**3. Results**

#### *3.1. ZIKV Infection Decreases the Pool of Peroxisomes in Primary HFAs and U251 Cells*

Given the important roles of peroxisomes in antiviral defense and brain development, we investigated how ZIKV infection of the most abundant cell type in the fetal brain affects these organelles. Primary HFAs were infected with four different ZIKV strains including two pandemic strains of Asian lineage; one isolated during the 2015/2016 outbreak in South America (PRVABC59) and one isolated from an outbreak in French Polynesia in 2013 (H/PF/2013). The other two strains included a third contemporary Asian strain (PLCal) isolated from a returning Canadian traveler [33] and the prototype African strain MR766. The effects of ZIKV infection on peroxisomes and peroxisomal proteins in HFAs were assessed by confocal microscopy analysis and immunoblotting, respectively.

Data in Figure 1A show that depending upon the viral strain, the numbers of peroxisomes as identified by staining with anti-PMP70 antibodies were reduced by 60–70% at 48-h post-infection. To rule out the possibility that the apparent loss of peroxisomes in ZIKV-infected HFAs was not due to decreased expression of PMP70 alone, the steady levels of other peroxisome-associated proteins were assessed by immunoblotting at 24- and 48-h post-infection. This included PEX3, PEX7, PEX11B, PEX13 and PEX19, all of which are critical for peroxisome biogenesis. The loss of multiple PEX proteins was evident at 24-h post-infection; however, by 48-h, the reduction in PEX3, PEX7, PEX11B, PEX13 and PEX19 was much more pronounced (Figure 1B). PEX19 was most sensitive to viral infection as its levels were decreased by as much as 90% depending upon the infecting ZIKV strain. Unlike peroxisome biogenesis factors, levels of the peroxisomal matrix protein catalase were not affected by ZIKV infection (Figure 1B). This was not unexpected, however, as a previous study showed that unlike many other cellular proteins that are degraded when not targeted to their proper location, catalase is quite stable in the absence of peroxisomes [23]. Infection with PRVABC59 and PLCal strains was associated with the greatest loss of PEX proteins from HFAs. This did not appear to be due to more robust replication as the infection of HFAs with PRVABC59, which consistently resulted in slightly lower titers than the other three strains (Figure 1C), generally had the greatest effect on peroxisomes (Figure 1A,B).

Given that primary HFAs have a finite lifespan and limited expansion capacity, we assessed whether the human astrocytoma U251 cell line could be used to further examine the interplay between ZIKV infection and peroxisome biogenesis. Similar to HFAs, the infection of U251 cells with PRVABC59 or MR766 strains resulted in a significant loss of peroxisomes and peroxisome biogenesis factors (Figure 2A,B). Moreover, the infection of U251 cells with the pandemic strain PRVABC59 resulted in a greater loss of peroxisomes than MR766.

**Figure 1.** Zika virus (ZIKV) infection decreases the pool of peroxisomes in primary human fetal astrocytes (HFAs). (**A**) HFAs were infected at the multiplicity of infection (MOI) of 3) with ZIKV (PRVABC59 (PR), H/PF/2013 (FP), MR766 (MR) or PLCal (Cal) strains) for 48 h and then processed for confocal microscopy. Peroxisomes were detected using a mouse monoclonal to PMP70 and donkey anti-mouse IgG conjugated to Alexa Fluor 488. Infected cells were detected using a goa<sup>t</sup> polyclonal antibody to ZIKV NS5 and chicken anti-goat IgG conjugated to Alexa Fluor 647. Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). Images were obtained using a spinning disc confocal microscope. The number of peroxisomes in mock- and ZIKV-infected cells were determined using Volocity image analysis software. Averages were calculated from three independent experiments, in which a minimum of 20 cells for each sample were analyzed. The average number of peroxisomes in mock-treated cells was normalized to 1.0. Bars represent standard error of the mean. \*\*\* *p* < 0.001, \* *p* < 0.05. (**B**) HFAs were infected with ZIKV strains (MOI = 5) for the indicated time periods, after which total cell lysates were processed for immunoblot analyses with antibodies to catalase, PEX3, PEX7, PEX11B, PEX13, PEX19, ZIKV NS5 and actin. The relative levels of peroxisomal proteins (compared to actin) from three independent experiments were averaged and plotted. The average levels of peroxisomal proteins in mock-infected cells were normalized to 1.0. Error bars represent standard error of the mean. \* *p* < 0.05. (**C**) HFAs were infected with ZIKV (MOI = 1) for the indicated time periods, after which the viral titers in the media were determined by plaque assay. The data are averaged from the results of three independent experiments. Bars represent standard error of the mean.

**Figure 2.** ZIKV infection decreases the number of peroxisomes in U251 cells. (**A**) U251 cells were infected with ZIKV PRVABC59 (PR) or MR766 (MR) using MOI = 3 for 48 h and then processed for confocal microscopy. Peroxisomes were detected using a mouse monoclonal to PMP70 and donkey anti-mouse IgG conjugated to Alexa Fluor 488. Infected cells were detected using a goa<sup>t</sup> polyclonal antibody to ZIKV NS5 and chicken anti-goat IgG conjugated to Alexa Fluor 647. Nuclei were stained with DAPI. Images were obtained using a spinning disc confocal microscope. The number of peroxisomes in mock- and ZIKV-infected cells were determined using Volocity image analysis software. Averages were calculated from three independent experiments in which a minimum of 20 cells for each sample were analyzed. The average number of peroxisomes in mock-treated cells was normalized to 1.0. Bars represent standard error of the mean. \*\* *p* < 0.01, \* *p* < 0.05. (**B**) U251 cells were infected with ZIKV PRVABC59 or MR766 (MOI = 5) for the indicated time periods, after which total cell lysates were processed for immunoblot analyses with antibodies to PEX11B, PEX19, ZIKV NS5 and actin. The relative levels of peroxisomal proteins (compared to actin) from three independent experiments were averaged and plotted. The average levels of peroxisomal proteins in mock-infected cells were normalized to 1.0. Error bars represent standard error of the mean. \* *p* < 0.05. h.p.i.=hours post-infection.

#### *3.2. ZIKV Capsid Protein Binds PEX19 and Induces Loss of Peroxisomes*

Similar to WNV and DENV capsid proteins, the ZIKV capsid protein formed a stable complex with PEX19 (Figure 3A). Of note, interaction between ZIKV capsid and PEX19 was reported in a recent interactome study [34]. However, this was not verified by reciprocal co-immunoprecipitation or assays. To determine if the ZIKV capsid protein behaved similarly to the analogous proteins from WNV and DENV [20], U251 were transfected with a plasmid encoding FLAG-tagged ZIKV capsid or vector alone. Forty-eight hours post-transfection, cells were processed for immunoblotting or confocal microscopy. Data in Figure 3B show that the expression of ZIKV capsid protein reduced PEX19 levels by 50%, indicating that ZIKV capsid protein may be the major viral determinant that impairs peroxisome biogenesis. We next investigated peroxisome numbers in ZIKV capsid expressing cells by confocal microscopy. The expression of capsid protein was detected using anti-FLAG, and peroxisomes were identified using an antibody to the tripeptide Ser-Lys-Leu (SKL), a targeting motif found at the carboxyl termini of many peroxisomal matrix proteins [35]. The quantification of SKL-positive structures showed that transient expression of ZIKV capsid protein reduced the number of peroxisomes by 30% versus the control (Figure 3C).

**Figure 3.** Expression of ZIKV capsid protein reduces peroxisome numbers. (**A**) U251 cells were infected with ZIKV PRVABC59 (MOI = 1). Forty-eight hours later, cell lysates were subjected to immunoprecipitation (IP) with rabbit anti-ZIKV capsid, rabbit anti-PEX19, or rabbit IgG followed by SDS-PAGE and immunoblotting (IB) with antibodies to PEX19 or ZIKV-capsid. WCL, whole-cell lysate. (**B**) HEK293T cells were transfected with a plasmid encoding FLAG-tagged ZIKV capsid or empty vector (pcDNA3.1) for 48 h. Cell lysates were subjected to SDS-PAGE and immunoblotting with antibodies to PEX19, ZIKV-capsid and actin. The relative levels of PEX19 (compared to actin) from three independent experiments were averaged and plotted. Error bars represent standard error of the mean. \* *p* < 0.05. (**C**) U251 cells were transfected with a plasmid encoding FLAG-tagged ZIKV capsid or empty vector (pcDNA3.1) for 48 h and then processed for confocal microscopy. Peroxisomes were detected with a rabbit polyclonal antibody to the tri-peptide SKL and donkey anti-rabbit IgG conjugated to Alexa Fluor 546. Transfected cells expressing capsid were detected with a mouse anti-FLAG epitope antibody and donkey anti-mouse IgG conjugated to Alexa Fluor 488. Nuclei were stained using DAPI. Images were obtained using spinning disc confocal microscopy. The relative numbers of peroxisomes (SKL-positive structures) in cells transfected with or without ZIKV capsid plasmid were determined using Volocity image analysis software. Averages were calculated from three independent experiments, in which a minimum of 20 cells for each sample were analyzed. The average number of peroxisomes in mock-treated cells was normalized to 1.0. Bars represent standard error of the mean. \*\* *p* < 0.01.

#### *3.3. Over-Expression of PEX11B Inhibits ZIKV Replication*

Since flavivirus infection depletes peroxisomes, likely as a mechanism to impair the innate immune response, we questioned whether expanding the cellular pool of peroxisomes would restrict ZIKV replication. PEX11B is a cellular protein that induces peroxisome proliferation by stimulating the division of these organelles, and its over-expression results in increased numbers of peroxisomes [36,37]. U251 cells and Vero cells were transduced with lentiviruses encoding the reporter protein AcGFP alone as a control or AcGFP plus myc-tagged PEX11B for 48 h. Lentiviral-mediated over-expression of PEX11B resulted in a 20% increase in the number of peroxisomes in U251 cells (Figure 4A).

**Figure 4.** Over-expression of PEX11B inhibits ZIKV replication. (**A**) U251 or Vero cells were transduced with lentiviruses encoding AcGFP alone or AcGFP plus myc-tagged PEX11B for 48 h and then processed for confocal microscopy. Peroxisomes were detected with a rabbit polyclonal antibody to the tri-peptide SKL and donkey anti-rabbit IgG conjugated to Alexa Fluor 546. Nuclei were stained using DAPI. Images were obtained using spinning disc confocal microscopy. The relative numbers of peroxisomes (SKL-positive structures) in cells were determined using Volocity image analysis software. Averages were calculated from three independent experiments, in which a minimum of 20 cells for each sample were analyzed. The average number of peroxisomes in control cells was normalized to 1.0. Bars represent standard error of the mean. \* *p* < 0.05. (**B**) U251, A549 or Vero cells were transduced with lentiviruses encoding the reporter protein AcGFP alone as a control or AcGFP plus myc-tagged PEX11B proteins for 48 h, after which the cells were infected with ZIKV PRVABC59 (MOI = 1) for another 48 h. Cell media were processed by plaque assay to determine viral titers. In parallel, cell lysates were also processed for RNA extraction and subsequent qRT-PCR to determine viral RNA level (**C**). U251 and Vero cell lysates were processed to determine cell viability (**D**). The data are averaged from the results of three independent experiments. Bars represent standard error of the mean. \*\*\* *p* < 0.001, \* *p* < 0.05. N.S. = not significant.

While the e ffect of PEX11B expression on peroxisome proliferation was modest, the e ffect on ZIKV replication was dramatic. Specifically, ZIKV titers were reduced by more than 80% in U251 cells over-expressing PEX11B (Figure 4B). This e ffect was not limited to U251 cells as over-expression of PEX11B in A549 cells also resulted in a significant inhibition of ZIKV replication and reduction in viral titers (Figure 4B,C). To determine if the antiviral e ffect increasing peroxisome numbers was related to the ability of cells to mount an IFN response, PEX11B was over-expressed in Vero cells, a monkey kidney cell line which does not secrete type I IFN in response to viral infection [38]. While the over-expression of PEX11B increased the number of peroxisome in Vero cells by an average of 35% (Figure 4A), unlike in U251 or A549 cells, this peroxisome biogenesis factor did not reduce ZIKV replication or viral titers (Figure 4B,C). Data in Figure 4D confirm that the antiviral e ffects of PEX11B over-expression were not due to cytotoxicity in U251 or A549 cells.

#### *3.4. Over-Expression of PEX11B Enhances the Innate Immune Response*

One possible scenario to account for the antiviral e ffect of PEX11B is a more robust innate immune response due to an increase in the surface area of peroxisomes, which have been termed antiviral signaling platforms [19]. However, the expansion of the peroxisome pool without a corresponding increase in the level of MAVS may not enhance antiviral signaling in response to the detection of viral genomes by RIG-I and MDA5. As such, we first investigated how the over-expression of PEX11B affected MAVS protein levels. Immunoblotting data in Figure 5 show that in cells transduced with lentiviruses encoding PEX11B, levels of MAVS protein were increased two-fold. PEX13 protein levels were also increased in response to PEX11B over-expression, suggesting that structural components of this organelle were induced by PEX11B expression. Whether the additional MAVS protein induced by PEX11B over-expression localizes exclusively to peroxisomes, mitochondria, or both is not known at this point.

**Figure 5.** Over-expression of PEX11B increases the expression of MAVS protein. U251 cells were transduced with lentiviruses encoding AcGFP alone or AcGFP plus myc-tagged PEX11B for 48 h and then transfected with poly(I:C) (+) or an empty plasmid vector (−) for 12 h. The cell lysates were processed for immunoblot analyses with a mouse monoclonal antibody to MAVS, rabbit polyclonal PEX13, goa<sup>t</sup> polyclonal antibody to GFP, and a mouse monoclonal antibody to the myc epitope. The relative levels of MAVS and PEX13 (compared to actin) from three independent experiments were averaged and plotted. Bars represent standard error of the mean. \*\* *p* < 0.01, \* *p* < 0.05.

To determine if peroxisome proliferation enhanced the IFN response following the detection of viral RNA mimics such as poly(I:C), levels of type I and III IFN and ISGs were assessed in cells transduced with lentiviruses encoding PEX11B or AcGFP alone. Data in Figure 6 show that the induction of type I (*IFN*β) and III (*IFN*λ*2*) IFNs was markedly elevated in cells over-expressing PEX11B. Similarly, the transcription of ISGs such as *Viperin*, *Mx2*, *IFIT1*, *RIG-I* and *MDA5* was significantly increased by PEX11B over-expression (Figure 6).

**Figure 6.** Over-expression of PEX11B enhances the innate immune response. U251 cells were transduced with lentiviruses encoding AcGFP alone or AcGFP plus myc-tagged PEX11B for 48 h and then transfected with poly(I:C) for 12 h. Cell lysates were processed for RNA extraction and subsequent qRT-PCR. Fold induction of selected ISG transcripts in response to poly(I:C) was determined. The mRNA levels of ISGs were normalized to *ACT-B* mRNA levels. The data represent the average from the results of three independent experiments. Bars represent standard error of the mean. \* *p* < 0.05.
