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. 2023 Nov 10;339:199264. doi: 10.1016/j.virusres.2023.199264

Fig. 1.

Fig 1

Construction of CiLV-C infectious cDNA clones using pJL89 and pLXAS binary vectors. (A) Schematic representation of CiLV-C infectious cDNA clone in binary plasmid. The pJL89 amplification strategy consists of plasmid amplification by inverse PCR with a set of primers that anneal to the HDV Rz region and the 35S CaMV promoter region. The arrows show the direction of amplification. This amplification strategy ensures that the viral fragments are assembled between the double 35S promoter (double arrow) and the HDV Rz (rectangular box), followed by the NOS terminator (square box). It is shown the genomic organization of CiLV-C RNA1 and RNA2. The boxes highlight the virus ORFs: polymerase and p29 (RNA1), and p15, p61, MP, and p24 (RNA2). The black, hot pink, and orange arrows indicate the direction and the primers used for the amplification of the two RNA1 (first -F1- and second -F2-) or the RNA2 (F3) fragments, respectively. On the right, a schematic representation of the pJL89 RNA1 CiLV-C and pJL89 RNA2 CiLV-C constructs assembled. F1 and F2 correspond to the first and second fragments of the RNA1, and F3 corresponds to the RNA2 fragment. The sizes in base pairs (bp) of the viral genome, amplified fragments, and assembled constructs are presented. (B) The pLXAS amplification strategy consists of plasmid amplification by inverse PCR with a set of primers that anneals to the HDV Rz region and the 35S CaMV promoter region. The arrows show the direction of amplification. This amplification strategy ensures that the viral fragments are assembled between the double 35S promoter (double arrow) and the HDV Rz (rectangular box), followed by the PoPit terminator (square box). The green arrows indicate the direction of amplification of the RNA 1 (F1) fragment for the assembly using BsmBI restrict enzyme with nts compatibles to the 3-end of the 35S promoter and 5-end of HDV Rz and the red arrows indicate the direction of amplification of the RNA2 (F2). (C) The stop codon insertion into the viral gene polymerase was performed by replacing a G-T at position 1.405 using a pair primer (Hpolymutstop fwd/Hpolymutstop rev) carrying the point mutation. “a, b, c, d, e, f, g, and h” correspond to the primers named in Supplementary Table 1.