Simple detection of point mutations associated with HIV-1 drug resistance
Introduction
Highly Active Anti-Retroviral Therapy (HAART) is the standard of care for HIV-1 infected patients (BHIVA Executive Committee, 2000). Some patients ‘fail’ HAART, as determined by an inability to achieve or maintain undetectable levels of plasma viral RNA, or ‘viral load’. ‘Undetectability’ is defined as a viral load below the lower limit of detection of the assay in use, for example less than 50 RNA copies/ml for the Chiron 3.0 bDNA assay (Emeryville, USA). A major reason for drug failure is the development of resistant viral strains (Condra et al., 1996).
Resistance-conferring mutations have been identified for the three major anti-retroviral drug classes: nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors. Some nucleoside reverse transcriptase inhibitors are susceptible to single point mutations in Reverse transcriptase (RT), such as the M184V mutation which confers loss of sensitivity to Lamivudine (3TC) (Tisdale et al., 1993). Other nucleoside reverse transcriptase inhibitors, such as Abacavir, require a number of mutations to confer significant resistance (Tisdale et al., 1997). For the non-nucleoside reverse transcriptase inhibitors, resistance can be conferred by single point mutations in RT, such as K103N or Y181C (Kleim et al., 1994, Richman et al., 1994). Multiple mutations are usually required in protease before resistance develops to protease inhibitors (Cabana et al., 1999, Schmit et al., 1996).
Resistance testing is established in many HIV clinics by sequencing — the ‘gold standard’ — using ‘in-house’ techniques or commercial kits (e.g. Visible Genetics, Applied Biosystems). There is also a probe-based point mutation assay (Lipa, Murex) which is limited to mutations in RT, although a kit for protease is currently under evaluation. Sequencing is limited by poor sensitivity for mixed populations of wild type and mutant virus (Schuurman et al., 1999) and clade-specific detection limitations. Other genotyping methods include the probe-based Affymetrix gene chip technology, for which poor sensitivity with non-B specimens has been reported (Vahey et al., 1999).
PCR-based assays are an alternative method of detecting point mutations, and have the advantage of increased sensitivity, low cost and high through-put. Allele-specific primer extension assays have been applied to HIV drug resistance (Richman et al., 1991), but have not been adequately specific for wide-spread application (Eastman et al., 1995). ‘Mutagenically-Separated PCR’ (MS–PCR) (Rust et al., 1993) is a PCR-based point mutation assay which overcomes these specificity limitations and has been clinically applied (Chang et al., 1995, Merryweather et al., 1997, Rust et al., 1993), but not to the detection of drug resistance in an infectious disease.
MS–PCR uses three primers: two allele-specific primers (mutant and wild type) derived from one template DNA strand and a third primer derived from the complementary strand (Fig. 1), as described elsewhere (Rust et al., 1993). Essentially, the mutant and wild type primers match their corresponding templates, differing from each other at the 3′ termini. Further mismatches are introduced at either the second, third or fourth base from the 3′ terminus, but at different sites in the mutant and wild type primers. The resulting PCR products from the wild type and mutant primers will, therefore, differ by three bases at the 3′ annealing sites. The primer–template mismatch is, thus, converted from a single base to three bases, and the probability of non-specific primer binding (e.g. mutant primer to wild type template) is almost eliminated after the first PCR cycle. The wild type and mutant primers, and therefore their final products, differ in length by 20 nucleotides and can be easily differentiated by 3% agarose gel electrophoresis.
Section snippets
Laboratory and patient viral isolates
Wild type laboratory clade B HIV-1 strains IIIB (Popovic et al., 1984), RF (Popovic et al., 1984), MN (Gallo et al., 1984), SF2 (Levy et al., 1984), SF-162 (Cheng-Mayer and Levy, 1988), Ba-L (Gartner et al., 1986) and Ada-M (Gendelman et al., 1988) were acquired from the AIDS Reagent Program (NIBSC, UK) as cell-free tissue-culture supernatants. Laboratory strain HIV-1 SQVR-GB8 is a protease inhibitor-resistant clade B strain obtained from NIBSC, containing the G48V and L90M substitutions in
Application of MS–PCR to laboratory HIV-1 isolates with known genotype
MS–PCR was applied to wild type and mutant laboratory isolates, containing mutations Y181C and K103N in RT and M36I, M46I, G48V, I54V, L63P, V82A, I84V and L90M in protease. Using a limiting dilution PCR to quantify viral copies (Simmonds et al., 1990), MS–PCR detected low copy numbers. For example, for codon 181, MS–PCR detected 1.14 copies of wild type and 4.85 copies of mutant laboratory strain (Fig. 2a.). Specificity was 100% for wild type and mutant strains for protease and RT for all
Discussion
The development of viral resistance to anti-retroviral therapy is a major cause of treatment failure. Where funds are available, resistance testing by gene sequencing is undertaken to identify patients who might not benefit from certain drug combinations. With the increasing use of anti-retroviral drugs in developing areas such as Africa and Asia, especially as mono- or dual-therapy, resistance is, increasingly, becoming an problem (Juntilla et al., 2000, Weidle et al., 2000). Outside the
Acknowledgements
Acknowledgements: AJF is funded by the Medical Research Council. JNW is supported by the Wellcome Trust.
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