key: cord-0014380-mgrbibju
authors: Victorio, Carla Bianca Luena; Novera, Wisna; Tham, Jing Yang; Watanabe, Satoru; Vasudevan, Subhash G.; Chacko, Ann-Marie
title: Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers for In Situ Live-Cell Molecular Imaging of Dengue Virus Replication
date: 2020-12-04
journal: Int J Mol Sci
DOI: 10.3390/ijms21239260
sha: 2b6fb94d42207e66259e572b97713da683ec1b9c
doc_id: 14380
cord_uid: mgrbibju

Current methods to detect and monitor pathogens in biological systems are largely limited by the tradeoffs between spatial context and temporal detail. A new generation of molecular tracking that provides both information simultaneously involves in situ detection coupled with non-invasive imaging. An example is antisense imaging that uses antisense oligonucleotide probes complementary to a target nucleotide sequence. In this study, we explored the potential of repurposing antisense oligonucleotides initially developed as antiviral therapeutics as molecular probes for imaging of viral infections in vitro and in vivo. We employed nuclease-resistant phosphorodiamidate synthetic oligonucleotides conjugated with cell-penetrating peptides (i.e., PPMOs) previously established as antivirals for dengue virus serotype-2 (DENV2). As proof of concept, and before further development for preclinical testing, we evaluated its validity as in situ molecular imaging probe for tracking cellular DENV2 infection using live-cell fluorescence imaging. Although the PPMO was designed to specifically target the DENV2 genome, it was unsuitable as in situ molecular imaging probe. This study details our evaluation of the PPMOs to assess specific and sensitive molecular imaging of DENV2 infection and tells a cautionary tale for those exploring antisense oligonucleotides as probes for non-invasive imaging and monitoring of pathogen infections in experimental animal models.

Real-time in situ molecular imaging [1] is a non-invasive imaging technique that surpasses traditional methods of pathogen detection and monitoring by providing both spatial and temporal information. Such methods provide spatial localization of the pathogen that circumvents the problem of spatial heterogeneity inherent in most diseases; and enable real-time monitoring of infection kinetics. These are both crucial in studies elucidating disease pathogenesis and evaluating the therapeutic efficacy of candidate vaccines and antimicrobials. Real-time in situ molecular imaging employs probes that bind to either the genome, transcribed genes, or infection-specific proteins expressed over time in live cells, tissues, or animals [2] . This approach relies on biocompatible, non-toxic pathogen-specific probes detectable in vitro, ex vivo, and/or in vivo by molecular imaging technologies ranging from optical imaging (e.g., fluorescence, and bioluminescence), to magnetic imaging (e.g., magnetic resonance imaging, MRI), and nuclear imaging (e.g., positron emission tomography, PET; single-photon emission

We evaluated the potential of antiviral 5 SL PPMO ( Figure 1A) , which was designed to bind the Dengue virus seroptype-2 (DENV2) genome ( Figure S1 ), as an imaging agent for DENV2 cellular infection; and compared its performance as a DENV-specific probe to a non-targeted control (CTRL) PPMO that was designed to not bind to the DENV2 genome ( Figure S2 ). These PPMOs were described as naturally stable and delivered into cells with the aid of a cell-penetrating peptide (RxR) 4 B ( Figure 1A ) [38] . The 5 SL PPMO sequence was also target-specific and did not target other mRNA sequences in the cell ( Figure S1 ). Moreover, the 5 SL and CTRL PPMOs were labeled with distinct fluorophore tags to facilitate simultaneous imaging in cells ( Figure 1A) . To determine the PPMO concentration most suitable for subsequent imaging experiments, we evaluated three different PPMO concentrations (1 µM, 10 µM, and 50 µM) for cytotoxicity and fluorescence imaging contrast. Monkey kidney (Vero) cells were incubated with either PPMO for 10 min and subsequently subjected to live fluorescence imaging. Acute cytotoxicity was observed in cells incubated with 50 µM 5 SL PPMO as early as 2 h after PPMO incubation, but not at lower concentrations (1 or 10 µM) ( Figure 1B ). Similar phenomena were observed with CTRL PPMO (data not shown).

To determine the PPMO concentration providing highest imaging contrast, we quantified the intensity of fluorescence signals contributed by PPMOs in the cytoplasm where viral replication was known to occur. At 2 h after PPMO incubation, we observed fluorescent green (5 SL PPMO) and red (CTRL PPMO) punctae scattered throughout the cytoplasm ( Figure 1C ), which confirmed cellular entry of these PPMOs. The punctae were attributed to PPMOs concentrated in cellular vesicles, possibly endosomes. Diffuse fluorescence signals were also observed in both the cytoplasm and nucleus ( Figure 1C ). To quantify these fluorescence signals, the Corrected Total Cell Fluorescence (CTCF) value was determined in individual cells by obtaining the integrated signal density within regions-of-interest (ROIs) drawn around the cytoplasm and subtracting the contribution of background within these ROIs ( Figure 1C ) (See Methods for full details of image analysis). Cells incubated with 10 µM PPMOs exhibited the highest CTCF values for both PPMOs ( Figure 1D ) and therefore afforded the best imaging contrast. On the other hand, cells incubated with 1 µM PPMOs exhibited low-intensity signals that were indistinguishable from background ( Figure 1C ,D). Hence, 10 µM PPMO concentration was selected for subsequent imaging assays due to low toxicity and superior imaging contrast associated with this PPMO concentration.

We next determined whether the 5 SL PPMO specifically tracked DENV2 cellular infection. DENV2-infected Vero and BHK-21 cells were incubated with both PPMOs (10 µM each, 10 min) at 48 h and 72 h post-infection and subsequently immunostained with antibodies targeting double-stranded RNA (dsRNA). DENV replication had been known to occur in membrane-bound multi-protein structures that assemble within vesicle packets and produces double-stranded RNA (dsRNA) as replication intermediates [39] . These vesicle packets were observed as intensely fluorescent punctae around nuclei in both time points assayed (Figure 2A ,B). To evaluate whether the 5 SL PPMO colocalized with viral replication vesicle packets, we calculated the Manders split coefficient (tM1) [40] between dsRNA (proxy for viral replication) and either 5 SL or CTRL PPMO. The tM1 dsRNA describes the fraction of total intensity from the dsRNA fluorescence channel located in pixels where the intensity from the other fluorescence channel exceeds a threshold automatically determined by the software. The amount of dsRNA fluorescence signals that colocalized with 5 SL PPMO (tM1 dsRNA + 5 SL PPMO ) was similar to the amount of dsRNA fluorescence colocalized with CTRL PPMO (tM1 dsRNA + CTRL PPMO ) in both DENV2-infected Vero and BHK-21 cells regardless of time post-infection ( Figure 2C ). This indicated that the extent of colocalization of dsRNA with 5 SL PPMO was comparable to the extent of colocalization of dsRNA with CTRL PPMOs. Moreover, the vesicle packets were not selectively enriched with 5 SL PPMOs over time. This was not entirely surprising, since 5 SL and CTRL PPMOs exhibited high colocalization with each other ( Figure 2D ,E), more than they colocalized with dsRNA. Similar observations were noted in cells infected with ZIKV ( Figure S3 ), where the PPMOs colocalized more with each other than with the ZIKV vesicle packets. These results suggested that the majority of PPMOs had been either occupying the same vesicles or trapped in endosomes and were not found within vesicle packets where DENV2 replication occurs. Figure 3C ). However, the CTRL PPMO was similarly enriched in BHK-21 cells regardless of infection status ( Figure 3C ). Despite differences in PPMO clearance kinetics in the two cell lines, the DENV2 replication kinetics in both cells were comparable ( Figure 3D) . Hence, these findings demonstrated that the kinetics of PPMO clearance varied independently of DENV2 replication kinetics in either cell line.

Due to the antiviral nature of 5 SL PPMO, we assessed whether it affected DENV2 infection at the intended imaging dose (10 µM final concentration), which would confound its application as a non-interfering imaging probe for DENV infection. Incubation of Vero cells with 5 SL PPMO (10 min duration) 1 h prior to DENV2 infection resulted in a precipitous drop in virus production to non-detectable levels at 3 days and 6 days post-infection ( Figure S5 ), which corroborated previous observations [31, 32] . However, this was not observed in cells inoculated with ZIKV ( Figure S5 ), which confirmed that the antiviral effect was specific to DENV2 infection. Indeed, when we assessed DENV2 replication in Vero cells at various time points post-infection and following short incubation with 5 SL PPMO, we observed a drastic reduction in cellular content of viral RNA ( Figure 3E ). This confirmed that 5 SL PPMO inhibited DENV2 replication as early as 24 h post-infection. Thus, 5 SL PPMO was deemed unsuitable as antisense imaging probe when used at 10 µM concentration because of its pharmacological effect on the phenomenon it was tracking. Interestingly, pre-treatment with CTRL PPMO also reduced DENV2 production by as much as 50% (p < 0.0001) ( Figure S5 ), which contradicted previous reports [30, 32] . We speculated that mechanisms other than the canonical Watson-Crick base pairing between the CTRL PPMO and DENV2 genome may have contributed to inhibition of viral replication. One possibility was electrostatic interactions of the CPP with viral proteins or host proteins assembled in the viral replication complex [41] . This study evaluated antiviral PPMOs as potential in situ molecular imaging probes for cellular-based infection assays using DENV2-targeted 5 SL and an unrelated non-targeted CTRL PPMOs. These PPMOs were used as live-cell fluorescence imaging agents to determine whether it tracked spatially and temporally DENV cellular infection in an initial validation test before further development as probes for in vivo PET/SPECT tracking. For the assays, we used Vero and BHK-21 cells-the two most widely used in vitro models of DENV cellular infection [30, 32, 34] . Based on low cytotoxicity and superior imaging contrast, 10 µM final PPMO concentration was selected as the optimal dose for live-cell fluorescence imaging. From immunofluorescence staining of DENV-infected cells incubated with PPMOs at 48 h or 72 h post-infection, the 5 SL PPMO was determined to not colocalize with viral vesicle packets where viral replication occurs. Instead, the majority of 5 SL PPMOs seemed to occupy the same cellular vesicles as CTRL PPMOs, and this could have been an artifact of the general route of PPMO cellular entry: PPMOs were generally delivered into cells by CPPs that nonspecifically triggered endocytosis. Another possibility was that many of these PPMOs become trapped in endosomes, never reaching their target replicating DENV RNA-a likely scenario that had been previously highlighted as a major barrier for ASOs to become successful cellular therapeutics [20, 42] . Though the DENV-specific targeting of these PPMOs could be evaluated independently of cellular entry (e.g., through transient electroporation), this approach would not be translatable to non-invasive preclinical and clinical imaging applications. Hence, future generations of antisense imaging probes would need to include alterations in the CPP to enhance endosomal escape [43] and improve delivery of the probe to the intended target [20] . In addition, the kinetics of PPMO clearance from cells did not coincide with the kinetics of viral replication. Instead, we found the 5 SL PPMO drastically inhibited DENV2 replication in cells at the imaging dose (10 µM). Due to the limited detection sensitivity of optical imaging, it was not possible to evaluate whether using the PPMO at lower concentrations (<10 µM) would better track DENV infection without inhibiting viral replication. This limitation could be circumvented in the future by tagging the PPMOs with probes amenable to more sensitive detection methods, such as PET or SPECT imaging, which could potentially reach optimal imaging dose in the nanomolar concentration range. However, radiolabeling of PPMOs with appropriate PET/SPECT radionuclides includes new chemical modifications which could affect both the chemical integrity, as well as the biological behavior and targeting of the radiolabeled PPMO probe relative to the original PPMO. Hence, radiolabeled PPMO probes will have to go through a similar battery of tests as those described herein to demonstrate their suitability for their intended use.

In conclusion, the potential of 5 SL PPMO as an antisense imaging probe for DENV cellular infection is largely limited by its nonspecific route of intracellular delivery, the need for additional requirement for endosomal escape, and its intrinsic potency in inhibiting viral replication. There is a misconception that because ASOs are designed to be target-specific, then it will specifically track the target (spatially and temporally). Our results demonstrate that this is not the case, and our study serves as a cautionary tale for those exploring the use synthetic ASOs as probes for non-invasive imaging and monitoring of viral infections in experimental animal models. As potential imaging agents, and as candidate therapeutics, several barriers and challenges need to be overcome, which have been discussed thoroughly in past reviews [42, 44] . With the increasing global risk of viral pandemics-such as the high transmissibility and virulence of the current SARS-CoV-2 outbreak-pathogen-specific non-invasive imaging probes are highly valued and urgently needed [45] . However, unless the inherent limitations of ASOs are circumvented, antisense imaging can take the back seat for now as we focus our attention on other candidate pathogen-specific imaging probes.

Vero (ATCC ® CCL-81; monkey kidney epithelial cells) and BHK-21 (ATCC ® CCL-10; baby hamster kidney epithelial cells) cell lines were purchased from the American Type Tissue Culture Collection (ATCC, Manassas, VA, USA) and confirmed free of Mycoplasma by PCR methods [46] prior to use. Vero cells were cultured in DMEM (Gibco BioSciences, Dublin, Ireland), and BHK-21 cells were cultured in RPMI-1640 (Gibco BioSciences, Dublin, Ireland), and media supplemented with 10% (v/v) Fetal Bovine Serum (FBS).

The mouse-adapted Dengue virus serotype-2 (DENV2) S221 strain was a gift from Prof. Sujan Shresta (La Jolla Institute for Immunology, La Jolla, CA, USA) [47] . The Zika virus (ZIKV) French Polynesia strain (H/PF/2013) was obtained from the European Virus Archive. Both virus strains were propagated in mosquito (Aedes albopictus) C6/36 cell line (ATCC ® CRL-1660) prior to use.

The DENV-targeted (5 SL) and non-targeted unrelated control (CTRL) morpholino oligomers (PMOs) conjugated with cell-penetrating peptides (PPMOs) were obtained from Prof. Hong Moulton and Dr. David Stein at Oregon State University (Corvallis, OR, USA). Both PPMOs were synthesized as previously described [37] . Briefly, PPMOs were conjugated at the 3 -end with fluorophores and at the 5 -end with the cell-penetrating peptide (RXR) 4 XB [48] , where R = arginine, X = aminohexanoic acid, and B = β-alanine. The DENV-targeted 5 SL PPMO has the following sequence: 5 GTC GGT CCA CGT AGA CTA ACA ACT 3 and was designed to bind to the stem-loop (SL) region at the 5 untranslated region (5 UTR) of DENV2 genomes [30] . The 5 SL PPMO was tagged with carboxyfluorescein (FAM) with excitation and emission peak wavelengths (λ Ex/Em ) of 495 nm and 519 nm, respectively. The CTRL PPMO has the following sequence: 5 CCT CTT ACC TCA GTT ACA ATT TAT A 3 and was tagged with lissamine rhodamine (LRB, λ Ex/Em = 560/583 nm).

Cells were seeded (2.5 × 10 4 cells/well) overnight in 24-well µ-plates (Ibidi, Munich, Germany) and inoculated with virus at a multiplicity of infection (MOI) of 1 (i.e., 1 plaque-forming unit (pfu) per cell). Cells were incubated with virus in serum-free media (either DMEM or RPMI-1640) for 1 h at 37 • C, 5% CO 2 . Afterwards, the virus inoculum was removed and replaced with PPMO solution in sterile PBS containing both the targeted (5 SL, 10 µM final concentration) and non-targeted control (CTRL, 10 µM final concentration) PPMOs, as well as Hoechst 33,342 (λ Ex/Em , 350/461 nm) nuclear counterstain (1 µg/mL). Cells were incubated in the PPMO solution for 10 min at 37 • C, 5% CO 2 and afterwards washed twice in sterile PBS solution. Cells were finally maintained in culture media supplemented with 2% (v/v) FBS and incubated at 37 • C, 5% CO 2 . For assays involving determining colocalization of double-stranded RNA (dsRNA) with PPMOs, PPMOs were incubated with cells at 48 h or 72 h post-infection. For live-cell imaging assays at various timepoints and determining viral replication kinetics in the presence of 5 SL PPMO, PPMOs were incubated with cells at 1 h post-infection. After PPMO incubation, cells were washed twice in sterile PBS solution and incubated in fresh culture media prior to live-cell imaging.

Cells were fixed in 4% paraformaldehyde for 20 min at room temperature (RT) at 48 h or 72 h post-infection and subsequent PPMO addition. Cells were permeabilized in 0.3% Triton X-100 (15 min, RT) and blocked with 5% BSA for 1 h. DsRNA (i.e., replicating DENV genome) was tagged using J2 mouse monoclonal antibody mAb (Scicons, Hungary) at 1:500 dilution (1 h, 37 • C). Cells were incubated with goat-anti-mouse IgG conjugated with Alexafluor 647 (Life Technologies, Carlsbad, CA, USA) at 1:1000 dilution (1 h, 37 • C). Cells were washed with PBS between antibody incubations and finally mounted in ProLong TM Gold anti-fade mountant (Molecular Probes, Eugene, OR, USA).

All imaging was done using Olympus Ix83 microscope set at 37 • C, 5% CO 2 . The plate was stabilized inside the live-cell imaging chamber for 1 h prior to start of image acquisition using Hamamatsu SCMOS V3 camera. Images were acquired using the Olympus CellSens © Dimension software, 60× oil immersion objective lens (N.A. 1.4), and XCite LED light source set at 30% power.

For visualization, fluorescence images were deconvoluted using the built-in 2D CI-Deconvolution algorithm for widefield fluorescence in CellSens © Dimension program (1 iteration, 512-pixel tile overlap). Prior to live-cell imaging at various time points, cells were washed with PBS and replaced with fresh culture media.

Viral replication kinetics after PPMO addition was determined with qRT-PCR as described elsewhere [49] . Briefly, viral RNA was extracted from the infected cells using QIAamp RNEasy mini kit (Qiagen, Hilden, Germany). The amount of viral RNA in cells was determined by using LunaScript RT one-step qRT-PCR kit (New England Biolabs, Ipswich, MA, USA). Absolute quantitation was performed by comparison of C T values with a standard curve generated from known concentrations of in vitro transcribed RNA. Primer sequences are described here [49] .

All image analyses were performed in ImageJ software, using the OlympusViewer plugin (NIH, MA, USA). A region-of-interest (ROI) was drawn around a cell, excluding the nucleus. The nucleus was excluded since viral replication is known to occur only in the cytoplasm. A similar ROI was drawn outside of the cells that corresponds to the "background." For each analysis, 50-100 ROIs were used. The corrected total cell fluorescence (CTCF) values were calculated using the formula:

The values for integrated density, area, and mean background density were obtained from the "measurement" function in ImageJ. The full procedure for determining CTCF values is detailed elsewhere [50] .

Signal colocalization analysis was performed in ROIs using the coloc2 and coloc threshold functions inbuilt in ImageJ. These functions include the thresholded Manders colocalization coefficient (tM1) as output [40, 51, 52] . The tM1 value describes the degree of colocalization of signals from channel 1 with signals from channel 2. For this analysis, 80-100 cells were used per sample.

Statistical analyses were performed using GraphPad Prism ver. 8.4.2 (San Diego, CA, USA). Means between two groups were compared using an unpaired t-test. Means between more than two groups were compared using two-way ANOVA.

Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/23/ 9260/s1. Figure S1 : Specificity of 5 SL peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) to Dengue virus RNA.; Figure S2 : Potential binding sites of control (CTRL) peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) within the cell.; Figure S3 : Colocalization of PPMOs with replicating ZIKV genome.; Figure S4 : Representative fluorescent live-cell images following incubation with 5 SL and CTRL PPMOs.; Figure S5 : Antiviral effect of 5 SL PPMO against Dengue virus replication. 

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We would like to thank Hong Moulton and David Stein at Oregon State University (Corvallis, OR, USA) for providing the PPMOs used in this study; Sujan Shresta at La Jolla Institute for Immunology (La Jolla, CA, USA) for providing the mouse-adapted DENV2 S221 strain; Alvin Guo (Duke-NUS Medical School, Singapore) for his technical expertise in live-cell image acquisition and image analysis; and Kitti Chan (Duke-NUS Medical School, Singapore) for reviewing the manuscript.