doi: 10.3967/bes2022.043
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Abstract:
Objective This study aimed to identify internal ribosome entry sites (IRESs) in the open reading frame (ORF) of the Coxsackievirus B3 (CVB3) genome. Methods The sequences of P1, P2, or P3 of the CVB3 genome or the truncated sequences from each antithymocyte globulin (ATG) to the end of the P1, P2, or P3 gene were inserted into the pEGFP-N1 vector. After transfection, possible IRES-dependent green fluorescent protein (GFP)-fused proteins were detected by anti-GFP western blotting. The sequences of possible IRESs were inserted into specific Fluc/Rluc bicistronic vectors, in which the potential IRESs were determined according to the Fluc/Rluc activity ratio. Expression of Fluc and Rluc mRNA of the bicistronic vector was detected by RT-qPCR. Results After transfection of full length or truncated sequences of the P1, P2, or P3 plasmids, six GFP-fused protein bands in P1, six bands in P2 and nine bands in P3 were detected through western blotting. Two IRESs in VP2 (1461–1646 nt) and VP1 (2784–2983 nt) of P1; one IRES in 2C (4119–4564 nt) of P2; and two IRESs in 3C (5634–5834 nt) and 3D (6870–7087 nt) of P3 were identified according to Fluc/Rluc activity ratio. The cryptic promoter was also excluded by RT-qPCR. Conclusion Five IRESs are present in the CVB3 coding region. -
Figure 1. Possible cap-independent translation of P1, P2, and P3 genes of CVB3 in vitro. After transfection of plasmids bearing P1, P2, or P3 genes fused with GFP, nine bands in P1, six bands in P2 and nine bands in P3 were identified by anti-GFP western blotting. The protein molecular weight markers are indicated at left. GFP, green fluorescent protein.
Figure 2. Screening of IRES translation in the coding region of CVB3. (A) Schematic diagram of IRES screening plasmids with sequences from each ATG to the end of P1 (A1), P2 (A2), or P3 (A3). (B) After transfection of plasmids pP1 (735–3304), pP1 (1235–3304), pP1 (1343–3304), pP1 (1478–3304), pP1 (1559–3304), pP1 (1697–3304), pP1 (1751–3304), pP1 (1811–3304), pP1 (2093–3304), pP1 (2183–3304), pP1 (2984–3304), and pP1 (3089–3304), potential IRES-dependent translation of P1 was identified by anti-GFP western blotting (B1). After transfection of plasmids pP2 (3284–5029), pP2 (3284–5029), pP2 (3671–5029), pP2 (3737–5029), pP2 (4028–5029), pP2 (4073–5029), pP2 (4364–5029), pP2 (4619–5029), and pP2 (4955–5029), potential IRES-dependent translation of P2 was identified by anti-GFP western blotting (B2). After transfection of plasmids pP3 (4754–7297), pP3 (4955–7297), pP3 (5390–7297), pP3 (5507–7297), pP3 (4765–7297), pP3 (6134–7297), pP3 (6344–7297), pP3 (6470–7297), pP3 (6581–7297), pP3 (6770–7297), pP3 (6881–7297), and pP3 (7088–7297), potential IRES-dependent translation ofP3 was identified by anti-GFP western blotting (B3). IRES, internal ribosome entry site; GFP, green fluorescent protein; ATG, antithymocyte globulin.
Figure 3. Identification of IRESs in the P1 coding region. (A) Schematic diagram of IRES identifying bicistronic vectors with a hairpin structure between the Rluc and putative IRES sequence. (B) Schematic diagram of identification of P1 IRESs. The potential IRES sequences of the P1 coding region were serially cloned into the bicistronic vectors (B1). pP1 (735–1234), pP1 (1197–1346), pP1 (1311–1810), pP1 (2376–2876), pP1 (2784–2983), and pP1 (2589–3088) were transfected into BHK cells. The Fluc/Rluc ratio of each vector was calculated and compared with that of the negative control (B2). The locations of three positive IRESs in P1 (B3). (C) Schematic diagram of shortened potential IRES sequences in VP2–VP3 region. The sequence at 1197–1810 nt was serially shortened with a 50 bp overlap between adjacent sequences (C1). Schematic diagram of shortened potential IRES sequences inserted into the bicistronic vectors (C2). IRES identification in VP2–VP3 through the constructed bicistronic vector. pP1 (1197–1346), pP1 (1297–1446), pP1 (1297–1446), pP1 (1397–1546), pP1 (1497–1546), pP1 (1597–1646), and pP1 (1697–1810) were transfected into BHK cells, and the Fluc/Rluc activity ratio of each vector was calculated and compared with that of the negative control (C3). (D) Schematic diagram of shortened potential IRES sequences in the VP1 region (D1). Schematic diagram of shortened P1 sequences inserted in bicistronic vectors (D2). IRES identification in the VP1 region through constructed bicistronic vectors. pP1 (2484–2683), pP1 (2654–2833), and pP1 (2784–2983) were transfected into BHK cells, and the Fluc/Rluc activity ratio of each vector was calculated and compared with that of the negative control (D3). (E) Schematic diagram of truncated IRES sequence at 1311–1810 nt in the VP2 region (E1). Schematic diagram of 50 bp deletion IRES sequences inserted into the bicistronic vectors (E2). pP1 (1311–1696), pP1 (1311–1646), pP1 (1311–1596, pP1 (1311–1546), pP1 (1311–1496), pP1 (1361–1696), pP1 (1411–1696), pP1 (1461–1696), or pP1 (1511–1696) was transfected into BHK cells, and the Fluc/Rluc activity ratioof each vector was compared with that of the negative control (E3). IRESs, internal ribosome entry site; EMCV, encephalomyocarditis virus; Rluc, Renilla luciferase; Fluc, Firefly luciferase.
Figure 4. Identification of IRESs in the P2 region. Schematic diagram of IRES bicistronic reportersfor P2. The sequence before the ATG start codon of the P2 region was serially cloned between the hairpin structure and Fluc of the bicistronic vectors (A). pP2 (3171–3670), pP2 (3528–4027), pP2 (3864–4363), pP2 (4065–4564), pP2 (4119–4618), and pP2 (4455–4954) were transfected into BHK cells, and the Fluc/Rluc activity ratio of each vector was compared with that of the negative control (B). Locations of two positive IRESs in P2 (C). IRESs, internal ribosome entry site; EMCV, encephalomyocarditis virus; Rluc, Renilla luciferase; Fluc, Firefly luciferase.
Figure 5. Identification of IRESs in the P3 region. (A) Constructs of bicistronic reportersfor P3 IRESs. Potential IRESs in the P3 region were serially cloned between the hairpin structure and Rluc in bicistronic vectors (A1). Identification of P3 IRESs with bicistronic vectors. pP3 (4890–5389), pP3 (5634–6133), pP3 (5844–6343), pP3 (5970–6469), pP3 (6270–6769), pP3 (6305–6880), pP3 (6583–7087), and pP3 (6786–7285) were transfected into BHK cells, and the Fluc/Rluc activity ratio was compared with that of the negative control (A2). Locations of five positive IRESs in 3A, 3C, and 3D (A3). (B) Mapping of potential IRESs in P3. The IRES sequences at 5634–7087 nt, 4890–5389 nt, and 5634–7087 nt were truncated to 150 bp with 50 bp overlap (B1). Construction of bicistronic reporters of potential P3 IRESs (B2). IRESs in the P3 region were identified with bicistronic vectors. pP3 (4890–5090), pP3 (5040–5240), pP3 (5190–5389), pP3 (5634–5834), pP3 (5784–5984), pP3 (5934–6133), pP3 (5970–6170), pP3 (6120–6320), pP3 (6270–6470), pP3 (6420–6620), pP3 (6570–6770), pP3 (6720–6920), and pP3 (6870–7087) were transfected into BHK cells, and the Fluc/Rluc activity ratio of each vector was compared with that of the negative control (B3). IRESs, internal ribosome entry site; EMCV, encephalomyocarditis virus; Rluc, Renilla luciferase; Fluc, Firefly luciferase.
Figure 6. Identification of the integrity of dual luciferase bicistronic mRNAs containing IRESs. The X-axis represents the dual-luciferase reporter vectors, and the Y-axis represents mRNA copies of Rluc and Fluc. Black histogram: Rluc mRNA; white histogram: Fluc mRNA. Experiments were repeated independently three times. Significant difference (P < 0.05). Rluc, Renilla luciferase; Fluc, Firefly luciferase
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