Novel hiv-based recombinant viral clones and use thereof in analytical methods

An HIV-based recombinant viral clone containing the following elements in 5′ to 3′ direction:

• a. LTR or redundant terminal sequences (R) of the proviral DNA of NL4.3;
• b. gag is the gene which codes the p55 capsid protein formed by the 3 protein subunits MA, CA and NC, said gag gene can comprise:
• b.1) NarI restriction site at position 637 made unique by directed mutagenesis;
• c. pol is the gene which codes the viral enzymes needed for the viral replication process, and whose 5′ end overlaps with gag element, if b.1) does not apply, said pol gene can comprise restriction sites generated by directed mutagenesis, said restriction sites being selected from the following group:
• c.1) NcoI-AgeI restriction sites at positions 2593 and 3485 respectively,
• c.2) ApaI-NcoI restriction sites at positions 2006 and 2593 respectively, and
• c.3) ApaI-AgeI restriction sites at positions 2006 and 3485 respectively, or
• if b.1) does apply, said pol gene can comprise:
• c.4) KspI restriction site generated by directed mutagenesis at position 4498, and
• c.5) ApaI-NcoI restriction sites generated by directed mutagenesis at positions 2006 and 2593 respectively;
• d. vif is the gene that codes the protein Vif, it's 5′ end overlaps with pol element and it's 3′ end overlaps vpr element;
• e. vpr is the gene that codes the protein Vpr and it's 5′ end overlaps vif element;
• f. tat is the gene that codes the protein Tat, it's second exon is contained inside env sequence;
• g. vpu is the gene that codes Vpu;
• h. env is the gene which codes the protein gp160 of the viral envelope, if b.1), c.1), c.2), c.3), c.4) and c.5) do not apply, said env gene can comprise:
• h.1) XbaI-NotI restriction sites generated by directed mutagenesis at positions 6114 and 8796 respectively;
• i. rev is the gene that codes the protein Rev, it's second exon is contained inside env sequence;
• j. NotI is a restriction site for NotI enzyme, that has been introduced by directed mutagenesis at position 8796 of the viral genome;
• k. XhoI is a restriction site for the XhoI enzyme, in position 8887 of the viral genome;
• l. nef is the gene that codes protein Nef, and is deleted at the bases encompassing the fragment between positions 8796 and 8887 of the viral genome by cutting with restriction enzymes NotI and XhoI;
• m. Renilla is the gene that codes the luciferase reporter protein Renilla, and that has been cloned in restriction sites NotI-XhoI in position 5′ and 3′, respectively;
• n. LTR, whose 5′ end overlaps with the 3′ end of nef element;
• and
• o. LacZ gene cloned in restriction sites generated by directed mutagenesis, substituting fragments of the viral genome in order to allow further replacement of such sequences with fragments from isolates coming from patients, said restriction sites being selected from the following group:
• o.1) ApaI-AgeI restriction sites, in positions 5′ and 3′, respectively, wherein c.3) applies, said LacZ gene is cloned between positions 2006 and 3485, substituting the fragment of pol gene that encode the protease and the reverse transcriptase regions of the viral genome,
• o.2) ApaI-NcoI restriction sites in positions 5′ and 3′, respectively, wherein c.2) applies or b.1), c.4) and c.5) applies, said LacZ gene is cloned between positions 2006 and 2593, substituting the fragment of pol gene that encode the protease region of the viral genome,
• o.3) NcoI-AgeI restriction sites in positions 5′ and 3′, respectively, wherein c.1) applies, said LacZ gene is cloned between positions 2593 and 3485, substituting the fragment of pol gene that encode the reverse transcriptase region of the viral genome, and
• o.4) XbaI-NotI restriction sites in positions 5′ and 3′, respectively, wherein h.1) applies, said LacZ gene is cloned between positions 6114 and 8796, substituting the fragment of env gene of the viral genome.