Alphavirus-based replicons demonstrate different interactions with host cells and can be optimized to increase protein expression

ABSTRACT

Alphaviruses have been used as vectors for the expression of heterologous genetic information for a few decades. Their genomes contain two open reading frames. One of these encodes viral nonstructural proteins (nsPs), which mediate the replication of viral genome (G RNA) and synthesis of subgenomic (SG) RNA. Viral structural proteins encoded by SG RNA are dispensable for G RNA replication and transcription of the SG RNA. Their genes can be replaced by heterologous genetic materials. Such defective viral genomes, replicons, can efficiently express the encoded heterologous genes of interest. Within recent years, alphavirus replicons, also termed self-amplifying RNAs (saRNAs), attracted additional attention as a tool to increase the expression level of mRNA vaccines. The results of this study demonstrate that replicons derived from both New World and Old World alphaviruses can efficiently express heterologous genetic information. However, the levels of expression are cell-specific and are strongly determined by the origin of the replicons. Moreover, replication of alphavirus replicons mimics many aspects of alphavirus infections in terms of virus-host interactions and induction of the innate immune response. They also express viral nsPs that may induce vector immunity. These characteristics need to be carefully examined before the wide application of saRNAs. On the other hand, the data show that alphavirus replicons are very flexible expression systems. The efficiencies of protein expression and interactions with host cells can be manipulated by introducing mutations into nsPs and other modifications in replicon genomes.

IMPORTANCE

Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon’s origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression.

INTRODUCTION

The Alphavirus genus in the Togaviridae family represents a group of viral pathogens, which are widely distributed on all continents (1). In natural conditions, most of them are transmitted by mosquito vectors between amplifying vertebrate hosts. In mosquitoes, alphaviruses develop a persistent infection, which apparently does not cause negative effects on mosquito biology (2). In vertebrates, the infections are acute and often result in high titer viremia and the development of debilitating diseases (3). Based on the circulation areas, alphaviruses are divided into the Old World (OW) and the New World (NW) species. However, human travel has led to a wider distribution of both susceptible mosquitoes and viruses themselves. For instance, the OW chikungunya virus (CHIKV) is now a public health threat not only in Africa and Asia but also on American continents and Caribbean islands (4).

Alphavirus virions have a similar structure, in which an icosahedral nucleocapsid is surrounded by a lipid envelope with embedded glycoprotein spikes. Viral genomes (G RNAs) are represented by single-stranded RNAs of positive polarity of ~11.5 kb in length (1). G RNA mimics cellular mRNAs in that it has a cap and a poly(A)-tail at the 5′ and 3′ ends, respectively. Upon delivery into the cells, G RNA is translated into two polyproteins P123 and P1234. After the first step of processing mediated by the encoded protease, the resulting P123 and nsP4 and virus-specific sets of cellular proteins form primary replication complexes (RCs) (5 – 7). They are capable of synthesizing the negative strands on G RNA templates to produce the double-stranded RNA (dsRNA) replication intermediates. These dsRNAs are compartmentalized in spherules initially formed at the plasma membrane (8). Further sequential processing of P123 polyproteins leads to the formation of mature RCs, containing individual nsP1, nsP2, nsP3, and nsP4 and host protein factors. They utilize dsRNA intermediates for transcription of G and subgenomic (SG) RNAs but no longer function in negative strand RNA synthesis (7, 9). SG RNA serves as a template for the translation of the structural polyprotein, which is co- and post-translationally processed into capsid protein and spike glycoproteins. These structural proteins ultimately package G RNA into infectious virions that are released at the plasma membrane of vertebrate cells (2). At early times post-infection (p.i.), the newly synthesized G RNAs serve as templates for the synthesis of nonstructural proteins (nsPs) and dsRNAs and thus promote exponential growth of viral RCs. Within a few hours p.i., concentrations of virus-specific G and SG RNAs expand from a single G RNA molecule delivered into the cell to a few orders of magnitude higher levels, and the newly formed virions are released at the rate of about 2,000 infectious units per cell per hour (2). As SG RNAs lack packaging signals, they are packaged into virions very inefficiently and remain translated in the cytoplasm (10, 11).

To date, infectious cDNA clones of G RNAs have been made for many NW and OW alphaviruses (12 – 16). They are widely used in molecular virology to study functions of viral structural and nonstructural proteins, and the cis-acting RNA elements and to further understand multiple aspects of viral biology and pathogenesis. Importantly, these studies have shown that alphavirus structural proteins are generally dispensable for G RNA replication and transcription of SG RNA (17, 18). Therefore, their genes in the G RNAs can be either deleted or replaced by heterologous sequences. These modified G RNAs that lack structural genes were termed replicons. Upon delivery into vertebrate or mosquito cells, they remain capable of self-amplification and expression of heterologous genes of interest replacing natural viral structural genes (18, 19). The in vitro synthesized replicon G RNAs can be delivered into the cells using numerous RNA transfection protocols. Alternatively, their genomes can be cloned under the control of promoters of cellular DNA-dependent RNA polymerase II and transfected in a plasmid form (20). Accordingly, the first copies of replicon genomes are synthesized in the nucleus by cellular RNA transcription machinery and then begin self-amplification in the cytoplasm as during viral infection. Replicons can be also packaged to very high titers into infectious alphavirus particles by co-transfection of their in vitro-synthesized genomes and so-called helper RNAs (hRNAs). The latter RNAs lack nonstructural genes but have all of the viral structural genes and the promoters driving RNA synthesis intact (18, 21). Virions released from the co-transfected cells contain only replicon genomes. They are dramatically more efficient in terms of replicon delivery, particularly in vivo, than any RNA or DNA transfection-based systems. However, the latter packaging-based delivery system demonstrates some drawbacks, such as the possibility of recombination between replicon and hRNAs (22). In vivo, after repeated use, packaged replicons may also induce vector immunity, an immune response to the virion’s structural proteins.

Despite being used in research and vaccine development for more than 30 years, recently, alphavirus replicon RNAs have attracted additional attention as a possible tool to increase the expression of genes of interest by mRNA vaccines (23, 24). Their ability for self-amplification is expected to dramatically reduce the doses of RNA and transfection reagents used in vaccination. To date, most of the research efforts have focused on Venezuelan equine encephalitis virus (VEEV)-based replicons, which have already been in use for several decades (23). The possibility of application of other alphavirus replicons for the development of self-amplifying RNA (saRNA)-based vaccines has been less investigated, if at all.

The results of this study demonstrate important differences between the OW and the NW alphaviruses, in terms of the levels of heterologous gene expression in the cell lines of different origins, inhibition of cellular translation, and induction of the innate immune response. These and other characteristics of the alphavirus replicons need to be considered in the development of saRNA-based vaccines. We also show that alphavirus replicons represent very flexible expression systems and additional modifications can greatly increase expression levels of the encoded heterologous genes.

RESULTS

Replicon design and GFP expression kinetics

The NW and OW alphaviruses have developed specific interactions with host cells at the molecular and cellular levels. They strongly differ in which sets of host factors they exploit for the replication of their genomes and transcription of SG RNAs (25 – 27). Moreover, profound differences were detected between viral species even inside the OW and NW alphavirus groups (26). Therefore, in this study, we compared the biological characteristics of replicons based on two OW and two NW alphaviruses, which need to be considered in the possible development of saRNA-based mRNA vaccines.

The OW alphavirus replicons were designed based on the Sindbis virus (SINV) and CHIKV genomes (SINrep and CHIKrep, respectively) (Fig. 1A). The NW alphavirus replicons were engineered using infectious cDNA clones of VEEV and eastern equine encephalitis virus (EEEV) (VEErep and EEErep, respectively) (Fig. 1A). In all replicons, the green fluorescent protein (GFP)-coding sequence was cloned under the control of natural virus-specific 5′ untranslated regions (UTRs) of the SG RNAs. GFP fluorescence was used as a simple means of quantitative analysis of the efficiency of expression of the heterologous gene (see Materials and Methods for details), because of a few orders of magnitude linear range of the plate reader. GFP expression was also used to monitor the completeness of infection of all cells in the monolayers by fluorescence microscopy. To simplify the delivery of replicons into vertebrate cells, SINrep/GFP, CHIKrep/GFP, and VEErep/GFP were packaged into structural proteins of SINV TE12, CHIKV 181/25, and VEEV TC-83 strains, respectively, by using corresponding hRNAs (see Materials and Methods for details). For safety reasons, EEErep/GFP was packaged using hRNA-encoding SINV TE12-specific structural proteins instead of homologous ones. In this case, even after potential recombination of replicon and hRNAs in co-electroporated cells, the resulting infectious virus would not be a highly pathogenic EEEV but an EEE/SINV chimera (27, 28). This chimeric virus does not encode proteins with transcription inhibitory functions, is poorly cytopathic, and demonstrates an attenuated phenotype. Thus, the work with packaged replicons, including EEErep/GFP, could be performed in BSL2 conditions. Depending on the type, replicons were packaged into infectious viral particles to titers between 1 × 10⁸ and 4 × 10⁹ infectious units per milliliter (inf.u/mL) if determined on BHK-21 cells (see Materials and Methods for details). The use of replicons packaged into infectious alphavirus particles allowed us to avoid problems with different transfection efficiencies of the in vitro-synthesized replicons’ RNAs in different cell lines and to infect 100% of the cells regardless of their type. Infection of all the cells was required in comparative studies of protein expression and was particularly important in the analysis of interactions of replicons with host cells (see the following sections).

Fig 1.

The kinetics of GFP accumulation in BHK-21 cells infected with alphavirus replicons. (A) The schematic representation of replicons engineered from the NW and OW alphaviruses. (B) BHK-21 cells were seeded in 6-well Costar plate (4 × 10⁵ cells/well) and infected with indicated replicons at an MOI of 20 inf.u/cell. At the indicated time points, cells were lysed, and GFP fluorescence was assessed as described in Materials and Methods. The experiment was repeated three times. SDs are not visible at this scale.

Initially, we assessed the rates of GFP accumulation after infecting BHK-21 cells with all four designed replicons (Fig. 1B). GFP expression was readily detectable at 4 h p.i. and, for the NW alphavirus replicons, continued to increase up to 28-30 h p.i. Based on these kinetics, the intermediate, 18 h p.i., time point was selected for the following comparative studies.

Replicons express GFP in cell type- and replicon type-dependent modes

Packaged CHIKrep/GFP, EEErep/GFP, VEErep/GFP, and SINrep/GFP were used to infect a variety of vertebrate cell lines, including BHK-21, Vero E6, HEK 293T, MRC-5, and NIH 3T3 cells and primary mouse embryonic fibroblasts (MEFs) (Fig. 2A). Thus, the efficiency of GFP expression was tested on hamster, mouse, primate, and human cells. The infectivity of viral particles strongly depended on the cell type and viral structural proteins used for packaging of the replicon genomes. Therefore, in the experiments presented in this and other sections, infectivities of each packaged replicon were initially evaluated on each cell line, and the cell type-specific multiplicities of infection (MOIs) of 20 infectious units per cell (inf.u/cell) were applied. Such MOI was sufficient for achieving infection of 100% of cells, and its completeness was also monitored at 18 h p.i. by fluorescence microscopy. BHK-21 cells were also electroporated with the in vitro-synthesized replicon RNAs. Based on GFP expression, or all the constructs, the transfection efficiencies were above 95%. The efficiencies of GFP expression by the transfected cells were very similar to those found in the cells infected with the same packaged replicons (Fig. 2A and B). Of note, at least in BHK-21 cells, a further increase of MOIs in SINrep/GFP and VEErep/GFP infections by 10-fold did not have any positive effect on GFP expression at 18 h p.i. time point (Fig. 2C). In the case of SINrep/GFP, higher MOI even led to detectably lower accumulation of GFP. Other replicons could not be tested because their titers were insufficient for achieving so high MOI of 200 inf.u/cell.

Fig 2.

GFP expression is dependent on the origin of replicon and the cell types. (A) The indicated cells were seeded into 6-well Costar plates (5 × 10⁵ cells/well) and, in 4 h, infected with alphavirus replicons at cell line-specific MOIs of 20 inf.u/cell, sufficient to achieve 100% infection. After incubation at 37°C for 18 h, cells were evaluated in terms of being 100% GFP-positive and then lysed, and GFP fluorescence was assessed. Means and SDs are presented, n = 3. (B) BHK-21 cells were electroporated with the in vitro-synthesized replicons’ RNAs. At 18 h post-transfection, cells were evaluated for efficiency of electroporation under a fluorescence microscope, and GFP fluorescence was assessed. Means and SDs are presented, n = 3. (C) BHK-21 cells were infected with VEErep/GFP and SINrep/GFP at the MOIs of 20 and 200 inf.u/cell. At 18 h p.i., cells were lysed, and GFP fluorescence was evaluated. Means and SDs are presented, n = 3. Significance of differences was determined by unpaired t test (***P < 0.001). (D) The NIH 3T3 cells were seeded into 6-well Costar plates (5 × 10⁵ cells/well) and, in 4 h, infected with alphavirus replicons at an MOI of 20 inf.u/cell. Images were acquired at 8 h p.i. under a fluorescence microscope. Of note, images of cells infected with SINrep/GFP and those mock-infected were acquired using an exposure threefold longer than that used for other replicons.

GFP expression from alphavirus replicons depends on several factors, such as the availability of host proteins involved in RC functioning, efficiency of RNA replication, transcription and translation of the GFP-encoding SG RNAs, and induction of antiviral response. Consequently, at 18 h p.i., GFP levels strongly depended on the cell line (Fig. 2A). Vero and BHK-21 cells, which are very permissive to many viral infections, produced the highest levels of GFP, while in human MRC-5 and HEK 293 cells, GFP accumulated to the lowest levels. NIH 3T3 cells demonstrated intermediate GFP expression. In all cell lines tested, GFP levels also depended on the type of replicons. CHIKrep/GFP, VEErep/GFP, and EEErep/GFP exhibited comparable levels of GFP expression, and SINrep/GFP produced 20- to 30-fold less GFP than other replicons. Thus, at least three replicons derived from both the NW alphaviruses (VEErep/GFP and EEErep/GFP) and the OW alphavirus (CHIKrep/GFP) demonstrate similar but, noticeably, cell-type-dependent expression of the encoded heterologous GFP gene.

Alphavirus replicons inhibit cellular translation

Alphavirus replicons generally mimic viral infections in terms of RNA replication mechanism and modification of the intracellular environment. Many alphaviruses globally inhibit cellular translation, which would strongly affect the expression of a heterologous gene. Thus, we have evaluated the inhibition of translation by different replicons using the labeling of translated proteins with puromycin (Pur) (29). Within 8 h p.i., all the replicons inhibited translation of cellular mRNA templates in both NIH 3T3 and MRC-5 cells (Fig. 3B) although with different efficiencies. Replication of SINrep/GFP resulted in dramatic translational shutoff, and in agreement with the above expression data (Fig. 1 and 2), in these conditions, even the GFP-encoding SG RNA was translated very inefficiently (Fig. 3B). Interestingly, in CHIKrep/GFP-, VEErep/GFP-, and EEErep/GFP-infected, but not in SINrep/GFP-infected, cells, the SG RNAs were efficiently translated and produced Pur-labeled proteins with molecular weight reminiscent of GFP. A similar band was also detected in the cells infected with SINrep/C-GFP, encoding SG RNA having translational enhancer in the capsid sequence. These bands had slightly higher mobility than the main GFP band detected by Western blot (WB) (Fig. 3C). Its lower position in the gel was likely the result of either Pur incorporation to the protein or synthesis of its unfinished shorter version. Since Pur incorporation terminates translation, the presence of almost full-size, puromycylated GFP suggests the delay at the end of GFP translation. Similar accumulation of distinct bands of Pur-labeled proteins was previously detected in CHIKV- and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells, but this phenomenon has not been investigated in detail (30, 31).

Fig 3.

Alphavirus replicons inhibit translation of cellular mRNA templates. (A) Schematic representation of the replicons used. (B) 5 × 10⁵ NIH 3T3 and MRC-5 cells were infected with the indicated replicons at an MOI of 20 inf.u/cell. At 8 h p.i., the translated proteins were labeled with Pur (see Materials and Methods for details) and analyzed by WB using Pur-specific MAb and secondary Abs labeled with infrared dye. The experiment was repeated three times with similar results. One of the representative WBs is presented. (C) The membrane presented in panel B was additionally incubated with GFP-specific Ab conjugated with DyLight 800. Membranes were scanned and analyzed on Odyssey CLx imager (LI-COR Biosciences).

GFP expression by alphavirus replicons is determined by different levels of eIF2α phosphorylation

Translation inhibition in replicon-infected cells depends on levels of phosphorylated eIF2α (p-eIF2α). Since replication of alphaviruses includes the synthesis of dsRNA replication intermediates, the activation of dsRNA-dependent protein kinase (PKR) likely plays an important role in this process. However, the function of three other protein kinases in eIF2α phosphorylation during viral and replicon infections also cannot be ruled out (32). Compared to mock-infected cells, at 8 h p.i., levels of p-eIF2α were found to be reproducibly higher in cells infected with any of the replicons. However, SINrep/GFP and SINrep/C-GFP induced eIF2α phosphorylation most efficiently (Fig. 4A). This suggested that during replication of SINrep/GFP, expression of GFP is determined more by eIF2α phosphorylation than during infections by other replicons. In agreement with these data, MEFs expressing only the S51A mutant form of the eIF2α (MEF/AA) (33) produced higher levels of GFP than MEFs producing wild-type (wt) eIF2α (MEF/SS) (Fig. 4B through F). However, the ratios of GFP expression by CHIKrep/GFP and the NW alphavirus replicons in MEF/AA versus MEF/SS were significantly lower (Fig. 4G). Similarly, the MEF/AA to MEF/SS GFP-expression ratio was very low for SINrep/C-GFP, which contains a translational enhancer upstream of the GFP-coding sequence.

Fig 4.

PKR and phosphorylation of eIF2α play critical roles in the regulation of heterologous gene expression by alphavirus replicons. (A) NIH 3T3 cells in 6-well Costar plates were infected at an MOI of 20 inf.u/cell. At 8 h p.i., after evaluating the infection under a fluorescence microscope, cell lysates were analyzed by WB using Abs specific to p-eIF2α and other indicated Abs and infrared dye-labeled secondary Abs. (B–F) Equal numbers of MEF/SS and MEF/AA in 6-well Costar plates were infected with the indicated replicons at an MOI of 20 inf.u/cell. At 18 h p.i., cells were lysed, and GFP expression was analyzed as described in Materials and Methods. (G) Ratios of GFP expression in MEF/AA versus MEF/SS in the experiments presented in panels B to F. (H) Lack of PKR expression in cloned Pkr KO cell lines assessed by WB using PKR-specific Abs. Clone 1 was used in further experiments. (I) The parental NIH 3T3 and NIH 3T3 Pkr KO cells were infected with the indicated replicons. At 18 h p.i., cells were analyzed under a fluorescence microscope for completeness of infection and lysed, and GFP expression was assessed. Ratios of GFP expression in NIH 3T3 Pkr KO versus wt NIH 3T3 cells are presented. Means and SDs are presented, n = 3. Significance of differences was determined by unpaired t test (****P < 0.0001) for panels B to F and by one-way ANOVA followed by Dunnett test (****P < 0.0001) for panels G and I.

Since translation in MEF/AA is determined by the mutation in eIF2α, the above results on these cells did not directly point to a critical role for PKR in translation regulation in replicon-infected cells and left a possibility that other kinases may be involved. Therefore, experiments were repeated on Pkr KO NIH 3T3 cells (Fig. 4H), which express all other kinases capable of eIF2α phosphorylation. For all of the replicons, GFP expression in Pkr KO NIH 3T3 cells was reproducibly higher than that in the parental NIH 3T3 cell line (Fig. 4I). The strongest effect was detected for SINrep/GFP, and it additionally confirmed a more critical role of PKR in SINrep/GFP-infected cells compared to cells infected with the other constructs. Of note, the effect of Pkr KO was not as strong as the expression of the S51A eIF2α mutant (Fig. 4G and I). This points to a possible role for kinases other than PKR in translation regulation within cells infected with alphavirus replicons. However, it could be due to some differences in the levels of host proteins, which are required for replication of virus-specific RNAs.

Alphavirus replicons differ in their ability to activate an innate immune response

The NW and OW alphaviruses have evolved different mechanisms of interfering with the development of an innate immune response, which can affect their replication by an autocrine mechanism (27, 28, 34 – 36). In agreement with data from virus-related experiments, infection of NIH 3T3 and MRC-5 cells, which are fully competent in type I interferon (IFN) induction and signaling, by VEEV- and EEEV-derived replicons resulted in IFN-β induction (Fig. 5). Since these replicons do not express capsid protein, which is responsible for inhibition of cellular transcription (27, 37), IFN-β was readily detectable in the media of infected NIH 3T3 cells at 8 h p.i. and reached high concentrations by 24 h p.i. in both NIH 3T3 and MRC-5 cells. In contrast to the NW alphavirus replicons, those derived from the OW alphaviruses, SINrep/GFP and CHIKrep/GFP, encode nsP2 exhibiting transcription-inhibitory functions (35, 36, 38, 39). Therefore, even at 24 h p.i., the NIH 3T3 cells were unable to induce IFN-β to a level detectable by enzyme-linked immunoassay (ELISA), and in MRC-5 cells, SINrep/GFP induced IFN-β barely at the level of detection.

Fig 5.

The NW and OW alphavirus replicons demonstrate different abilities to induce IFN-β. NIH 3T3 cells (A) and MRC-5 (B) cells were infected with the indicated replicons at an MOI of 20 inf.u/cell, and levels of the released IFN-β were assessed by ELISA (see Materials and Methods for details). LOD indicates the limit of detection; n.d. indicates not detectable. These experiments were reproducibly performed multiple times, and the results of one of them are presented.

Modifications of replicons can strongly increase expression of encoded heterologous genes

The above sections presented the basic characteristics of alphavirus replicon-cell interactions that determine the expression level of encoded heterologous proteins. This knowledge can be used to increase the expression level of such replicons including the underperforming SINrep/GFP. For example, the translational enhancer, the stable stem-loop structure located downstream of the initiating AUG, can increase the translation of SINrep-specific SG RNA in conditions of virus-induced translation inhibition (40). Since this enhancer made translation dramatically less dependent on translation inhibition caused by eIF-2α phosphorylation (Fig. 3B, Fig. 4G and I), the GFP expression by SINrep/C-GFP increased 20- to 30-fold (Fig. 6B), compared to parental SINrep/GFP. Similarly, insertion of a SINV-specific translational enhancer upstream of the GFP-coding sequence in CHIKV replicon (Fig. 6C) (CHIKrep/C_sin-GFP) also caused an increase in translation of the SG RNA, albeit the positive effect was not strong (Fig. 6D).

Fig 6.

Translational enhancer strongly increases GFP expression by CHIKV- and SINV-derived replicons. (A) The schematic representation of the SINV-based replicons. (B) Cells were infected with SINrep/GFP and SINrep/C-GFP at an MOI of 20 inf.u/cell. At 18 h p.i., they were evaluated for completeness of infection by fluorescence microscopy and lysed, and GFP fluorescence was assessed. (C) The schematic representation of the CHIKV-based replicons used. (D) Cells were infected with CHIKrep/GFP and CHIKrep/C_sin-GFP and analyzed as described in panel B. Means and SDs are presented, n = 3. Significance of differences was determined by unpaired t test (****P < 0.0001).

Positioning a translational enhancer upstream of the coding sequence of the gene of interest is not the only means of increasing the expression of the heterologous, replicon SG RNA-encoded genes. Another approach is to introduce attenuating mutations into the replicons’ backbones. In the OW alphaviruses, nsP2 and nsP3 play important roles in transcription inhibition of cellular genes, induction of translational shutoff during viral replication, and development of cytopathic effect (CPE) (35, 41 – 43). Some mutations have previously been found capable of making SINV replicons no longer cytopathic (41, 44). Although such mutations decrease the rates of RNA replication and transcription of the SG RNA (35, 45), their presence in SINV nsP2 strongly enhances the expression of genes of interest. The results presented in Fig. 7 show that SINrep/G/GFP replicon, which is identical to SINrep/GFP, except for having a previously described P726G mutation in nsP2 (Fig. 7A) (45), expressed GFP in BHK-21 and NIH 3T3 cells with a few fold higher efficiency (Fig. 7B). The P726G mutation had no positive effect on GFP expression in MRC-5 cells. However, the presented expression level, which was similar to that of SINrep/GFP, was achieved by SINrep/G/GFP despite the strong negative effect of the nsP2-specific mutation on RNA replication (44).

Fig 7.

Attenuating mutations in SINV nsP2 have a positive effect on GFP expression. (A) The schematic representation of SINV-based replicons. (B) Cells were infected with packaged replicons at an MOI of 20 inf.u/cell. The levels of GFP were assessed at 18 h p.i. Means and SDs are presented, n = 3. Significance of differences was determined by unpaired t test (****P < 0.0001).

A third method to increase the level of heterologous gene expression is to decrease the eIF2α phosphorylation. This can be achieved through a high level of expression of the K296R PKR mutant (mutPKR). To show this, the coding sequence of mutPKR was cloned into GFP-expressing SINV replicons under the control of an additional SG promoter. We tested the effects of mutPKR expression in the context of the original replicon (SINrep/mutPKR/GFP) and the replicon encoding the mutated form of nsP2 (SINrep/G/mutPKR/GFP). Packaged SINrep/GFP, SINrep/mutPKR/GFP, and SINrep/G/mutPKR/GFP replicons were used to infect a variety of widely used human cell lines, which included HEK 293T, HeLa, and A549 cells. The results presented in Fig. 8 demonstrate that the expression of mutPKR had strong stimulatory effects on GFP expression by SINrep/mutPKR/GFP in all cell lines used. However, the P726G mutation in SINrep/G/mutPKR/GFP caused a further threefold to fourfold increase in GFP production.

Fig 8.

Expression of mutPKR and attenuating mutation in nsP2 synergistically increase GFP expression by SINV replicon. (A) The schematic representation of the used replicons. (B) Cells were infected with the replicons at an MOI of 20 inf.u/cell. At 18 h p.i., they were evaluated in terms of being 100% GFP-positive, and then levels of GFP were assessed. Means and SDs are presented, n = 3. Significance of differences was determined by one-way ANOVA followed by Dunnett test (****P < 0.0001) independently for each cell type.

DISCUSSION

In recent years, mRNA vaccination has become widely used for prophylactic vaccination in the setting of the SARS-CoV-2 pandemic. BNT162b2 and mRNA-1273 vaccines have received authorization for emergency use and were used for the immunization of hundreds of millions of people worldwide. The main advantages of the mRNA vaccines include (i) a lack of integration into the host genome, (ii) high safety in terms of the inability to develop infection, (iii) rapid development of the vaccine candidates against emerging pathogens and new viral variants, and (iv) reasonably high efficiency in eliciting immunity. The drawbacks are the high instability of the in vitro-synthesized RNAs, low efficiency of the RNA delivery in vivo, the short half-life of mRNA in the cells, and thus, a short-time availability for cellular translational machinery. As a result, high doses (30–100 μg) of the in vitro-synthesized mRNAs are currently being applied for vaccination.

saRNAs have the potential to overcome some problems associated with mRNA-based vaccination. They make cells to express higher levels of RNAs encoding the proteins of interest and probably for a longer time. Genomes of RNA(+) viruses are natural saRNAs. They can be used as starting materials for further modification and expression of heterologous genes. Members of the Alphavirus genus in the Togaviridae family are particularly attractive, because of their robust replication and accumulation of the SG RNA encoding viral structural proteins to tens of thousands of copies per cell (1). Alphaviruses were one of the first viral groups for which the reverse genetic system was developed and used to manipulate viral genomes (13). Its application has led to the identification of viral and cellular proteins and RNA elements required for G RNA amplification and transcription of the SG RNA. Since structural proteins have been found to be dispensable for the synthesis of viral RNAs, their genes in G RNAs can be replaced by heterologous genetic materials (18, 46). After delivery into cells, these modified G RNAs remain capable of self-amplification and serve as templates for the transcription of SG RNAs, which are ultimately translated into the proteins of interest. Another benefit of the alphavirus genome coding strategy is that more than one SG promoter can be engineered and multiple genes of interest can be cloned into the same replicon (Fig. 8). Alternatively, the additional open reading frames can be cloned into the same SG RNA under the control of internal ribosome entry sites (IRESes) or fused with each other through ubiquitin- and/or picornavirus 2A protease-coding sequences.

At first glance, the results of this study demonstrate that both OW and NW alphavirus replicons are capable of expressing heterologous genes in a variety of cells and have a potential for application in RNA-based vaccination. All of the tested constructs expressed heterologous proteins, albeit replicons derived from the NW alphaviruses appeared to produce them more efficiently than the OW counterparts. Without additional modifications, SINV-based replicons are the least productive. This difference results from more efficient phosphorylation of eIF2α during RNA replication and robust translational shutoff (Fig. 3) induced by the accumulation of dsRNA replication intermediates. Inhibition of translation has a strong negative effect on the translation of SG RNA-encoded heterologous genes. However, expression from SINV- and even from CHIKV-based constructs can be dramatically improved by using a translational enhancer, which is present at the beginning of the SINV capsid-coding RNA sequence. Moreover, the artificially designed enhancers work as efficiently as the wt capsid-specific RNA sequences (40). Other options to achieve higher expression levels are (i) to use SINrep and CHIKrep constructs with mutated nsP2 or nsP2/nsP3, which do not affect cellular transcription and translation as much as constructs encoding the wt counterpart (45), (ii) to express a K296R mutant form of human PKR from the same replicon, or (iii) to apply a combination of the above-described modifications. Moreover, the expression cassettes can be re-designed to produce SG RNAs, which are also capable of additional self-amplification by viral nsPs and, thus, are more efficient in expression of the encoded proteins of interest (47).

The level of protein expression is not the only important characteristic of the alphavirus replicons. Despite encoding only four nsPs, replicons mirror many critical aspects of viral infection. They employ virus-specific sets of host factors and induce specific modifications of the intracellular environment (26, 48). Thus, the efficiency of their self-amplification depends on the availability of such factors, is cell-specific, and needs further investigation. The results presented in Fig. 2 and 8 demonstrate that GFP expression in the human cells utilized in this study was not as high as in murine cells, and it will likely be even lower in the RNA transfection-based experiments in vivo. Accordingly, immunogenicities of saRNAs expressing SARS-CoV-2 spike in humans and nonhuman primates are not as high as could be expected (49, 50) and are different from those previously detected in mice.

The NW and OW alphaviruses also fundamentally differ in their abilities to induce an antiviral response. The NW alphavirus replicons (VEErep and EEErep) do not encode homologous capsid proteins, which play critical roles in the development of transcriptional shutoff and, ultimately, in the inhibition of the innate immune response during the NW alphavirus replication (28, 37). This makes such replicons very potent inducers of type I IFN and, most likely, other pro-inflammatory cytokines (Fig. 5) (51, 52). Activation of innate immunity was previously proposed to be beneficial for the induction of a more efficient adaptive immune response. Thus, the NW alphavirus replicons potentially have self-adjuvant activity (53). However, on the other hand, it remains unclear whether additional inflammation is a desired characteristic of potential vaccine candidates. Moreover, since the VEEV- and EEEV-based replicons do not interfere with cellular transcription, the release of type I IFN downregulates their replication by inducing interferon-stimulated genes (ISGs). Their products have negative effects on RNA replication and expression of SG RNA-encoded proteins of interest (52, 54). Thus, an open question remains whether the lack of IFN induction is beneficial for the development of antibody and T-cell responses. However, EEEV- and VEEV-specific capsid proteins contain short peptides that bind CRM1 and importin-α/β at the same time (37). These large complexes block nuclear pores and ultimately cause transcription inhibition. Thus, the expression of capsids or their nuclear receptor-binding peptides by the NW alphavirus replicons may have positive effects on the production of proteins of interest and the development of adaptive immunity.

In contrast to the NW alphaviruses, nsP2 proteins of the OW species are not only involved in ns polyprotein processing and RNA synthesis, but they also accumulate in the nuclei of infected cells. They induce the degradation of Rpb1, the catalytic subunit of cellular DNA-dependent RNA polymerase 2 (35, 36). The dramatic decrease in Rpb1 level results in the rapid development of transcriptional shutoff and makes cells incapable of mounting an antiviral response. Human and mouse cells containing the OW alphavirus replicons (CHIKrep and SINrep) do not induce type I IFN (Fig. 5) and cannot respond to its presence by induction of ISGs (43). Thus, they may be more efficient in vivo in terms of induction of adaptive immunity. However, it is possible that as was proposed for the NW alphavirus replicons (53), induction of type I IFN and pro-inflammatory response during SINrep and CHIKrep replication may be beneficial for the development of an adaptive immune response to encoded protein. If this is the case, the OW alphavirus-based constructs can be easily modified through the introduction of previously identified attenuating mutations into nsP2 and nsP3 (35, 41, 44, 55). Such mutations not only increase the expression levels of the proteins of interest but also make replicons incapable of interfering with cellular transcription. In contrast to the wt constructs, such nsP2 mutants become strong type 1 IFN inducers and are dramatically less cytopathic.

Another important characteristic of all alphavirus-derived replicons is that besides the genes of interest, they encode four viral nsPs, nsP1–4. In virus-infected cells, these proteins are efficiently synthesized at early times p.i., when the concentration of capsid protein is relatively low. Since capsid is not synthesized in replicon-containing cells, the synthesis of nsPs can continue for a longer time. Their concentrations may become above the threshold required for T-cell and B-cell activation and sufficient for induction of nsP-specific antibody and T-cell responses. Thus, repeated vaccination with the replicon derived from the same alphavirus can potentially lead to the development of vector immunity by targeting viral nsPs and activating the cytotoxic T-cell response. Moreover, the alphavirus replicon-induced CPE likely contributes to a release of nsPs and development of antibody and T-cell responses. The levels and roles of such adaptive immune response during repeat applications of replicons in vivo need to be evaluated. The use of more than one replicon in repeated saRNA vaccinations is probably one of the ways to avoid or at least slow the development of vector immunity.

Interestingly, alphavirus replicons expressing glycoproteins derived from other viruses often function as pseudoviruses (56 – 59). They can develop spreading infection, at least in cultured cells, and further evolve in the direction of more efficient spread. Thus, the expressed glycoproteins need to be carefully designed to avoid the formation and spread of pseudoviruses and syncytia formation.

Taken together, the previously published data and the results of this study demonstrate that all alphavirus replicons have a potential to improve the mRNA vaccines in terms of more efficient and longer expression of heterologous proteins of interest. Moreover, some additional modifications may have strong positive effects on protein expression levels and additionally increase or decrease induced innate immune response. However, replicons mimic many aspects of viral infections. Their self-amplification is determined not only by the encoded viral nsPs but also by the availability of host factors and by other replicon-cell interactions. The levels of protein expression strongly depend on the replicon’s origin and cell types. Thus far, many aspects of the interactions between replicons and the host remain insufficiently understood at the cellular and organismal levels. Further studies are needed to help to understand the biology of the replicons before they are applied for the development of saRNA-based vaccines.

MATERIALS AND METHODS

Cell cultures

The BHK-21 cells were kindly provided by Paul Olivo (Washington University, St. Louis, Missouri, USA). The NIH 3T3, MRC-5, HEK 293T, Vero E6, HeLa, and A549 cells were obtained from the American Tissue Culture Collection (Manassas, Virginia, USA). MEF/SS and MEF/AA cells (33) were provided by Nancy L. Kedersha (Brigham and Women’s Hospital, Boston, Massachusetts, USA). NIH 3T3 Pkr KO cells were generated as described elsewhere (60) and used as cloned cell lines. MRC-5 cells and MEFs were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS). Other cell lines were maintained in alpha minimum essential medium supplemented with 10% FBS and vitamins.

Plasmid constructs

Plasmids encoding SINrep/GFP, CHIKrep/GFP, VEErep/GFP, and EEErep/GFP were engineered from infectious cDNA of SINV Toto1101 (13), CHIKV 181/25 (16), VEEV TC-83 (61), and EEEV (62), respectively. In all plasmids, GFP was positioned under the control of natural 5′ UTR of SG RNA, including the initiating ATG. The 3′ UTRs in all encoded replicons also remained intact. SINrep/G/GFP was identical to SINrep/GFP, except for containing a P726G mutation in the nsP2 sequence. pSINrep/C-GFP encoded not only the 5′ UTR but also a SINV capsid-coding sequence upstream of the GFP gene. pCHIKrep/C_sin-GFP contained both 5′ UTR of SG RNA and the capsid-coding sequence derived from SINV. SINrep/mutPKR/GFP and SINrep/G/mutPKR/GFP encoded K296R mutant form of human PKR under the control of an additional SG promoter. Plasmids encoding defective helper genomes of VEEV, SINV, and CHIKV contained 5′ fragments of corresponding viral genomes followed by virus-specific SG promoters and the entire sequences of the SG RNA (18, 21). The genes of nsPs were mostly deleted. Helper for packaging EEErep/GFP had the same design, except that the EEEV structural genes in the SG RNA were replaced by those derived from SINV TE-12. The 3′ UTR remained EEEV-specific. In the plasmids, all of the cDNAs of replicon and helper genomes were cloned under the control of the SP6 promoters.

Packaging of replicons

Plasmid DNAs encoding helper and replicon genomes were purified by ultracentrifugation in CsCl gradients. They were linearized using unique restriction sites located immediately downstream of the poly(A) tails. RNAs were in vitro-synthesized by SP6 RNA polymerase in the presence of a cap analog (New England Biolabs) according to the manufacturer’s recommendations (New England Biolabs). The synthesized RNAs were analyzed by electrophoresis in nondenaturing agarose gels and further used without additional purification. Replicons’ and helpers’ RNAs were mixed and used for electroporation of BHK-21 cells under previously described conditions (14). Packaged replicons were harvested at 24 h post-electroporation and stored at −80°C.

To determine infectious titers, the cell lines indicated in the figures were seeded into 6-well Costar plates (5 × 10⁵ cells/well) and, in 4 h, infected with different dilutions of stocks of packaged replicons. After incubation for 18 h at 37°C, numbers of GFP-positive cells were evaluated and used for calculating infectious titers, which were specific for each cell line.

Analysis of GFP expression by alphavirus replicons

The cell lines were infected with packaged replicons in 6-well Costar plates at an MOI of 20 inf.u/cell, calculated based on the infectious titers determined for each replicon on each used cell line. This MOI was sufficient to infect all of the cells in monolayers. Infected cells were further incubated in complete media at 37°C for times indicated in the figures. The completeness of infection, the presence of GFP-expressing replicons in 100% of cells, was always evaluated by fluorescence microscopy, and then cells were lysed in 0.8 mL of buffer (50-mM Tris-HCl, pH 7.5; 100-mM NaCl; 2-mM β-mercaptoethanol; and 1% Triton X-100). 0.1-mL aliquots were used to assess GFP fluorescence on Cytation V plate reader (BioTek Instruments, Inc.).

Analysis of cellular protein synthesis

The cell lines indicated on the figures were plated in 6-well Costar plates and infected with packaged replicons at an MOI of 20 inf.u/cell. After incubation for 8 h at 37°C, media were replaced by that supplemented with Pur (10 µg/mL). After 15-min incubation at 37°C, cells were harvested. Then, they were lysed directly in protein gel-loading buffer, and the lysates were analyzed by WB using Pur-specific MAb (MABE343MI, MilliporeSigma) and the infrared dye-labeled secondary Abs. In one of the experiments, the same membranes were additionally incubated with GFP-specific Ab conjugated with DyLight 800, acquired from Rockland (600-145-215). Images were acquired on Odyssey CLx imager (LI-COR Biosciences) and processed using the manufacturer’s software.

WB analysis of PKR and eIF2α phosphorylation

Equal numbers of cells infected with packaged replicons were harvested and lysed directly in protein gel-loading buffer. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The following antibodies were used for detection: anti-p-eIF2α (3398, Cell Signaling Technology), anti-PKR (sc-1702, Santa Cruz), and anti-β-actin (66009-1-Ig, Proteintech). Secondary infrared dye-labeled IRDye 680RD or IRDye 800CW were acquired from LI-COR Biosciences. Membranes were scanned and analyzed on Odyssey CLx imager (LI-COR Biosciences).

IFN-β assay

Concentrations of murine and human IFN-β in the media were assessed by using the VeriKine Human and Mouse IFN-β ELISA Kits (PBL Assay Science).