Identification of Mutations that Encode Drug Resistance in the Polymerase Gene of the Human Immunodeficiency Virus
In vitro selection in MT-4 cells was used to generate human immunodeficiency virus-type 1 (HIV 1) variants that are resistant to 2',3'-dideoxycytidine (ddC), 2',3'-didcoxyinosine (ddI) and the (-) enantiomer of 2' ,3'-dideoxy-3'-thiacytidine (3TC). The complete rev...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
1994-01-01
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| Series: | Canadian Journal of Infectious Diseases |
| Online Access: | http://dx.doi.org/10.1155/1994/826340 |
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| Summary: | In vitro selection in MT-4 cells was used to generate human immunodeficiency virus-type 1
(HIV 1) variants that are resistant to 2',3'-dideoxycytidine (ddC), 2',3'-didcoxyinosine (ddI) and the (-) enantiomer
of 2' ,3'-dideoxy-3'-thiacytidine (3TC). The complete reverse transcriptase open reading frames of these
viruses, and portions of flanking protease and integrase within the pol gene, were cloned and sequenced
by polymerase chain reaction (PCR) techniques. Mulalions were observed at each of amino acid sites 65 (Lys
→ Arg: AAA → AGA) and 184 (Met → Val: ATG → GTG) when ddC was used in this protocol, and at site 184
only when either 3TC or ddl was employed. These mutations were introduced into the pol gene of infectious
recombinant HXB2-D DNA by site-directed mutagenesis to confirm, by viral replication assay, their
importance in conferring resistance against these drugs. A recombinant virus containing the site 65
mutation only possessed greater than 10-fold resistance against ddC compared with parental HXB2-D.
Moreover, cross-resistance of about 20-fold and threefold, respectively, was delectable against 3TC and dell
but not against 3'-azido-3'-deoxthymidine (AZT). When the 65 and 184 mutations were combined into
HXB2-D, the resultant construct did not possess higher levels of resistance lo any of these drugs than
observed with the site 65 or 184 mutation alone. These mutations were further demonstrated by PCH analysis
of peripheral blood mononuclear cells from 10 patients on long term ddC therapy, although variable patterns
were observed in terms of which of the two mutations or both were present. Sometimes, the wild-type site
65 codon was also detected, indicating the presence of mixtures of viral quasi-species. Direct cloning and
sequencing revealed the site 65 mutation in viruses isolated from patients on prolonged ddC therapy. |
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| ISSN: | 1180-2332 |