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

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 meas

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

DENV-2 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

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, 3 and 7 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

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 7 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

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

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

Figure 3-3 Linear regression analysis between RNA copy number and MID 50 (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

A

B

C

Table 3-1 Comparative titration of C6/36 cell culture virus supernatants by PCR and mosquito inoculation (N=3)

qRT-DENV serotype Copy number/mL MID 50 /mL

Log Difference (p-value)

DENV-1 EDEN2928 5.81E+10 5.88E+08 2 (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)

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 2 to 5 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 non-

infectious 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 qRT-PCR to quantify infectious virus

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

Figure 3-4 Comparative titration of ten viremic (DENV-2) human sera by qRT-PCR and mosquito inoculation technique.

3.2 Investigate the role of the UPP in DENV life cycle

3.2.1 Functional UPP is required for infectious DENV-2 production in mosquito midguts

Although many studies have identified the UPP to be important for successful DENV production, how the UPP contributes to DENV life cycle as host factors remains ill

defined Consequently, none of the licensed proteasome inhibitors have been tested in vivo due to 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, 2002) Upon the establishment of a successful infection in these organs, the infection is life-long and the continued production of DENV, especially in the salivary glands, enables

infection of new susceptible human hosts and thus ensures the survival of DENV In the midgut, however, infectious DENV titers decrease after their peak at 7-10 days post blood meal (dpbm) despite continued replication of its RNA genome (Salazar et

al, 2007)

We first tested if the observation described previously (Salazar et al, 2007) could be

replicated in our hands by characterizing virus replication in the midgut of Ae aegypti

following ingestion of blood spiked with DENV-2 DENV-2 infection was detected in midgut epithelial cells as early as 2 dpbm, peaked at 8 dpbm and decreased thereafter Paradoxically, measurement of the DENV-2 genomic material by qRT-PCR revealed

no significant reduction in the viral RNA copy number as late as 21 dpbm (Figure 5A) This observation recapitulated previously reported findings (Salazar et al, 2007)

3-In contrast, both DENV-2 titers and viral RNA copy number in the heads/thoraces

were positively correlated through to the limit of the lifespan of Ae aegypti in the

laboratory (Figure 3-5B) This observation recapitulated previously reported findings (Salazar et al, 2007) and guides our experimental design to study proteasome function

8 days post infection (dpi)

To demonstrate a functional requirement of proteasome on the virus life cycle besides

viral entry, RNAi-mediated gene silencing of the catalytic subunits of the proteasome, β1 (capase-like activity), β2 (trypsin-like activity) and β5 (chymotrypsin-like

activity), was performed in vivo as described elsewhere (Garver & Dimopoulos,

2007) Female mosquitoes were orally infected with DENV-2 before inoculation of dsRNA at 3 dpbm to preclude the possibility that knockdown of these genes could interfere with endocytosis and hence DENV entry in the midgut epithelial cells

(Hicke, 2001; Krishnan et al, 2008) DENV-2 infected mosquitoes inoculated with dsRNA targeting sequences from pGEM T easy vector served as a control for these experiments The mosquito midguts were then harvested and analyzed at 6 dpbm (Figure 3-6A), the time-point before infectious DENV-2 titers decreased Efficacy of RNAi-mediated knockdown was assessed by gene-specific qRT-PCR (Figure 3-6B) and the effect of their knockdown on viral propagation was measured by both plaque assay and DENV-specific qRT-PCR Compared to the dsRNA control, silencing of

the β1, β2 and β5 subunits of the proteasome resulted in a significant reduction of

infectious DENV-2 titers (Figure 3-6C) but not viral genomic RNA (Figure 3-6D) Correspondingly, ratio of PFU to RNA copy number per midgut decreased

significantly (Figure 3-6E) Moreover, the proportions of infected mosquitoes were also significantly reduced with β1, β2 and β5 knockdown respectively (Table 3-2) Altogether, the data suggests a post-entry role for the UPP in the DENV life cycle

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 increase in both infectious particles and viral RNA are coupled over time Error bar ± SEM, N=8-10

Figure 3-6 Proteasome inhibition decouples infectious DENV-2 production from viral RNA

replication in mosquito midguts (A) Workflow of gene silencing assay in Ae aegypti

mosquitoes (B) Silencing efficiencies of β subunits of the proteasome were determined by specific qPCR, and expression values were normalized against control N=10 (C) Virus titers per midgut declined significantly after knockdown of β1, β2 and β5 subunits at 6 dpbm Student’s t test, *p < 0.05, **p<0.01 (D) No statistically significant differences were observed in DENV-2 viral RNA levels per midgut 6 dpbm after gene knockdown (E) log(PFU/Copy Number) was

gene-significantly lower after β1, β2 and β5 knockdown N=7-16 Student’s t test, **p<0.01

Table 3-2 Percentage of DENV-2-infected mosquitoes after knockdown of proteasome subunits (p value; Fischer’s Exact Test)

Percentage with infectious particles detected (%)

3.2.2 Regulation of UPP-specific genes decouples infectious DENV-2 production from viral RNA replication in mosquito midguts

The observed decoupling of infectious virus production despite persistent DENV genome replication after proteasome β-subunit knockdown is reminiscent of the

observed outcome in the Ae aegypti midgut naturally following an infectious blood

meal We hypothesized that differential regulation of the proteasome or other genes in the UPP could be involved in the observed reduction in infectious DENV-2 titers in the midgut but not the head/thorax To test this, we utilized RNAseq to analyze the

transcripts of the female Ae aegypti midgut at 8 dpbm This approach overcomes the

limitation of mosquito protein-specific reagents as well as difficulty in designing oligonucleotide primers that accurately complement outbred, field-collected

mosquitoes supplemented monthly (10%) to the Ae aegypti colony in our insectary

This methodology also enabled us to characterize genes regulated at the

post-transcriptional level (Sessions et al, 2013) and serves as a reference for future studies The experimental workflow is depicted in Figure 3-7A Mosquitoes that were fed on blood without spiked DENV-2 served as control RNAseq analysis was performed for

a pool of 100 dissected midguts and compared to a similarly pooled control using Cufflinks v13.0 (Trapnell et al, 2010) The quality of our libraries and sequencing performance was assessed using Partek Genomic Suite v6.6 (Partek Incorporated) (Table 3-3)

Our results showed no differential regulation in any components of the proteasome Instead, several genes within the UPP were differentially regulated (Figure 3-7B)

Blood meal spiked with DENV-2

Starve 3-4 days old

female Ae aegypti

Midgut dissection Total RNA extraction

Sequencing

Partek, TopHat and Cufflinks

8 days

1 day

Figure 3-7 RNA-sequencing of Ae aegypti midgut (A) Experimental workflow of

transcriptomic analysis of Ae aegypti midgut 8 dpbm (B) Differentially regulated genes (red for down-regulation, blue for up-regulation) belonging to the UPP in KEGG pathway (Ae aegypti)

P-value is lesser than the FDR < 0.1 after Benjamini-Hochberg correction for multiple-testing

A

B

Table 3-3 Summary of Illumina HighSeq 2000 RNA-sequencing using Partek Genomic Suite v6.6

Percentage of transcripts with reads

Total reads with junctions

Reads with junctions that are compatible with a transcript

A subset of these were validated by qRT-PCR in a separate experiment using

individual midguts from mosquitoes fed with either DENV-2 infected or uninfected blood meal (Figure 3-8A) Interestingly, the 3 genes that were down-regulated in the midgut were significantly up-regulated in the head/thorax at 21 dpbm (Figure 3-8A), when peak viral titers and RNA levels were observed (Figure 3-5B) These

observations suggest that down-regulation of the 3 UPP-related genes, UBE2A (AAEL002118), DDB1 (AAEL002407), UBE4B (AAEL006910) in the midgut may be

responsible for the decoupling of infectious DENV-2 production from viral RNA replication

If expression of these 3 UPP-related genes were required for DENV production, then

it follows that these genes must be up-regulated prior to the peak of virus replication

at 8 dpbm, to allow for systemic spread of DENV2 to the salivary glands This must occur before virus production is suppressed in the midgut We thus examined the kinetics of the expression of these 3 genes Expression levels of these 3 UPP genes

were measured at 2, 4, 6, 8, 11 and 14 dpbm (Figure 3-8B) UBE2A, DDB1 and UBE4B were significantly up-regulated at 6 dpbm compared to uninfected blood-fed

control, but were all down-regulated from 8 dpbm onwards

To demonstrate a functional requirement of UBE2A, DDB1 and UBE4B on DENV

production in the mosquito, RNAi-mediated gene silencing was performed Gene

silencing was also performed for UBE2M (AAEL009026), which was not detected in

our RNAseq analysis as a negative control Efficacy of RNAi-mediated knockdown was assessed by gene-specific qRT-PCR (Figure 3-9A) When compared to the

dsRNA control, knockdown of UBE2A and DDB1 produced a similar outcome and