Mutation sites in the nsP1-4 region of repRNA
It has been reported that mutations in nsP1-4 may affect many aspects of replicon biology such as expression levels of GOI and host immune responses against repRNA-encoded immunogens(.Frolova et al. 2002, Kääriäinen and Ahola 2002, Li et al. 2019). To enhance the transgene expression mediated by TC-83 VEE-derived replicon vectors, we introduced several point mutations at multiple sites in their nsP1-4 region that is the key component responsible for repRNA replication. As shown in Fig. 1, these mutation sites include G357C, G1569A, A1572C and C1575T in nsP1; A3821T, G3892C and T3922C in nsP2; A4714G in nsP3. Furthermore, we constructed the following repRNA constructs, each bearing multiple mutations in a single replicon vector: (1) the combination of G357C, G1569A, A1572C and C1575T in nsP1 (nsP1GGAC); (2) the combination of nsP1GGAC and T3922C in nsP2 (nsP1GGAC-nsP2T); (3) the combination of nsP1GGAC-nsP2T, G3892C in nsP2 and A4714G in nsP3 (nsP1GGAC-nsP2GT-nsP3A); (4) the combination of G3892C in nsP2 and A4714G in nsP3 (nsP2G-nsP3A).
Vee Nsp1ggac-nsp2t Promotes Transgene Expression
We first subcloned interleukin (IL)-15 or IL-12 sequences into T7-VEE plasmids bearing either wild type (WT) nsP1-4 sequence or the above mutant nsP1-4 sequences. The resulting T7-VEE plasmids were linearized by restriction endonuclease digestion and the linearized DNA templates were in vitro transcribed into repRNAs encoding IL-15 or IL-12 (repRNA-IL15 or repRNA-IL12). We then evaluated IL-15 or IL-12 expression in cells and cell culture supernatants after transfecting 293T cells with repRNA-IL15 or repRNA-IL12 for 36 h. Compared with WT repRNA-IL15, repRNA-IL15 bearing nsP1GGAC-nsP2T mutations significantly up-regulated IL-15 expression in cells and IL-15 secretion in cell culture supernatants after transfection using Lipofectamine 2000 (Fig. 2A). While repRNAs-IL15 bearing other mutation(s) failed to up-regulate IL-15 expression in cells and decreased IL-15 secretion in cell culture supernatants (Fig. 2A). In addition, compared with WT repRNA-IL12, transfection with repRNA-IL12 bearing nsP1GGAC-nsP2T or nsP1GGAC mutations significantly increased IL-12 expression in cells and IL-12 secretion in cell culture supernatants (Fig. 2B). While repRNAs-IL12 bearing other mutation(s) led to undetectable IL-12 expression in cells and little IL-12 secretion in cell culture supernatants (Fig. 2B). These data indicate that VEE-derived repRNAs bearing nsP1GGAC-nsP2T mutation could enhance transgene expression under control of the SGP. On the contrary, the introduction of A4714G mutation in nsP3 sequence could potentially compromise the expression of target genes downstream of SGP.
The A3821 Site Of Nsp2 Is Important For Subgenome Expression
Furthermore, we mutated A to T at position 3821 of nsP2 in nsP1 GGAC-nsP2T mutant to generate a new nsP1GGAC-nsP2AT mutant (Fig. 1). We investigated if the introduction of A3821T mutation in nsP2 (nsP2 A3821T) could affect the transgene expression mediated by replicons bearing nsP1GGAC-nsP2AT mutation. As shown in Fig. 3A, compared with repRNA-IL15 bearing nsP1GGAC-nsP2T mutations, repRNA-IL15 bearing nsP1GGAC-nsP2AT mutations further increased IL-15 expression in 293T cells and IL-15 secretion in cell culture supernatants at 36 h after transfection. Similar gene expression profile was observed when 293T cells were transfected with repRNA-IL12. Compared with repRNA-IL12 bearing nsP1GGAC-nsP2T mutations, repRNA-IL12 bearing nsP1GGAC-nsP2AT mutations further increased IL-12 expression in cells and IL-12 secretion in cell culture supernatants at 36 h after transfection (Fig. 3B). These results suggest that the introduction of nsP2 A3821T mutation in nsP1GGAC-nsP2T mutant (nsP1GGAC-nsP2AT) could further up-regulate the expression of target genes downstream of SGP.
In addition, we synthesized WT replicons and the above mutants (replicons bearing nsP1GGAC-nsP2T or nsP1GGAC-nsP2AT mutations) encoding GM-CSF, IFN-γ or IL-2, and evaluated their transfection efficiency. Compared with WT repRNA-GMCSF, repRNA-GMCSF bearing either nsP1GGAC-nsP2T or nsP1GGAC-nsP2AT mutations enhanced GM-CSF expression in cells and GM-CSF secretion in cell culture supernatants at 36 h after transfection (Fig. 4A). In addition, compared with WT repRNA-IFNγ, repRNA-IFNγ bearing nsP1GGAC-nsP2AT mutations enhanced IFN-γ expression in cells and IFN-γ secretion in cell culture supernatants; and repRNA-IFNγ bearing nsP1GGAC-nsP2T mutations led to enhanced IFN-γ expression in cells and similar IFN-γ secretion in cell culture supernatants (Fig. 4B). RepRNA-IFNγ bearing nsP1GGAC-nsP2AT mutations showed improved IFN-γ expression in cells and similar IFN-γ secretion in cell culture supernatants compared to repRNA-IFNγ bearing nsP1GGAC-nsP2T mutations (Fig. 4B). We also analyzed the effects of mutation(s) in the nsP1-4 region on the expression of repRNA encoding IL-2. Compared with WT repRNA-IL2, repRNAs-IL2 bearing nsP1GGAC-nsP2T, nsP1GGAC-nsP2AT and nsP2G-nsP3A enhanced IL-2 secretion by ~ 1.5-fold, ~ 17.0-fold and ~ 3.0-fold in cell culture supernatants respectively, and increased IL-2 expression by ~ 1.5-fold, ~ 28.0-fold and ~ 3.0-fold in cells respectively (Fig. 4C). Above all, VEE-derived replicons bearing nsP1GGAC-nsP2AT mutations lead to optimal expression of target genes such as IL-2, IL-12, IL-15, GM-CSF and IFN-γ.
RdRp regulates the replication and amplification of replicon genomic RNA and subgenomic RNA through a complex and orderly mechanism. The nsP1 protein is responsible for mRNA capping and has both guanylyltransferase (GTase) and guanine-7-methyltransferase (MTase) activities, thereby guiding the capping and methylation of viral genomic and subgenomic RNAs(.Ahola and Kääriäinen 1995, Cross 1983). The nsP2 protein plays a variety of functions during alphavirus infection. The N-terminal of nsP2 protein contains a helicase structure which performs the first viral RNA capping reaction as an RNA triphosphatase and promotes RNA helicase activity as a nucleotide triphosphatase (NTPase)(.Karpe et al. 2011, Vasiljeva et al. 2000). The C-terminal of nsP2 protein is identified as a papain-like cysteine protease similar to known cathepsins, which processes viral non-structural polyproteins(.Russo et al. 2006). The nsP3 protein is necessary for repRNA synthesis and replication, but its specific role is not clear. The nsP3 protein contains the N-terminal macrodomain with both nucleic acid binding and phosphatase capabilities, the alphavirus unique domain with a strong homology in alphavirus, and the C-terminal hypervariable region(.Aaskov et al. 2011, Malet et al. 2009, Shin et al. 2012). Since the nsP4 protein, the most highly conserved region in alphaviruses, contains the core RdRp domain and motifs, it is directly responsible for RNA synthesis of the viral replicase complex(.Lello et al. 2021, Rubach et al. 2009). The nsP1GGAC-nsP2T and nsP1GGAC-nsP2AT mutant VEE replicons could significantly enhance the expression of transgenes encoding inflammatory cytokines, which may be related to the capping activity of nsP1 and the activity of helicase, triphosphatase or protease of nsP2. In this study, we discovered that VEE replicons bearing nsP1GGAC-nsP2T and nsP1GGAC-nsP2AT mutations in the nsP1-4 region could significantly enhance the transgene expression of repRNA.