AAACH is a conserved motif in a cis-acting replication element that is artificially inserted into Senecavirus A genome

Highlights

• •A₁A₂A₃C₄A₅ is a wild-type motif in cre of Senecavirus A (SVA).
• •SVA can tolerate two mutations (A5C and A5U) in an artificial cre.
• •A5C and A5U mutations in artificial cre affect grow kinetics of SVA.
• •Both A5C and A5U mutations are stable with serial viral passaging.
• •A SDM-modified natural cre shows two mutations with serial passaging.

Abstract

Cis-acting replication element (cre) is required for generating a diuridylylated VPg that acts as a protein primer to initiate the synthesis of picornaviral genome or antigenome. The cre is a stem–loop structure, dependent of different picornaviruses, located in different genomic regions. The AAACA motif is highly conserved in the apical loop of cre among several picornaviral members, and plays a key role in synthesizing a diuridylylated VPg. We previously demonstrated that senecavirus A (SVA) also possesses an AAACA-containing cre in its genome. Its natural cre (Nc), if functionally inactivated through site-directed mutagenesis (SDM), would confer a lethal impact on virus recovery, whereas an artificial cre (Ac) is able to compensate for the Nc-caused functional inactivation, leading to successful rescue of a viable SVA. In this study, we constructed a set of SVA cDNA clones. Each of them contained one functionally inactivated Nc, and an extra SDM-modified Ac. Every cDNA clone had a unique SDM-modified Ac. The test of virus recovery showed that only two SVAs were rescued from their individual cDNA clones. They were AAACU- and AAACC-containing Ac genotypes. Both viruses were serially passaged in vitro for analyzing their viral characteristics. The results showed that both AAACU and AAACC genotypes were genetically stable during twenty passages, implying when the Nc was functionally inactivated, SVA could still use an AAACH-containing Ac to complete its own replication cycle.

1. Introduction

Senecavirus A (SVA), formerly named Seneca Valley virus (SVV), causes vesicular lesions in pigs, clinically indistinguishable from other vesicular diseases, such as foot-and-mouth disease and vesicular stomatitis (Zhang et al., 2018). Gross lesions include multifocal, round, discrete erosive and/or ulcerative lesions on distal limbs, especially around the coronary bands. Crusting and sloughing of the hoof wall may also be observed. The SVA-infected cases were initially found in dozens of pigs at a Canada market in 2007 (Pasma et al., 2008). However, the prototype strain (SVV-001) of SVA was originally identified as a contaminant in cell culture during cultivation of PER.C6 cells in 2002 (Hales et al., 2008). SVA is additionally an oncolytic virus with selective tropism for some tumors with neuroendocrine characteristics, but not pathogenic for human beings (Morton et al., 2010; Poirier et al., 2013; Reddy et al., 2007).

SVA is classified into the genus Senecavirus in the family Picornaviridae. To date, this virus has been the only member of the genus Senecavirus. Mature virion is an icosahedral-shaped particle without envelope, approximately 30 nm in diameter (Strauss et al., 2018). The viral capsid is composed of a densely-packed icosahedral arrangement of 60 protomers. The viral genome is one positive-sense, single-stranded RNA, approximately 7 300 nt in length. It contains 5′ and 3′ untranslated regions (UTRs), 3′ poly(A) tail, and a single open reading frame (ORF) of polyprotein precursor. The 5′ UTR possesses a hepatitis C virus-like internal ribosome entry site, structurally and functionally similar to those of pestiviruses, allowing for initiating the polyprotein translation in a cap-independent manner (Willcocks et al., 2011). The polyprotein precursor, after translation, will be cleaved into various proteins stepwise: L, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C and 3D (Liu et al., 2021).

The picornaviral genome is actually one mRNA, but without 5′-capped structure. As a substitute, the viral genome-linked protein (VPg) is covalently linked to the 5′ terminus of RNA genome (Gavryushina et al., 2011; Paul and Wimmer, 2015). Initiating such a covalent linkage is concerned with two major steps. The first step is involved in the VPg that serves as a primer to synthesize a diuridylylated VPg, namely, VPg-pUpU, in a reaction catalyzed by a picornaviral polymerase. The VPg-pUpU is characterized by covalent linkage of two uridine monophosphate molecules to the hydroxyl group of the third amino acid (tyrosine) in the VPg. In the second step, the VPg-pUpU is transferred to 3′ termini of positive- and negative-strand RNAs, and then functions as a primer for initiating the synthesis of full-length antigenome and genome, respectively (Pathak et al., 2008; Paul et al., 1998; Sun et al., 2014). The molecular characteristics of VPg-pUpU indicate that two consecutive U resides are the first two nucleotides at the 5′ terminus of picornaviral genome.

The process of VPg uridylylation is carried out in a template-dependent manner via a stem–loop structure (Thiviyanathan et al., 2004), known as cis-acting replication element (cre), which has been identified within different genomic regions of picornaviruses, like 2C (Goodfellow et al., 2000), 2A (Gerber et al., 2001), VP1 (McKnight and Lemon, 1998), VP2 (Lobert et al., 1999) and 3D (Yang et al., 2008). Almost all reported picornaviruses possess their own cres in encoding regions, except that of foot-and-mouth disease virus, demonstrated to be located in the 5′ UTR (Mason et al., 2002). The apical loop of cre structure harbors an adenosine-rich motif, AAACD, in which the letter “D” is a degenerate base that means A, G or U. The AAACD, especially the AAACA, is phylogenetically conserved among picornaviral members. The AAACA motif is widely identified in enterovirus (Rieder et al., 2000), cardiovirus (Lobert et al., 1999), aphthovirus (Mason et al., 2002) and more recently in senecavirus (Meng et al., 2023). The AAACG and AAACU motifs, albeit less reported, have been recognized in hepatovirus (Yang et al., 2008) and parechovirus (Al-Sunaidi et al., 2007), respectively.

The AAACA motif has been extensively characterized in poliovirus cre that as a template triggers the uridylylation of VPg via a slide-back mechanism. The slide-back process is briefly described below: (i) the viral 3CD^pro binds to the cre, and enhances binding of the 3D^pol–VPg complex to the loop; (ii) the first U residue is linked to the hydroxyl group of tyrosine in VPg using the first A residue as a template nucleotide; (iii) the nascent VPg-pU slides back to hydrogen bond with the second A residue, followed by the addition of the second U residue again on the first A residue (Paul et al., 2003). The slide-back event leads to two U residues covalently linked to the tyrosine of VPg, resultantly generating the protein primer, VPg-pUpU. Subsequently, both VPg-pUpU and 3D^pol will be recruited to the negative-sense RNA to prime the synthesis of a positive-strand viral genome (Paul and Wimmer, 2015).

We recently identified a 52-nt-long cre, named natural cre (Nc), located in the VP2-encoding region of SVA. This cre is composed of an 18-nt-long apical loop, and a stem region that harbors three single mismatches (G–G, U–U and G–G). A typical A₁A₂A₃C₄A₅ motif was found in the 5′ half of the cre loop (Meng et al., 2023). Although the A₁A₂A₃C₄A₅, especially the first four residues, have been demonstrated to be highly conserved among picornaviral members, it is unclear whether viable SVA can tolerate a single point mutation occurring in the A₁A₂A₃C₄A₅ motif. In the present study, we used the reverse genetics to demonstrate that the A₁A₂A₃C₄ residues were absolutely conserved, whereas the fifth reside A could tolerate two single mutations (A5C and A5U) occurring in a cre artificially inserted into the SVA genome.

2. Materials and methods

2.1. Cell line, plasmid and virus

The BSR-T7/5 cell line (BHK-21 cell clone) (Buchholz et al., 1999) was cultured at 37 °C with 5 % CO₂ in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10 % fetal bovine serum (VivaCell, Shanghai, China), penicillin (100 U/mL), streptomycin (100 µg/mL), amphotericin B (0.25 µg/mL) and G418 (500 µg/mL). A genetically modified SVA (GenBank: KX751945.1) cDNA clone, renamed cDNA-wt here, was previously constructed in our laboratory (Meng et al., 2023). The cDNA-wt contained a site-directed mutagenesis (SDM)-modified Nc (SNc), and an extra cre without modification. The extra cre, named artificial cre (Ac), was located between encoding sequences of the enhanced green fluorescent protein (eGFP) and the Thosea asigna virus 2A (T2A). A wild-type recombinant SVA (rSVA-wt) had been rescued previously from the cDNA-wt in our laboratory.

2.2. Construction of cDNA clone with SDM-modified Ac

The apical loop of Ac also harbored the A₁A₂A₃C₄A₅ motif, in which each site was subjected to SDM, amounting to thirteen single-site mutations, namely, A1T, A1G, A2T, A2C, A2G, A3T, A3C, A3G, C4A, C4G, A5T, A5C and A5G. Each of them was referred to as a Ref. No., used to name a cDNA clone, such as cDNA-A1T. A total of thirteen point-mutated cDNA clones were constructed through SDM and In-Fusion® assembly techniques. At first, a template plasmid was constructed. In brief, the cDNA-wt served as a PCR template for amplifying a stretch of sequence using a pair of primers, forward primer 1 (FP1) and reverse primer 2 (RP2) (Table 1), covering PshA I and Fse I sites of the cDNA-wt, respectively. The PCR product was purified, identified, and subcloned into the TA/Blunt-Zero cloning vector (Vazyme, Nanjing, China) for constructing a template plasmid, which then served as the PCR template to amplify two fragments for the overlap extension PCR (OE-PCR).

Table 1.

OE-PCR primers for constructing SVA cDNA clones with SAc.

cDNA clones	FP1	RP1 (5′ to 3′)*	FP2 (5′ to 3′)*	RP2
cDNA-A1U	#	gattgcgTGTTAatggcagtgtt	aacactgccatTAACAcgcaatc	##
cDNA-A1G	#	gattgcgTGTTCatggcagtgtt	aacactgccatGAACAcgcaatc	##
cDNA-A2U	#	tgattgcgTGTATatggcagtgt	acactgccatATACAcgcaatca	##
cDNA-A2C	#	tgattgcgTGTGTatggcagtgt	acactgccatACACAcgcaatca	##
cDNA-A2G	#	tgattgcgTGTCTatggcagtgt	acactgccatAGACAcgcaatca	##
cDNA-A3U	#	atgattgcgTGATTatggcagtg	cactgccatAATCAcgcaatcat	##
cDNA-A3C	#	atgattgcgTGGTTatggcagtg	cactgccatAACCAcgcaatcat	##
cDNA-A3G	#	atgattgcgTGCTTatggcagtg	cactgccatAAGCAcgcaatcat	##
cDNA-C4A	#	gatgattgcgTTTTTatggcagt	actgccatAAAAAcgcaatcatc	##
cDNA-C4G	#	gatgattgcgTCTTTatggcagt	actgccatAAAGAcgcaatcatc	##
cDNA-A5U	#	tgatgattgcgAGTTTatggcag	ctgccatAAACTcgcaatcatca	##
cDNA-A5C	#	tgatgattgcgGGTTTatggcag	ctgccatAAACCcgcaatcatca	##
cDNA-A5G	#	tgatgattgcgCGTTTatggcag	ctgccatAAACGcgcaatcatca	##

#5′-AACTCTAGTTGGACCTTTGTCAT-3′; ^##5′-AATTAGGAAGGTCCGGCCGGCCA-3′: two 16-nt italic sequences that are recombination fragments for In-Fusion® assembly with the PshA I/Fse I-digested cDNA-wt. FP2 and RP1 are reverse-complement sequences. *Uppercase sequences: 5-nt motifs; uppercase bold letters: SDM-modified sites. FP: forward primer; RP: reverse primer.

The OE-PCR was composed of two rounds of PCR. The first round was conducted independently using two pairs of primers (Table 1), FP1/RP1 and FP2/RP2, amplifying fragment I and II, respectively. Fragment I and II were purified, and subsequently used as two templates in a single tube for the second round of PCR using FP1/RP2. The second round of PCR made the two fragments be fused into a longer one, afterwards subjected to homologous recombination with the PshA I/Fse I-digested cDNA-wt using the In-Fusion® Kit (Takara, Dalian, China). The infusion product was transformed into TOP10 competent cells (AngYuBio, Shanghai, China) for screening a positive mutant, which contained both the SNc and an SDM-modified Ac (SAc). All mutants were identified by Sanger sequencing.

2.3. Rescue of recombinant SVA from SDM-modified cDNA clone

Thirteen cDNA clones were independently transfected into BSR-T7/5 cells to rescue recombinant SVAs (rSVAs). In brief, BSR-T7/5 cells were seeded into a 24-well plate for incubation at 37 °C. The cDNA clones were independently transfected into thirteen cell monolayers at 70 % confluency (1000 ng/well) using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA) as per the manufacturer's instructions. The 24-well plate was cultured at 37 °C, and observed under a fluorescence microscope at 72 h post transfection (hpt), followed by one freeze-and-thaw cycle to collect supernatants for five blind passages (48 h/passage) in vitro. The rSVAs, if successfully rescued, was named “rSVA-Ref. No.”. The recovery tests were repeated three times.

2.4. RT-PCR detection of rSVAs at passage-5

Cell cultures were harvested at 48 h post infection (hpi) with rSVAs at passage-5 (P5). Total RNAs were extracted using the Viral DNA/RNA Mini Kit (Vazyme, Nanjing, China) as per the manufacturer's instructions. The extracted products served as templates for one-step RT-PCR analysis separately using two pairs of primers, FP3 (5′-AATGATTTTGATTCCCGCGGCAA-3′)/RP3 (5′-ATGTAGGGGATGACAAAGGTCCA-3′) and FP4 (5′-ACTACCTGAGCACCCAGTCCGCC-3′)/RP4 (5′-TTCCAGAGAGAGGCCAGAGTGAC-3′), amplifying SNc- and SAc-containing sequences, respectively. The PrimeScript™ High Fidelity One Step RT-PCR Kit (Takara, Dalian, China) was used for RT-PCR reaction, which underwent 45 °C for 10 min, 94 °C for 2 min and then 30 cycles at 98 °C (10 s), 55 °C (15 s) and 68 °C (30 s). RNA samples were simultaneously analyzed by the conventional PCR using FP3/RP3 primers and the Spark Fast Taq Master Mix (Shandong Sparkjade Biotechnology Co., Ltd.). The PCR analysis aimed to identify whether plasmid contamination existed still in cell cultures at P5. The RT-PCR- and PCR-amplified products were subjected to agarose gel electrophoresis. All RT-PCR-positive products underwent Sanger sequencing.

2.5. Growth Kinetics of replication-competent rSVAs

BSR-T7/5 cells were seeded into four 24-well plates (5 × 10⁵ cells/well) for incubation at 37 °C for 2 h. The P5 viruses, including the rSVA-wt, were separately inoculated (3 wells/progeny, MOI = 0.001) into cell monolayers for incubation at 37 °C. A plate was removed at random from the incubator at 0, 24, 48 and 72 hpi, and then subjected to one freeze-and-thaw cycle to harvest supernatant for viral titration by TCID₅₀ analysis, as described previously (Meng et al., 2022). Kinetic curves of virus growth were drawn using the GraphPad Prism software (Version 8.0). Data at each time point were representative of three independent experiments.

2.6. Genetic stabilities of SNc and SAc with viral passaging

All replication-competent rSVAs totally underwent twenty serial passages (2 d/passage) in BSR-T7/5 cells. The P10 and P20 progenies were detected through RT-PCR separately using the FP3/RP3 and the FP4/RP4, as described in subheading 2.4, followed by Sanger sequencing.

3. Results and discussion

SVA causes vesicular lesions in pigs, still regarded as an emerging virus now. As the only member of the genus Senecavirus within the family Picornaviridae (https://ictv.global/taxonomy), it shares the highest homology with cardioviruses at the genomic level (Hales et al., 2008). SVA was even once believed to be a potential member in the genus Cardiovirus (Venkataraman et al., 2008). The cardioviral cre was proven to be in the viral VP2-encoding region (Lobert et al., 1999). The cre of SVA is not only similar to that of cardiovirus in structure, but also located in the VP2-encoding region (Fig. 1A). We recently demonstrated that SVA could tolerate neither A1C nor C4U point mutation in its Nc, despite both of them being synonymous mutations. SVA was also unable to tolerate the concurrence of A1C and C4U, unless another wild-type cre was artificially inserted into the viral genome to compensate for the functional inactivation of the SNc. These results suggested that the two synonymous mutations (A1C and C4U), irrespective of occurrence alone or in combination, would exert a lethal impact on SVA propagation (Meng et al., 2023).

Fig. 1.

Construction of rSVA cDNA clone with SAc. Schematic representation of full-length genome of foreign sequence-free SVA (A). The viral Nc is located in the VP2 sequence. The wild-type AAACA residues are marked with red circles. Map of cDNA-wt plasmid (B). Schematic representation of cDNA-wt genome (C). The cDNA-wt genome contains a SNc, and a fused sequence “eGFP–Ac–T2A”. Two SDM-modified sites in the SNc are marked with blue crosses and circles in the normal and enlarged rectangles, respectively. Profile of SDM-modified sites in A₁A₂A₃C₄A₅ motif of Ac (D). A total of thirteen single-site mutations (A1T, A1G, A2T, A2C, A2G, A3T, A3C, A3G, C4A, C4G, A5T, A5C and A5G) are designed for independently constructing thirteen SDM-modified cDNA clones. Mutated sites are shown with blue letters.

Due to the Nc located in the viral encoding region, a missense mutation in the AAACA motif would cause a codon that codes for a different amino acid. If a given missense mutation results in the failure of SVA recovery, the reason would be attributed either to the substitution of amino acid, or to the functional inactivation of AAACA motif. In our previous study, the cDNA-wt plasmid had been constructed, as shown in Fig. 1B. The cDNA-wt sequence (Fig. 1C) contained the SNc with A1C (Cross-marked) and C4U (Cross-marked), and one Ac between the eGFP and T2A sequences. The Ac played a role in compensating for the functional inactivation of the SNc. Because the fused sequence “eGFP–Ac–T2A” is a foreign fragment for the SVA genome, when a missense mutation arises in the Ac, the corresponding SVA would still maintain the integrality of its own ORF. In other words, missense mutations in the Ac would theoretically affect only the functional role of cre in synthesizing the Vpg-pUpU, but not interfere with the protein translation.

To uncover the conservation of Ac, a total of thirteen SDM-modified cDNA clones, excluding cDNA-A1C and -C4U, were constructed here in an attempt to rescue replication-competent viruses. Fig. 1D schematically showed a profile of SDM-modified sites in the Ac′s A₁A₂A₃C₄A₅ motif. All plasmids were independently transfected into BSR-T7/5 cell monolayers for rescuing viable rSVAs. All plasmid-transfected cell cultures were harvested at 72 hpt, followed by five blind passages in vitro. Only two groups, cDNA-A5U and -A5C, always emitted green fluorescence during serial passaging (Fig. 2A). The eGFP serves as a marker to indicate preliminarily whether a certain SVA has been rescued from its cDNA clone.

Fig. 2.

Recovery, detection and characterization of replication-competent rSVAs. Cell monolayers transfected with cDNA clones and serial blind passaging (A). BF: bright field; Bar = 20 μm. Green fluorescence is observable always in cDNA-A5U and -A5C groups. RT-PCR detection of virus recovery at P5 (B). Two pairs of primers are separately used for amplifying SNc- and SAc-containing fragments. Only cDNA-A5U and -A5C groups show RT-PCR-positive results. Sanger sequencing chromatograms of SDM-modified motifs at P5 (C). Multi-step growth curves of rSVA-wt, -A5U and -A5C (D). Data at 0, 24, 48 and 72 hpi are representative of three independent experiments. Error bar indicates standard deviation.

The two groups were separately collected at P5 for extracting total RNAs, further used for RT-PCR detection on the SNc and SAc. The fluorescence-free groups were mixed also for RT-PCR detection. The agarose gel electrophoresis showed two RT-PCR-positive progenies, rSVA-A5U and -A5C (Fig. 2B). The fluorescence-free groups revealed negative RT-PCR results on the gel. PCR analysis indicated no plasmid residue affecting the RT-PCR detection. Four RT-PCR products were subjected to Sanger sequencing, all exhibiting their original SDM-modified statuses (Fig. 2C). The rSVA-wt, -A5U and -A5C were measured to quantify their growth kinetics at P5. Three growth curves were drawn and compared with one another. The result showed that rSVA-A5U and -A5C grew much slower than the rSVA-wt (Fig. 2D), implying that both of the point mutations, especially the A5C, conferred a robust inhibitory effect on SVA replication, possibly resulting from the mutated sites that interfered with the slide-back process. The exact mechanism remains to be elucidated.

The rSVA-A5U and -A5C continued to be passaged in vitro. The virus-inoculated cell monolayers always emitted green fluorescence with passaging (Fig. 3A). The P10 and P20 progenies were harvested for RT-PCR analyses using two pairs of primers, displaying eight bands of interest on the gel (Fig. 3B and C). All eight RT-PCR products were subjected to Sanger sequencing. The sequencing results revealed that both SNc and SAc retained their original SDM-modified statuses at P10 (Fig. 3D) and P20 (Fig. 3E). Interestingly, we found two point mutations, mut-1 and 2 (Fig. 3F), arising in the SNc of rSVA-A5C at P10. Both mut-1 and 2 displayed a dual-peak phenotype at P10 through Sanger sequencing (Supplementary 1, SNc of rSVA-A5C at P10), suggesting each site at which wild-type and mutated bases coexisted. Such a coexistence phenomenon was referred to as single-nucleotide polymorphism (SNP) (Ni et al., 2016). The mut-1 unexpectedly disappeared at P20, whereas, at the mut-2 site, the wild-type base (G) was almost replaced with the mutant (A) at P20 (Supplementary 1, SNc of rSVA-A5C at P20).

Fig. 3.

Genetic stabilities of SNc and SAc with serial viral passaging. Cell monolayers infected with P10, P15 and P20 viral progenies (A). BF: bright field; Bar = 20 μm. RT-PCR analyses of SNc- and SAc-containing fragments at P10 (B) and P20 (C). Sanger sequencing chromatograms of SDM-modified motifs at P10 (D) and P20 (E). Two SNP sites in SNc of rSVA-A5C at P10 (F). The mut-1 and -2 sites exhibit A–U and A–G coexistences, respectively (as shown in Supplementary 1).

In our previous study, a foreign sequence-free SVA prototype underwent eighty serial passages in cells. The P10, P20, P30, P40, P50, P60, P70 and P80 progenies were subjected to next-generation sequencing. All eight sequencing results displayed no point mutation occurring in the Nc region (Liu et al., 2021), implying that the Nc was an extremely conserved region. In the present study, although the Nc was functionally inactivated, its role was compensated by the artificial one. Therefore, it is unnecessary for the SNc to maintain the wild-type stem–loop structure with viral passaging. To put it differently, the SNc of Ac-containing rSVA can theoretically tolerate some point mutations, which however cannot be adopted by an Ac-free SVA. Indeed, such a postulation was jointly confirmed by our present and previous studies. The former demonstrated that the SNc of Ac-containing rSVA was able to tolerate a particular mutation; the latter demonstrated that the same mutation in the Nc was lethal for an Ac-free SVA (Meng et al., 2023).

A picornaviral cre is generally embedded within the encoding region and far away from the genomic ends, but plays an essential role in regulating genomic replication (Liu et al., 2009). The AAACD motif is a conserved fragment among picornaviral cres (Steil and Barton, 2009). Nevertheless, in the present study, we found that AAACH (AAACA, AAACU or AAACC) were conserved residues to function in the Ac of rSVA. Due to the Ac located between the eGFP and T2A sequences, it can be also concluded that the cre is a position-independent functional element in SVA genome.

CRediT authorship contribution statement

Mengyao Wang and Di Zhao: Methodology. Jing Li and Lijie Zhu: Data analysis. Xiaoxiao Duan: Methodology and software. Youming Zhang and Yan Li: Project administration. Fuxiao Liu: Writing and project administration.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

• Data will be made available on request.