Development of Zika Virus Mini-Replicon Based Single-Round Infectious Particles as Gene Delivery Vehicles

Zika virus (ZIKV) is a type of RNA virus that belongs to the Flaviviridae family. We have reported the construction of a DNA-launched replicon of the Asian-lineage Natal RGN strain and the production of single-round infectious particles (SRIPs) via the combination of prM/E virus-like particles with the replicon. The main objective of the study was to engineer the ZIKV replicon as mammalian expression vectors and evaluate the potential of ZIKV mini-replicon-based SRIPs as delivery vehicles for heterologous gene expression in vitro and in vivo. The mini-replicons contained various genetic elements, including NS4B, an NS5 methyltransferase (MTase) domain, and an NS5 RNA-dependent RNA polymerase (RdRp) domain. Among these mini-replicons, only ZIKV mini-replicons 2 and 3, which contained the full NS5 and NS4B-NS5 genetic elements, respectively, exhibited the expression of reporters (green fluorescent protein (GFP) and cyan fluorescent protein–yellow fluorescent fusion protein (CYP)) and generated self-replicating RNAs. When the mini-replicons were transfected into the cells expressing ZIKV prM/E, this led to the production of ZIKV mini-replicon-based SRIPs. ZIKV mini-replicon 3 SRIPs showed a significantly higher yield titer and a greater abundance of self-replicating replicon RNAs when compared to ZIKV mini-replicon 2 SRIPs. Additionally, there were disparities in the dynamics of CYP expression and cytotoxic effects observed in various infected cell types between ZIKV mini-replicon 2-CYP and 3-CYP SRIPs. In particular, ZIKV mini-replicon 3-CYP SRIPs led to a substantial decrease in the survival rates of infected cells at a MOI of 2. An in vivo gene expression assay indicated that hACE2 expression was detected in the lung and brain tissues of mice following the intravenous administration of ZIKV mini-replicon 3-hACE2 SRIPs. Overall, this study highlights the potential of ZIKV mini-replicon-based SRIPs as promising vehicles for gene delivery applications in vitro and in vivo.


Introduction
Flaviviruses, including Zika virus (ZIKV), dengue virus, Japanese encephalitis virus (JEV), West Nile virus, and yellow fever virus, belong to a group of RNA viruses [1,2]. The flaviviral genome encodes a polyprotein consisting of ten proteins, including three structural proteins (C, prM, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The E protein is positioned within the lipid membrane surrounding the capsid and plays a crucial role in binding to receptors and attachment factors

Construction of ZIKV Mini-Replicons Comprising the Green Fluorescent Protein Gene
The ZIKV Natal RGN replicon (ZIKV rep, as shown in Figure 1) consisted of several components cloned into the pBR322 plasmin, including CMVp, cDNA fragments of the ZIKV Natal RGN genome (GenBank accession number KU527068) with the prM-E genes removed, GFP, F-2A, HDVr, and BGH-pA. The construction process of ZIKV Natal RGN replicon was previously described in our publication [10,11]. To create the ZIKV mini-replicon 1-GFP (ZIKV mini-rep 1-GFP, as shown in Figure 1), Fragment 1 (F1) containing RdRp-3 UTR-HDVr-BGH-pA-pBR322-5 UTR-C was firstly amplified using PCR with DreamTaq Green PCR Master Mix, the ZIKV Natal RGN replicon template, the forward primer NotI_Rsr_ZIKV nt7830-For, and the reverse primer ZIKV C_Kpn1_Asc1-Rev (Supplemental Table S1). Next, Fragment 2 (F2) containing the Thosea asigna virus 2A-like peptide (T2A), GFP, and Foot-and-Mouth Disease virus (FMDV) 2A peptide was amplified using PCR with the Asc1_T2A_GFP-For forward primer and the F2A_Not1-Rev reverse primer (Supplemental Table S1). Both F1 and F2 contained AscI and NotI restriction enzyme sites at the 5 and 3 ends, respectively. After digesting F1 and F2 with AscI and NotI, the fragments in a 1:3 or 1:5 ratio were mixed and ligated using T4 DNA Ligase reaction overnight at 16 • C (Thermo Scientific, Waltham, MA, USA). A total of 1 µL of the ligation reaction was then transformed into E. coli 10B competent cells and selected on LB agar plates containing ampicillin at 37 • C for 16 to 18 h. After checking the size of the clones via gel electrophoresis, the recombinant clones of ZIKV mini-rep 1-GFP were sequenced to confirm successful construction and stored at −70 • C. To construct the ZIKV mini-replicon 2-GFP (ZIKV mini-rep 2-GFP, as shown in Figure 1), Fragment 3 (F3), which contained the MTase of NS5, was amplified by PCR with the primer pair ZIKV NS5 MTase_Not1-For and the reverse primer ZIKV NS5(8406) AvrII-Rev (Supplemental Table S1). The resulting fragments of ZIKV mini-rep1-GFP and F3 that were digested using Not1 and AvrII were ligated using a T4 DNA Ligase reaction overnight at 16 • C. The ligation reaction was then transformed into E. coli 10B competent cells, and the transformed cells were selected on LB agar plates containing ampicillin overnight. To confirm the successful construction of the ZIKV mini-rep 2-GFP clones, the clones' sizes were determined by gel electrophoresis, and they were subjected to sequencing analysis to validate the construction. A similar approach was used to construct the ZIKV mini-rep 3-GFP ( Figure 1); Fragment 4 (F4), which contained C-terminal NS4A, NS4B, and MTase NS5, was amplified by PCR with the primer pair NS4A2 _NotI For and the reverse primer ZIKV NS5(8406) AvrII-Rev (Supplemental Table  S1) and then cloned into ZIKV mini-rep1-GFP. After transformation, antibiotic selection, and sequencing analysis, the recombinant clones of ZIKV mini-rep 3-GFP were successfully constructed and then stored at −70 • C.  Figure 1. The construction and gel electrophoresis analysis of ZIKV mini-replicons contain GFP reporter. Fragments F1 and F2 were amplified, digested, ligated, and transformed into cells, resulting in the creation of ZIKV mini-rep 1-GFP. Additional fragments F3 and F4, enco ing NS5 MTase and partial NS4A, NS4B, and NS5 MTase domain, were also amplified, di and ligated with ZIKV mini-rep 1-GFP, leading to the generation of ZIKV mini-rep 2-GFP an mini-rep 3-GFP clones.

Construction of ZIKV Mini-Replicons Comprising the Cyan Fluorescent Protein-Link Yellow Fluorescent Protein (CFP/YFP, CYP) Fusion Gene or Human ACE2 Gene
To generate mini-rep 2-CYP and mini-rep 3-CYP (Supplemental Figure S1), mini-rep 1-no reporter was first constructed. This was achieved by performing PCR the mini-rep 1-GFP template and the primer pairs Cla1_EcoRV_3′GFP-Fo 5′GFP_Mlu1_Cla1-Rev (Supplemental Table S1). The PCR product, also called Fra F5, was then subjected to digestion by ClaI enzyme. Subsequently, the resulting frag were self-ligated using T4 DNA Ligase, and a clone of ZIKV mini-rep 1-no report selected from this ligation reaction. Next, the CFP/YFP gene fragment (F6) was am through PCR using a previously reported template [17] and the primer pairs KpnI For and YFP_AscI-Rev (Supplemental Table S1). This fragment was then inserte ZIKV mini-rep 1-no reporter, resulting in a clone named ZIKV mini-rep 1-CYP. quently, the F3 and F4 fragments were individually cloned into the Not1/AvrII s ZIKV mini-rep 1-CYP. These clones were designated as mini-rep 2-CYP and mini CYP, respectively. Sequencing analysis was performed to confirm the successful con tion of the recombinant clones of ZIKV mini-replicons containing the CYP gene, an firmed clones were stored at −70 °C for further use. In addition, ZIKV mini-replicon taining the human ACE2 gene were created. The human ACE2 gene was amplified b using a reported template [17] and the primer pairs KpnI-ACE2-For and ACE2 As (Supplemental Table S1). The PCR products or the human ACE2 (hACE2) gene wer inserted into the KpnI/AscI sites of ZIKV mini-rep 2-GFP and ZIKV mini-rep 3-G sulting in mini-replicons named ZIKV mini-rep 2-hACE2 and ZIKV mini-rep 3-h respectively. Sequencing analysis was performed to confirm the successful constr of the recombinant clones of ZIKV mini-replicons containing the CYP or hACE2 gen confirmed clones were stored at −70 °C for further use. The construction and gel electrophoresis analysis of ZIKV mini-replicons containing the GFP reporter. Fragments F1 and F2 were amplified, digested, ligated, and transformed into E. coli cells, resulting in the creation of ZIKV mini-rep 1-GFP. Additional fragments F3 and F4, encompassing NS5 MTase and partial NS4A, NS4B, and NS5 MTase domain, were also amplified, digested, and ligated with ZIKV mini-rep 1-GFP, leading to the generation of ZIKV mini-rep 2-GFP and ZIKV mini-rep 3-GFP clones.

Construction of ZIKV Mini-Replicons Comprising the Cyan Fluorescent Protein-Linker-Yellow Fluorescent Protein (CFP/YFP, CYP) Fusion Gene or Human ACE2 Gene
To generate mini-rep 2-CYP and mini-rep 3-CYP (Supplemental Figure S1), ZIKV mini-rep 1-no reporter was first constructed. This was achieved by performing PCR using the mini-rep 1-GFP template and the primer pairs Cla1_EcoRV_3 GFP-For and 5 GFP_Mlu1_Cla1-Rev (Supplemental Table S1). The PCR product, also called Fragment F5, was then subjected to digestion by ClaI enzyme. Subsequently, the resulting fragments were self-ligated using T4 DNA Ligase, and a clone of ZIKV mini-rep 1-no reporter was selected from this ligation reaction. Next, the CFP/YFP gene fragment (F6) was amplified through PCR using a previously reported template [17] and the primer pairs KpnI_CFP-For and YFP_AscI-Rev (Supplemental Table S1). This fragment was then inserted into ZIKV mini-rep 1-no reporter, resulting in a clone named ZIKV mini-rep 1-CYP. Subsequently, the F3 and F4 fragments were individually cloned into the Not1/AvrII sites of ZIKV mini-rep 1-CYP. These clones were designated as mini-rep 2-CYP and mini-rep 3-CYP, respectively. Sequencing analysis was performed to confirm the successful construction of the recombinant clones of ZIKV mini-replicons containing the CYP gene, and confirmed clones were stored at −70 • C for further use. In addition, ZIKV mini-replicons containing the human ACE2 gene were created. The human ACE2 gene was amplified by PCR using a reported template [17] and the primer pairs KpnI-ACE2-For and ACE2 AscI-Rev (Supplemental Table S1). The PCR products or the human ACE2 (hACE2) gene were then inserted into the KpnI/AscI sites of ZIKV mini-rep 2-GFP and ZIKV mini-rep 3-GFP, resulting in mini-replicons named ZIKV mini-rep 2-hACE2 and ZIKV mini-rep 3-hACE2, respectively. Sequencing analysis was performed to confirm the successful construction of the recombinant clones of ZIKV mini-replicons containing the CYP or hACE2 gene, and confirmed clones were stored at −70 • C for further use. TE671-ZIKV prM-E cell line, which co-expresses ZIKV prM and E proteins, was established in our previous study [10]. TE671-ZIKV prM-E cells were grown in 6-well plates until 90% confluence and then transfected with mini-replicons of ZIKV that contained either GFP, CYP, or hACE2 genes using Lipofectamine LTX. The expression of mini-replicondriven proteins, including GFP, CYP, hACE2, and ZIKV NS5 proteins, was examined by immunofluorescence microscopy and immunofluorescence assay using primary antibodies against ZIKV NS5 and human ACE2 (GeneTex, Hsinchu, Taiwan) and secondary AF546 goat anti-rabbit IgG antibodies (from Thermo Fisher Scientific). The ZIKV prME/minirep SRIPs (ZIKV mini-rep P0 SRIPs) were collected from the media of the transfected TE671-ZIKV prM-E cells 72 h post-incubation. Passage 1 (P1) of the ZIKV prME/mini-rep SRIPs (ZIKV mini-rep P1 SRIPs) was collected from the media of TE671-ZIKV prM-E cells infected with the ZIKV mini-rep P0 SRIPs, condensed using the PEG Virus Precipitation Kit (Sigma-Aldrich, St. Louis, MO, USA), and stored at −80 • C.
To confirm the presence of mini-replicon-driven self-replicating RNA genomes within the ZIKV mini-rep P1 SRIPs, real-time RT-PCR assays were conducted using specific primer pairs for ZIKV NS5 and human ACE2 (listed in Supplementary Table S2). Additionally, the E, prM/M, and C proteins present on the surface of the ZIKV mini-rep P1 SRIPs were analyzed using Western and dot blotting assays with specific antibodies against E, prM/M, and C proteins, as described in our previous studies [10,18]. Furthermore, the infectious titers of the P1 SRIP stock were determined through a median tissue culture infectious dose (TCID50) assay. Serial dilutions of the P1 SRIP stock were added to 96-well plates containing a 90% confluent monolayer of TE671-ZIKV prM-E cells. Following incubation for 72 h at 37 • C, any cytopathic effects (CPE) in each well were observed and recorded to determine the TCID50 titer of the P1 SRIP stock.

Transient Reporter Expression Kinetics and Cytopathic Ability by ZIKV Mini-rep 2-CYP and 3-CYP SRIPs
The CYP reporter expression driven by ZIKV mini-rep 3-CYP SRIPs was evaluated in HEK293T, TE671, A549, and SF268 cells at MOIs of 0.1, 1, and 2. To quantify the FRET signal of CYP fluorescence, cell lysates were prepared from the infected cells and transferred to a 96-well black plate on Days 1, 2, and 3 post-infection to examine the expression of the CYP reporter, which is a fusion protein of CFP and YFP. The FRET signal of CYP was measured using a SpectraMax Multi-Mode Microplate Reader (Molecular Devices San Jose, CA, USA) with excitation and emission wavelengths set at 436-528 nm. Additionally, the viability of cells infected with ZIKV mini-rep 3-CYP SRIPs at different MOIs was determined on Day 3 post-infection using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay (Sigma-Aldrich). The absorbance of the mock-infected cells was used as a control to represent 100% cell viability, as described in our previous study [18].

In Vivo Expression of hACE2 in the Mice with an Intravenous Injection of ZIKV prME/Mini-rep 3-hACE2 SRIPs
A study was conducted at China Medical University using a mouse model to investigate the effects of an intravenous injection of ZIKV prME/mini-rep 3-hACE2 SRIPs. The Institutional Animal Care and Use Committee (IACUC) approved the study protocol on June 9th, 2020 (Animal Use Protocol No. CMUIACUC-2020-293). Female BALB/c mice, aged 6 weeks, were divided into two groups. Group I received a 50 µL intravenous injection of PBS on Day 1. Group II received a 50 µL intravenous injection of ZIKV prME/mini-rep 3-hACE2 SRIPs (1.5 × 10 6 TCID50/mL) on Day 1. On Day 6, the mice were euthanized, and their lungs and brains were collected for further analysis. The collected tissues were fixed in 10% neutral buffered formalin overnight. Subsequently, they underwent a series of steps for histopathology assays. The tissues were dehydrated using different concentrations of ethanol and then embedded in paraffin. Thin sections (ranging from 4 to 15 µm) were obtained from the paraffin-embedded tissues using a microtome. These sections were deparaffinized, rehydrated, and stained with SPECTRA H&E Stains to visualize tissue morphology. The staining process was performed using the BOND-III Fully Automated IHC and ISH Stainer by Leica. After staining, the sections were dehydrated, cleared, air-dried, and mounted. For the immunohistochemistry assay, the sections underwent deparaffinization, rehydration, and heat-induced antigen retrieval using SPECTRA H&E Stains by Leica on the BOND-III Fully Automated IHC and ISH Stainer. Primary antibodies against hACE2 were applied to the sections and incubated overnight. To block endogenous peroxidase activity, the sections were treated with 3% H 2 O 2 . Biotinylated universal antibodies from the Bond Enzyme Pretreatment Kit (Leica, Wetzlar, Germany) were added, followed by treatment with the Bond Enzyme Pretreatment Kit reagent. The presence of immune complexes in the sections was visualized by the development of a dark brown-black precipitate using the Bond Enzyme Pretreatment Kit. Finally, the sections were counterstained with hematoxylin and mounted. High-resolution digital images of the tissue sections were captured using the Pathology Scanner Second Generation SG300 by Philips. These images were then analyzed using the PMA.start software provided by Pathomation for whole-slide imaging analysis.

Statistical Analysis
The data presented in this study were obtained from three independent experiments. To analyze the data, a one-way analysis of variance (ANOVA) was performed, followed by Scheffe's post-hoc test. Statistical significance was determined by considering a p-value of less than 0.05.

The Involvement of ZIKV NS4B and MTase in RdRp-Mediated Replication of Mini-Replicon-Driven RNAs
To investigate the self-replication of Zika virus (ZIKV) mini-replicon-driven RNAs, we utilized TE671-ZIKV prM-E cells, which were previously established in our other study [10], to express ZIKV prM and E proteins. These cells were transfected with ZIKV mini-replicons individually, and subsequent analysis was conducted to examine the cytopathic effect, as well as the expression of green fluorescent protein (GFP) and ZIKV RdRp protein in the transfected cells (Figure 2A). After 72 h of transfection, noticeable cytopathic changes and robust green fluorescence spots were observed in the TE671-ZIKV prM-E cells transfected with ZIKV mini-rep 2-GFP and ZIKV mini-rep 3-GFP, respectively ( Figure 2A). In contrast, the cells transfected with ZIKV mini-rep 1-GFP showed only slight levels of cytopathic effect and green fluorescence spots compared to the mock-transfected cells. Immunofluorescent staining analysis revealed similar patterns of ZIKV RdRp protein expression in the cells transfected with the above-mentioned mini-replicons. (Figure 2A). To evaluate the self-replication of mini-replicon-driven RNAs, passage 0 (P0) of the ZIKV mini-replicon-based self-replicating infectious particles (ZIKV mini-rep P0 SRIPs) was collected from the supernatant of the mini-replicon-transfected prM-E co-expressing cells 72 h post-transfection. The prM-E co-expressing cells were then infected with these P0 SRIPs to generate passage 1 (P1) of the ZIKV mini-replicon-based SRIPs (ZIKV mini-rep P1 SRIPs). Subsequently, the yield titer of ZIKV mini-rep 2-GFP and 3-GFP P1 SRIPs was determined using a TCID50 assay with prM-E co-expressing cells. The titer was found to be 5.5 × 10 5 TCID50/mL for ZIKV mini-rep 2-GFP P1 SRIPs and 1.3 × 10 6 TCID50/mL for ZIKV mini-rep 3-GFP P1 SRIPs ( Figure 2B). Additionally, Western blotting and dot-blotting assays confirmed the presence of the structural proteins E, (pr)M, and C in the presence of ZIKV mini-rep 2-GFP and 3-GFP P1 SRIPs ( Figure 2C-E).
Viruses 2023, 15, x FOR PEER REVIEW 7 of 16 ( Figure 2A). In contrast, the cells transfected with ZIKV mini-rep 1-GFP showed only slight levels of cytopathic effect and green fluorescence spots compared to the mock-transfected cells. Immunofluorescent staining analysis revealed similar patterns of ZIKV RdRp protein expression in the cells transfected with the above-mentioned mini-replicons. (Figure 2A). To evaluate the self-replication of mini-replicon-driven RNAs, passage 0 (P0) of the ZIKV mini-replicon-based self-replicating infectious particles (ZIKV mini-rep P0 SRIPs) was collected from the supernatant of the mini-replicon-transfected prM-E co-expressing cells 72 h post-transfection. The prM-E co-expressing cells were then infected with these P0 SRIPs to generate passage 1 (P1) of the ZIKV mini-replicon-based SRIPs (ZIKV mini-rep P1 SRIPs). Subsequently, the yield titer of ZIKV mini-rep 2-GFP and 3-GFP P1 SRIPs was determined using a TCID50 assay with prM-E co-expressing cells. The titer was found to be 5.5 × 10 5 TCID50/mL for ZIKV mini-rep 2-GFP P1 SRIPs and 1.3 × 10 6 TCID50/mL for ZIKV mini-rep 3-GFP P1 SRIPs ( Figure 2B). Additionally, Western blotting and dot-blotting assays confirmed the presence of the structural proteins E, (pr)M, and C in the presence of ZIKV mini-rep 2-GFP and 3-GFP P1 SRIPs ( Figure 2C-E).

Engineering ZIKV Mini-Replicon Vectors
To create the multiple-cloning sites and clone the other reporter gene in ZIKV minireplicon vectors, ZIKV mini-rep 1-no reporter and ZIKV mini-rep 1-CYP were constructed by ligating F5 (ZIKV mini-rep 1-GFP with the GFP gene removed) alone or with F6 (CFP/YFP fusion gene) (Figure 3). The resulting recombinant clones were confirmed for size and sequenced (Supplemental Figure S1B). Fragments F3 and F4 were subsequently inserted into the NotI/AvrII sites of the ZIKV mini-rep 1-CYP vector, leading to the development of ZIKV mini-rep 2-CYP and ZIKV mini-rep 3-CYP. Their sizes were verified, and sequencing confirmed their construction (Supplemental Figure S3). Overall, the process involved amplification, digestion, ligation, transformation, and confirmation through size analysis and sequencing to create the ZIKV mini-replicons. A similar process was followed to generate the P0 of ZIKV mini-rep 2-CYP and 3-CYP SRIPs from prM-E co-expressing cells transfected with mini-rep 2-CYP and 3-CYP, respectively. These P0 SRIPs were then used to infect prM-E co-expressing cells, generating P1 SRIPs (Figure 4). Similar to ZIKV mini-rep 3-GFP P0 SRIPs, ZIKV mini-rep 3-CYP P0 SRIPs induced higher levels of cytopathic effect, CFP-YFP, and ZIKV RdRp expression in infected cells, resulting in a superior yield of P1 SRIPs compared to ZIKV mini-rep 2-CYP P0 SRIPs. The findings exhibited the ability of ZIKV mini-rep 3-GFP and 3-CYP RNAs, under the control of NS4B, MTase, and RdRp, to replicate themselves autonomously. This led to a significant generation of ZIKV mini-rep 3-GFP and 3-CYP SRIPs.

Engineering ZIKV Mini-Replicon Vectors
To create the multiple-cloning sites and clone the other reporter gene in ZIKV minireplicon vectors, ZIKV mini-rep 1-no reporter and ZIKV mini-rep 1-CYP were constructed by ligating F5 (ZIKV mini-rep 1-GFP with the GFP gene removed) alone or with F6 (CFP/YFP fusion gene) (Figure 3). The resulting recombinant clones were confirmed for size and sequenced (Supplemental Figure S1B). Fragments F3 and F4 were subsequently inserted into the NotI/AvrII sites of the ZIKV mini-rep 1-CYP vector, leading to the development of ZIKV mini-rep 2-CYP and ZIKV mini-rep 3-CYP. Their sizes were verified, and sequencing confirmed their construction (Supplemental Figure S3). Overall, the process involved amplification, digestion, ligation, transformation, and confirmation through size analysis and sequencing to create the ZIKV mini-replicons. A similar process was followed to generate the P0 of ZIKV mini-rep 2-CYP and 3-CYP SRIPs from prM-E co-expressing cells transfected with mini-rep 2-CYP and 3-CYP, respectively. These P0 SRIPs were then used to infect prM-E co-expressing cells, generating P1 SRIPs (Figure 4). Similar to ZIKV mini-rep 3-GFP P0 SRIPs, ZIKV mini-rep 3-CYP P0 SRIPs induced higher levels of cytopathic effect, CFP-YFP, and ZIKV RdRp expression in infected cells, resulting in a superior yield of P1 SRIPs compared to ZIKV mini-rep 2-CYP P0 SRIPs. The findings exhibited the ability of ZIKV mini-rep 3-GFP and 3-CYP RNAs, under the control of NS4B, MTase, and RdRp, to replicate themselves autonomously. This led to a significant generation of ZIKV mini-rep 3-GFP and 3-CYP SRIPs.   PCR-amplified fragment F5 using the mini-rep 1-GFP template and specific primers. Subsequently, a CFP/YFP gene fragment (F6) was amplified and inserted into ZIKV mini-rep 1-no reporter, resulting in ZIKV mini-rep 1-CYP. The F3 and F4 fragments were individually cloned into specific sites of ZIKV mini-rep 1-CYP, generating ZIKV mini-rep 2-CYP and ZIKV mini-rep 3-CYP, respectively.

Comparative Analysis of Heterologous Gene Expression in Infected Cells with ZIKV SRIPs Carrying Mini-Replicon-Driven RNAs
To investigate the expression kinetics of reporter genes driven by self-replicating RNAs of mini-replicons 2 and 3, we conducted experiments using HEK239T, TE671, A549, and SF268 cells. These cells were infected with ZIKV mini-rep 2-CYP and 3-CYP SRIPs at MOIs of 0.1, 1, and 2. The detection of FRET signals allowed us to assess the expression levels of the reporter gene CYP. We observed that the peak of the CYP signals driven by mini-replicons 2 and 3 was found in HEK239T and TE671 cells 72 h post-infection with ZIKV mini-rep SRIPs ( Figure 5A,B,E,F). In A549 cells, the infection with ZIKV mini-rep SRIPs at MOIs of 1 and 2, but not at an MOI of 0.1, led to a decrease in the CYP signals driven by mini-replicon 3 at 72 h post-infection ( Figure 5C,D). Furthermore, in SF268 cells, lower CYP signals driven by mini-replicons 2 and 3 were observed 48 h post-infection compared to 24 h post-infection with ZIKV mini-rep SRIPs at MOIs of 0.1, 1, and 2 ( Figure  5G,H). A survival analysis of the infected cells revealed that the infection with ZIKV minirep 2 and 3 SRIPs resulted in noticeable cytotoxic effects in TE671, A549, and SF268 cells ( Figure 6). The cells infected with ZIKV mini-rep 2 SRIPs exhibited a higher survival rate compared to cells infected with ZIKV mini-rep 3 SRIPs at the same MOI ( Figure 6). In addition, when cells were infected with ZIKV mini-rep 3 SRIPs, the survival rate of infected cells showed a dependence on the MOI. Notably, at an MOI of 2, ZIKV mini-rep 3 SRIPs resulted in a reduction of over 30% in the cell survival rate of SF268 cells ( Figure  6D). The experiments involving the infection of cells with ZIKV mini-replicon 2-CYP and

Comparative Analysis of Heterologous Gene Expression in Infected Cells with ZIKV SRIPs Carrying Mini-Replicon-Driven RNAs
To investigate the expression kinetics of reporter genes driven by self-replicating RNAs of mini-replicons 2 and 3, we conducted experiments using HEK239T, TE671, A549, and SF268 cells. These cells were infected with ZIKV mini-rep 2-CYP and 3-CYP SRIPs at MOIs of 0.1, 1, and 2. The detection of FRET signals allowed us to assess the expression levels of the reporter gene CYP. We observed that the peak of the CYP signals driven by minireplicons 2 and 3 was found in HEK239T and TE671 cells 72 h post-infection with ZIKV mini-rep SRIPs ( Figure 5A,B,E,F). In A549 cells, the infection with ZIKV mini-rep SRIPs at MOIs of 1 and 2, but not at an MOI of 0.1, led to a decrease in the CYP signals driven by mini-replicon 3 at 72 h post-infection ( Figure 5C,D). Furthermore, in SF268 cells, lower CYP signals driven by mini-replicons 2 and 3 were observed 48 h post-infection compared to 24 h post-infection with ZIKV mini-rep SRIPs at MOIs of 0.1, 1, and 2 ( Figure 5G,H). A survival analysis of the infected cells revealed that the infection with ZIKV mini-rep 2 and 3 SRIPs resulted in noticeable cytotoxic effects in TE671, A549, and SF268 cells ( Figure 6). The cells infected with ZIKV mini-rep 2 SRIPs exhibited a higher survival rate compared to cells infected with ZIKV mini-rep 3 SRIPs at the same MOI ( Figure 6). In addition, when cells were infected with ZIKV mini-rep 3 SRIPs, the survival rate of infected cells showed a dependence on the MOI. Notably, at an MOI of 2, ZIKV mini-rep 3 SRIPs resulted in a reduction of over 30% in the cell survival rate of SF268 cells ( Figure 6D). The experiments involving the infection of cells with ZIKV mini-replicon 2-CYP and 3-CYP SRIPs provided valuable insights into the expression kinetics of the reporter gene CYP and the cytotoxic effects induced by mini-replicons 2 and 3 in various cell types. The results shed light on the dynamic patterns of CYP expression and the potential harm caused by these mini-replicons when introduced into the cells by ZIKV mini-rep SRIPs.
3-CYP SRIPs provided valuable insights into the expression kinetics of the reporter ge CYP and the cytotoxic effects induced by mini-replicons 2 and 3 in various cell types. T results shed light on the dynamic patterns of CYP expression and the potential har caused by these mini-replicons when introduced into the cells by ZIKV mini-rep SRIPs

In Vivo Expression of hACE2 Delivered by ZIKV Mini-rep 3 SRIPs
To investigate the expression of foreign genes delivered by ZIKV mini-replicon SRIPs, a ZIKV mini-rep 2-hACE2-and 3-hACE2 SRIP-based approach was employed (see Figures 7 and 8). The initial step involved generating the P0 of the ZIKV mini-rep 2-hACE2 and 3-hACE2 SRIPs by transfecting mini-replicons 2-hACE2 and 3-hACE2 into the cells expressing ZIKV prM-E, respectively. The P0 hACE2 SRIP infection caused mild cytopathic effects in prM-E co-expressing cells, in which mini-rep 3-hACE2 SRIP infection showed high levels of hACE2 mRNA expression ( Figure 7B), resulting in a higher yield of P1 mini-rep 3-hACE2 SRIPs compared to P1 mini-rep 2-hACE2 SRIPs. To further evaluate in vivo hACE2 expression, an assay was conducted using BALB/c mice ( Figure 8). The mice were divided into two groups: Group I received intravenous injections of PBS on Day 1 as the control group, while Group II received intravenous administration of ZIKV prME/mini-rep 3-hACE2 SRIPs on Day 1. On Day 6, comprehensive analyses were performed on lung and brain samples collected from each group. IHC staining using hACE2specific antibodies ( Figure 8A) was carried out on lung and brain tissues from different groups to examine the expression of hACE2 mediated by ZIKV mini-rep 3-hACE2 SRIPs. The IHC results revealed that hACE2 expression was detected in the perivascular and alveolar regions of lung tissues in mice from Group II but not Group I. Additionally, the presence of the hACE2 protein was exclusively observed in the brain capillaries of mice from Group II. A histological examination using H&E staining was performed on mouse lung and brain tissues to further investigate the impact of ZIKV mini-rep 3-hACE2 SRIPs on pulmonary and brain inflammation ( Figure 8B). Group II mice, which received intravenous injections of ZIKV mini-rep 3-hACE2 SRIPs, exhibited mild inflammation in the lungs and brain. In contrast, no significant alterations were observed in the lung tissues of Group I mice injected with PBS intravenously. These findings demonstrate that the intravenous injection of ZIKV mini-rep 3-hACE2 SRIPs in mice resulted in hACE2 expression in the lungs and brain. Thus, it suggests that ZIKV mini-replicon SRIPs have the potential to serve as in vivo gene delivery vehicles.

In Vivo Expression of hACE2 Delivered by ZIKV Mini-rep 3 SRIPs
To investigate the expression of foreign genes delivered by ZIKV mini-replicon SRIPs, a ZIKV mini-rep 2-hACE2-and 3-hACE2 SRIP-based approach was employed (see Figures 7 and 8). The initial step involved generating the P0 of the ZIKV mini-rep 2-hACE2 and 3-hACE2 SRIPs by transfecting mini-replicons 2-hACE2 and 3-hACE2 into the cells expressing ZIKV prM-E, respectively. The P0 hACE2 SRIP infection caused mild cytopathic effects in prM-E co-expressing cells, in which mini-rep 3-hACE2 SRIP infection showed high levels of hACE2 mRNA expression ( Figure 7B), resulting in a higher yield of P1 mini-rep 3-hACE2 SRIPs compared to P1 mini-rep 2-hACE2 SRIPs. To further evaluate in vivo hACE2 expression, an assay was conducted using BALB/c mice ( Figure 8). The mice were divided into two groups: Group I received intravenous injections of PBS on Day 1 as the control group, while Group II received intravenous administration of ZIKV prME/mini-rep 3-hACE2 SRIPs on Day 1. On Day 6, comprehensive analyses were performed on lung and brain samples collected from each group. IHC staining using hACE2-specific antibodies ( Figure 8A) was carried out on lung and brain tissues from different groups to examine the expression of hACE2 mediated by ZIKV mini-rep 3-hACE2 SRIPs. The IHC results revealed that hACE2 expression was detected in the perivascular and alveolar regions of lung tissues in mice from Group II but not Group I. Additionally, the presence of the hACE2 protein was exclusively observed in the brain capillaries of mice from Group II. A histological examination using H&E staining was performed on mouse lung and brain tissues to further investigate the impact of ZIKV mini-rep 3-hACE2 SRIPs on pulmonary and brain inflammation ( Figure 8B). Group II mice, which received intravenous injections of ZIKV mini-rep 3-hACE2 SRIPs, exhibited mild inflammation in the lungs and brain. In contrast, no significant alterations were observed in the lung tissues of Group I mice injected with PBS intravenously. These findings demonstrate that the intravenous injection of ZIKV mini-rep 3-hACE2 SRIPs in mice resulted in hACE2 expression in the lungs and brain. Thus, it suggests that ZIKV mini-replicon SRIPs have the potential to serve as in vivo gene delivery vehicles.

Discussion
This study represents the first investigation into the use of ZIKV mini-replicons as viral vectors for in vitro and in vivo expressing various genes, including reporters (such as GFP and CFP) and hACE2 (Figures 1-8). The study demonstrated the successful construction of ZIKV mini-replicons and their application in ZIKV prM-E co-expressing cells to generate ZIKV mini-replicon based SRIPs. Transfection of these mini-replicons or infection with ZIKV mini-rep SRIPs revealed the crucial involvement of ZIKV MTase domain of NS5 and NS4B in facilitating efficient RdRp-mediated self-replication of the ZIKV mini-replicondriven RNAs, leading to a significant expression of the heterogeneous genes. The physical linkage between the MTase and RdRp domains within NS5 confers specificity for viral cap methylation during flavivirus RNA synthesis [19]. The MTase domain not only performs methylations but also plays a significant role in stimulating both the de novo initiation and elongation phases of RNA synthesis [20,21]. Additionally, MTase enhances the affinity of NS5 for the single-stranded RNA template. This study highlighted the involvement of MTase in RdRp-mediated self-replication of Zika virus mini-replicon-driven RNAs, further supporting the intricate interplay between the MTase and RdRp domains of NS5 [20,21]. NS4B, a transmembrane protein, assumes a critical role in establishing the viral replication complex and modifying intracellular membranes to create an ideal environment for efficient viral replication. Additionally, NS4B actively participates in viral RNA replication and maintains a close association with NS5 [22]. The findings in our previous study potentially correlate with the results of this study, demonstrating that MTase and NS4B enhance the self-replication of Zika virus mini-replicon-driven RNA synthesis mediated by RdRp.
This study provides valuable insights into the dynamics of foreign gene expression driven by ZIKV mini-replicon-based RNAs in cells infected with ZIKV mini-replicon-based SRIPs, as depicted in Figures 1-8. The results reveal distinct expression patterns of the reporter gene CYP in various infected cell lines, with some patterns being dependent on both MOI and the duration of infection. Notably, the infection had deleterious effects on SF268 and A549 cells but not on HEK293T cells, as shown in Figures 5 and 6. This study also highlights the significant potential of ZIKV mini-replicon-based SRIPs as highly efficient gene delivery vehicles capable of targeting a wide range of host cells, based on the analysis of self-replicating RNA levels, virus yield, and cell susceptibility, as illustrated in Figures 2, 4 and 7. The development of replicons from various flaviviruses, such as Kunjin virus (KUNV), Yellow Fever virus (YFV), Dengue virus, Japanese Encephalitis virus (JEV), Tick-borne Encephalitis virus, and West Nile virus, has been previously reported [7,9,23,24]. Some of these replicons have been used as both homologous and heterologous vaccines in animal studies [25][26][27]. For instance, the KUNV replicon system can be delivered as DNA, RNA, or virus-like particles, enabling efficient cell division and the stable expression of foreign genes [8,26]. These findings hold immense promise for various applications of ZIKV mini-replicon-based SRIPs, including enhanced protein production, advanced gene therapy, and the development of novel vaccines. The versatility and effectiveness of these SRIPs make them invaluable tools for advancing research and applications in these fields.
The lack of compatibility between the mouse ortholog of ACE2 and the SARS-CoV-2 virus poses a challenge for conducting pre-clinical studies in mice. To overcome this hurdle, scientists have employed various strategies, including mouse adaptation of the virus or introducing human ACE2 (hACE2) expression in mice. Previous studies have utilized transgenic mouse models expressing hACE2 driven by the K18 promoter, as well as mice that received hACE2 delivery through adenovirus (AdV) or adeno-associated virus (AAV) administration to study SARS-CoV-2 infection [28][29][30][31][32]. In this study, we have demonstrated the potential of ZIKV mini-rep 3-hACE2 SRIPs as gene delivery vehicles, in which the intravenous injection of these SRIPs led to hACE2 expression driven by ZIKV mini-replicon 3 in the lungs and brains of the injected mice, as illustrated in Figures 7 and 8. Therefore, this study successfully established a mouse model with ZIKV mini-replicon-mediated hACE2 expression, which can be utilized to investigate SARS-CoV-2 infection. Similar to the AdV-hACE2 and AAV-hACE2 models described in previous studies [28][29][30][31][32], the in vivo hACE2 expression model using ZIKV mini-replicon-based SRIPs offers the advantage of flexibility. It allows for immediate studies in multiple mouse strains without the need for the time-consuming breeding of mice with hACE2 transgenic or knock-in backgrounds. This flexibility facilitates faster research and enables the evaluation of different mouse strains in the context of SARS-CoV-2 infection.

Conclusions
The findings of this study emphasize the significance of the NS5 MTase domain and NS4B proteins in promoting the effective self-replication of ZIKV mini-replicon-driven RNAs through the RdRp mechanism. Additionally, this study highlights the potential of ZIKV mini-replicons as valuable tools for gene expression in mammalian cells. Furthermore, it showcases the use of ZIKV mini-replicon-based SRIPs as a highly efficient approach for delivering genes, offering promising applications both in vitro and in vivo.