Characterization of the Reverse Transcriptase of a Human Immunodeficiency Virus Type 1 Group O Isolate

Abstract

The catalytic properties and sensitivity to different inhibitors have been determined for the reverse transcriptase (RT) of group O human immunodeficiency virus type 1 (HIV-1). The RT-coding region was cloned from a new HIV-1 group O isolate from Spain, expressed in Escherichia coli, and purified by affinity chromatography. This new RT showed 79% amino acid sequence identity with the corresponding enzyme of group M subtype B strain BH10. The two enzymes showed very similar kinetics of RNA-dependent DNA polymerization using homopolymeric template-primers and RNase H specific activity. Inhibitor sensitivity to ddTTP and 3'-azido-2',3'-dideoxythymidine triphosphate (AZTTP) was also similar for both enzymes. However, the two enzymes differed dramatically in their sensitivity to several inhibitors. While the RT of the BH10 isolate was sensitive to nevirapine and loviride (IC50 ranged from 0.16 to 8.2 microM, depending on the substrates used), the enzyme of the Spanish HIV-1 group O isolate showed high-level resistance to those compounds (IC50 > 200 microM). The amino acid sequence of the RT of group O HIV-1 contains three amino acids (Cys-181, Glu-179, and Gly-98), which are found in group M subtype B strains resistant to nonnucleoside RT inhibitors. The recombinant group O HIV-1 RT should be useful for studies aimed at discovering and designing drugs directed toward group O isolates of HIV-1.

Functional Characterization of Chimeric Reverse Transcriptases with Polypeptide Subunits of Highly Divergent HIV-1 Group M and O Strains

“…21). In a preliminary study, we reported on the expression and characterization of an HIV-1 group O RT (ESP1 isolate) (22). ESP1 RT shares 79% amino acid sequence identity with the RT of group M:subtype B strain BH10.…”

“…nevirapine, delavirdine, and the TIBO derivative R82913) (23). The presence of Cys-181 in the group O RT-coding region, instead of the Tyr-181 found in sensitive group M:subtype B (M:B) strains, appears to confer NNRTI resistance (22,23). Unfortunately, detailed studies on the enzymatic activity of HIV-1 ESP1 and related M:B/O RT chimeras was hampered by the poor level of HIV-1 ESP1 group O subunit expression, reconstitution, and purification.…”

“…In this study, we initially screened for optimum Escherichia coli expression of the group O p66 subunit using 66 group O RT variants derived from quasi-species of six different isolates (24 (24). Genomic regions encoding the 66-kDa subunit of the RT were PCR-amplified from patient samples, cut with the NcoI and EcoRI restriction enzymes, and then cloned into the pRT6 or pTrcHisB (Invitrogen) expression vector (22,24). The pRT6 clones were used for large-scale purification of p66, whereas constructs made with pTrcHisB were used for screening the expression levels of RT variants.…”

“…To obtain an expression vector for p51 with the histidine tag for reconstitution and purification, the coding region of the 51-kDa subunit of HIV-1 RT was cloned in the expression vector pTrcHisB. Briefly, primers RTO7 and RTO8 (22) were used to PCR-amplify the p51-coding region from the pRT6-derived plasmid (see above) and to introduce the NheI and EcoRI endonuclease restriction sites for subsequent cloning of the p51 region into the pTrcHisB plasmid.…”

“…The RT activity of each RT variant-producing clone was determined by substracting the average RT activity of the background clones (bacteria containing the expression vector without the HIV-1 RT-coding region). The polymerase activity of each clone was then compared with that of the previously characterized HIV-1 ESP1 group O RT clone (22 (22). C represents a control using bacteria bearing the plasmid pTrcHisB without the RT-coding sequence.…”