Investigation of the role of the ubiquitin proteasome pathway in dengue virus life cycle 2

  Chapter 3: Results 3.1 Establish a mosquito infection model at Duke-NUS Graduate Medical School To investigate the role of UPP during DENV infection in mosquitoes, our first aim was to establish a mosquito infection model in our laboratory. As the colony of Ae. aegypti was only recently established in 2010 from field-caught specimens in Singapore, an important preliminary work would be to set up assays to measure DENV concentration in mosquitoes, as well as their individual organs. To this end, Ae. aegypti mosquitoes were intra-thoracically inoculated with DENV-2 to ensure all mosquitoes were infected, and virus kinetics was measured. Two low passage DENV2 strains (PR1940 and PR6913) with contrasting virus replication kinetics (Data from G. Manokaran) isolated during a 1994 epidemic in Puerto Rico were used. Infected mosquitoes were harvested at 3, 7, 10, 14 and 17 days post infection (dpi). Infectious titers were measured using both the mosquito inoculation technique (mosquito infectious dose 50, MID50) and plaque assay (plaque forming unit, PFU), while RNA copy number was measured using qRT-PCR. Three individual mosquitoes at each time point were triturated and titrated. 3.1.1 Comparison of mosquito inoculation technique and qRT-PCR to measure DENV concentration in mosquitoes, vertebrate and mosquito cell cultures, and human sera Although qRT-PCR has been compared to plaque assay (Bae et al, 2003; Colton et al, 2005; Richardson et al, 2006), the actual ratio of RNA copy number to infectious viral titer remains unclear. Moreover, it is not known to what degree the infected host, the virus strain, or time of infection may influence that ratio. In order to better define the   67     quantitative and biological relationships between RNA copy number and infectious DENV, we compared qRT-PCR with the mosquito inoculation technique and plaque assay using the data we have while optimizing these assays to measure DENV concentrations in mosquitoes. As a control, quantitative comparisons were performed using vertebrate and mosquito cell cultures. Both Ae. albopictus derived C6/36 and African green monkey derived Vero cells were inoculated with 0.1 multiplicity of infection of each virus. Cell culture supernatants were harvested on 1, and dpi. The replication kinetics of DENV in live Ae. aegypti mosquitoes, C6/36 and Vero cell cultures showed that RNA copy number was typically 2–3 logs greater than the MID50 titer, regardless of the host tissue or cell culture from which the virus was harvested (Figures 3-1 and 3-2). When titers per whole mosquito were compared, the RNA copy number was 100 to 1,000 times higher than the MID50 titer, which was 100 to 1,000 times higher than the PFU measured by plaque assay (Figure 3-1). This difference was evident for both DENV-2 strains (PR1940 and PR6913), regardless of the maximum titers observed in all assay platforms. In general, linear regression showed that the RNA copy number was correlated with MID50 titers for DENV-2 in mosquitoes (P < 0.0001, R2 = 0.567) and cell cultures (P < 0.0001, R2 = 0.950) (Figure 3-3A). However, the slopes differ significantly (P < 0.001, F = 13.95), showing that the ratio of RNA copy number to infectious virus may differ when using different host systems to grow DENV. Although there is a relatively good general correlation between the MID50 titers and RNA copy number using the same host systems to grow DENV, the accuracy of measuring infectious DENV using RNA copy number may vary based on the virus   68     strain or time of infection as the ratio may be significantly different from one another (Figures 3-3B and C). Different conversion ratios were also shown for different serotypes of DENV, with day old C6/36 virus supernatants for DENV-1, DENV-3, and DENV-4 showing 2.0, 0.7, and 2.5 logs higher concentrations by qRT-PCR, respectively (Table 3-1). Of interest, DENV-3 concentrations varied by only 0.7 log between the two methods. This small difference could be a unique replication characteristic of that virus strain or result from the specific time in viral growth when it was sampled.   69     A B       Figure 3-1 Replication kinetics of DENV-2 (A) PR1940 and (B) PR6913 in adult female Ae. aegypti mosquitoes. Virus titers are measured by plaque assay (PFU/mosquito ), mosquito inoculation technique (MID50/mosquito n) and qRT-PCR (RNA copy number/mosquito Ÿ). Error bar ± SD. N=3.                       70     A B C D       Figure 3-2 Replication kinetics of DENV-2 PR1940 and PR6913 derived in cell cultures measured by the mosquito inoculation technique (MID50/mL n) and qRT-PCR (RNA copy number/mL Ÿ). (A) PR1940 in Vero cell culture. (B) PR1940 in C6/36 cell culture. (C) PR6913 in Vero cell culture. (D) PR6913 in C6/36 cell culture. Error bar ± SD. N=3.                                   71     A B                                                   C   Figure 3-3 Linear regression analysis between RNA copy number and MID50. (A) RNA copy number vs MID50 in Ae. aegypti mosquitoes (Ÿ) and cell cultures (ο). The regression equations are DENV-2 copies = 0.653 MID50 + 4.93 (R2 = 0.567) and DENV-2 copies = 1.05 MID50 + 2.14 (R2 = 0.950) respectively. The two slopes are significantly different. (B) Ratio of genomic equivalents (GE) to MID50 at different time-points for PR1940 and PR6913 in mosquitoes. (C) Ratio of genomic equivalents (GE) to MID50 at different time-points for PR1940 and PR6913 in cell cultures. Error bar ± SD. N=3.       72       Log Difference DENV serotype Copy number/mL MID50/mL (p-value) DENV-1 EDEN2928 5.81E+10 5.88E+08 (0.0019) DENV-3 Indon1219 1.57E+09 2.88E+08 0.7 (0.0006) DENV-4 EDEN2270 1.83E+10 5.88E+07 2.5 (0.0009)                   Table 3-1 Comparative titration of C6/36 cell culture virus supernatants by qRTPCR and mosquito inoculation (N=3).   73     DENV-2 RNA copy number and MID50 values were also compared in viremic human sera, obtained from patients during a 2011 epidemic in Pakistan (Khan et al, 2013). A greater variation in virus concentration was observed when measuring DENV-2 viremias in 10 human sera (Figure 3-4). The difference in serum viremia level as measured by the two methods, varied from to logs, depending on the individual serum. No correlation was observed for RNA copy number and MID50 titers for human sera (p=0.3109). This is the first direct comparison of qRT-PCR with the mosquito inoculation technique in the measurement of DENV concentration. Our results agree with previous studies, which show positive correlations between flavivirus RNA copy number and infectious particles in cell cultures and Ae. aegypti mosquitoes (Bae et al, 2003; Colton et al, 2005; Richardson et al, 2006). A consistently higher, but variable RNA copies to infectious virus titer ratio is likely due to the presence of noninfectious immature virions or defective viral particles. However, the differences in ratio could also be due to intrinsic variation in virus replication or translational efficiencies in different host tissues. Of importance was the lack of correlation between RNA copy number and MID50 titers in human sera. Viremia in humans is influenced by the strain of virus, the day of infection the serum was collected from the patient, and the individual’s previous dengue experience, which influences the innate and adaptive immune response and thus, the production of noninfectious defective virus particles. As it is not accurate to quantitate infectious DENV in human tissues with the commonly used plaque assay, qRT-PCR is often used as a surrogate to measure viremia in patients. Clearly, our results question the relevance of using qRTPCR to quantify infectious virus.   74     From Section 3.1, a good general correlation exists between infectious DENV and genomic equivalents. However, the host, the virus strain, and time of infection may also influence the ratio of genomic equivalents to infectious DENV. An accurate measure of infectious virus is critical to understanding dengue virus biology and pathogenesis, as well as for the development of effective diagnostic tests, vaccines and therapeutics. Although qRT-PCR is a highly sensitive and useful DENV diagnostic tool, it measures only viral RNA and cannot replace the mosquito inoculation assay, which is arguably one of the most sensitive biological assays for measuring the infectious potential of low passage DENVs. Realizing that this will not be possible in most dengue diagnostic and research laboratories, qRT-PCR may be used as a valid and convenient proxy but the data should be interpreted with caution under specific experimental conditions. In summary, data from Section 3.1 indicate that a mosquito infection model for DENV has been established in the insectary in Duke-NUS and this will be used for subsequent investigations reported in Section 3.2.   75     Figure 3-4 Comparative titration of ten viremic (DENV-2) human sera by qRT-PCR and mosquito inoculation technique.     76     B A C Scale bar = 10 µm Figure 3-11 Proteasome inhibition with β-lactone did not alter virus entry at non-toxic levels. (A) No cytotoxicity was observed in THP-1 cells in the presence of β-lactone and genistein for 48 h using a cell viability assay. (B) Using flow cytometry, β-lactone does not block uptake of DENV-2 immune complexes. Genistein, an inhibitor of Fc receptor-mediated entry blocked uptake. (C) Alexa Fluor 647 labeled DENV-2 opsonized with h3H5 were internalized in cells pre-treated with DMSO or β-lactone, while genistein prevented the uptake of virus at hpi.     92     3.2.4 DENV-2 egress is dependent on proteasome function Infection of THP-1 cells with DENV opsonized with enhancing levels of h3H5 monoclonal antibody (Chan et al, 2011) resulted in the up-regulation of UBE2A and DDB1 at 48 hpi (Figure 3-12A), similar to the observation in mosquitoes. Moreover, inhibition of UPP in THP-1 cells recapitulated the decoupling of infectious virus production from viral RNA replication. Treatment with β-lactone resulted in a significant dose-dependent reduction in infectious DENV production (Figure 3-12B) without observable decrease in viral genomic RNA levels 48 hpi (Figure 3-12C). Correspondingly, the ratio of PFU to intracellular DENV genome decreased after βlactone treatment (Figure 3-12D). The observed decoupling of infectious DENV production from RNA replication in βlactone treated cells has two possible explanations. Firstly, newly formed DENV were released from the cells, but as immature and non-infectious particles. Alternatively, normal proteasome function may be critical for virus assembly or egress from infected cells. To test the first possibility, we measured viral RNA in the cell culture supernatant by qRT-PCR and compared it with the amount of infectious virions. Ratio of viral genomic RNA to PFU showed no statistically significant difference regardless of any drug concentration used (Figure 3-12E), indicating that treatment with βlactone did not result in the release of proportionately more immature DENV compared to DMSO treated cells.   93     A B D C E Figure 3-12 β-lactone decouples infectious DENV-2 production from viral RNA replication in THP-1 cells. (A) UBE2A and DDB1 in THP-1 cells were up-regulated 48 hours after DENV infection. (B) Proteasome inhibition showed a dose-dependent decrease in plaque titers 48 hpi. (C) DENV-2 RNA copy number per GAPDH showed no significant difference between β-lactone treated and DMSO control 48 hpi. (D) Ratio of infectious DENV-2 to genomic copies showed a dose-dependent decrease after β-lactone treatment. (E) Ratio of viral genomic RNA from supernatant to infectious DENV showed no significant differences after β-lactone treatment at different drug concentrations, compared to DMSO control, suggesting that the treatment with βlactone did not result in reduced DENV maturation. Error bar ± SD. N=4. Student’s t test, *p < 0.05, **p[...]... translation is the ER stress-induced eIF2αmediated translational repression of cellular mRNA eIF2α is an effector of the PKRlike ER kinase (PERK) pathway in the unfolded protein response (UPR) and phosphorylation of this protein prevents GDP-GTP exchange on eIF2α by the guanine nucleotide exchange factor eIF2B, thereby inhibiting recycling of the ternary complex that contains the initiator methionine Met-tRNAi... post-entry role for the UPP in the DENV life cycle   79     A B Figure 3-5 Characterization of DENV -2 replication in the midguts and head/thoraces of Ae aegypti following ingestion of an infectious blood meal (A) In the midgut, viral titers increased linearly until 8 dpbm and declined thereafter In contrast, viral RNA remained stable between 8 to 21 dpbm Error bar ± SEM, N=8-10 (B) In the heads/thoraces, the. .. uncertainty on their mechanism of action First, we sought to investigate the role of the proteasome in DENV infection in Ae aegypti mosquitoes, a natural insect host for DENV DENV first establishes infection in the midgut of the female mosquito after a viremic blood meal It then spreads systemically to the other organs, such as the head and salivary glands of the mosquito (Black et al, 20 02) Upon the. .. To further validate the function of UBE2A and DDB1 in the DENV life cycle, we tested if silencing of these two genes would also decouple infectious DENV -2 production from RNA replication in the head/thorax of Ae aegypti Mosquitoes were infected via intra-thoracic inoculation to ensure that all mosquitoes have disseminated infections by bypassing the midgut At 3 dpi, dsRNA was inoculated into the thorax... genistein prevented the uptake of virus at 2 hpi     92     3 .2. 4 DENV -2 egress is dependent on proteasome function Infection of THP-1 cells with DENV opsonized with enhancing levels of h3H5 monoclonal antibody (Chan et al, 20 11) resulted in the up-regulation of UBE2A and DDB1 at 48 hpi (Figure 3-12A), similar to the observation in mosquitoes Moreover, inhibition of UPP in THP-1 cells recapitulated the. .. β1, 2 and β5 knockdown N=7-16 Student’s t test, **p . (%) Infected/Total p-value Virus Control 83.3 10/ 12 - dsControl 80.0 16 /20 - dsβ1 31.8 7 /22 0.0 023 ds 2 40.0 8 /20 0. 022 5 dsβ5 45.8 10 /22 0. 028 9 ! ! 83! 3 .2. 2 Regulation of. humans is influenced by the strain of virus, the day of infection the serum was collected from the patient, and the individual’s previous dengue experience, which influences the innate and. mechanism of action. First, we sought to investigate the role of the proteasome in DENV infection in Ae. aegypti mosquitoes, a natural insect host for DENV. DENV first establishes infection in the