pan-specific monoclonal antibody 4G2. Cells were lysed in 200 μl cell lysis buffer (1×PBS; 50 mM Tris. HCl; 150 mM NaCl; 2 mM EDTA; 1% Triton-X; protease inhibitor cocktail), spun briefly to remove cellular debris, and analyzed by BCA following the manufacturer’s protocol (BCA Protein Assay Kit—Pierce). Roughly 50 μg of total protein was analyzed by SDS-PAGE under non-reducing conditions and immunoblotted with 4G2 at 1 μg/ml.

Quantitative real-time PCR

The genomic RNA content of RVP populations was measured using a modification of a previously described protocol (Hanna et al., 2005). Briefly, RVP-containing supernatants were treated with 100 U recombinant DNAse I, followed by RNA isolation using the QiaAmp Viral RNA kit per the manufacturers instructions (Qiagen, Hilden, GE). Amplification of viral genomic RNA was accomplished using the Superscript III one-step RT-PCR system (Invitrogen, Carlsbad, CA) and primers specific for either the 3′ untranslated region of WNV lineage II replicon (Geiss et al., 2005) or dengue 2 replicon (Houng et al., 2001).

This work was supported by the Intramural Research Program of the NIH, National Institutes of Allergy and Infectious Diseases (NIAID) and by the Pediatric Dengue Vaccine Initiative (PDVI). We are grateful to Qing Xu for technical support and Dr. Bridget Puffer for providing the DENV C-prM-E expression vectors used throughout this study, for excellent technical assistance, and productive discussions. We thank Drs. Christopher Buck, Subhajit Poddar, and Michael Diamond for useful discussions and their comments on the manuscript.