Principles of Resistance to Nucleoside Reverse Transcriptase Inhibitors
The nucleoside reverse transcriptase inhibitor (NRTI) class remain a key component of the backbone of most antiretroviral regimens used in current HIV clinical practice. The medications include abacavir (Ziagen), didanosine (Videx), stavudine (Zerit), tenofovir (Viread), zidovudine (Retrovir), and zalcitabine (Hivid). The NRTI drugs exert their action by inhibiting HIV reverse transcription, the key step that generates the conversion of HIV RNA to HIV DNA (Figure 1).[,] Specifically, the NRTIs are incorporated by HIV into the elongating DNA strand, but act as chain terminators because the NRTIs lack the 3'-hydroxyl group on the deoxyribose moiety, which is present on the naturally occurring deoxynucleotides and critical for the binding of the next incoming deoxynucleotide (Figure 2).[,] The reverse transcription process is generated by the enzyme reverse transcriptase, but this enzyme does not have proof-reading functionality, a property that lead to error prone DNA synthesis and increased drug-resistant mutation frequency. Drugs in the NRTI class include a heterogenous group, but all are considered competitive inhibitors (via competition with the natural deoxynucleotides). The mechanism of action and mechanisms of resistance with the NRTI class are distinct from those with the non-nucleoside reverse transcriptase inhibitors (NNRTIs). The NRTI resistance involves one of two biochemical mechanisms: (1) decreased incorporation (discriminatory) and (2) excision (primer unblocking) (Figure 3).[,,,,,] The discriminatory mutations allow the reverse transcriptase enzyme to preferentially select the naturally occurring deoxynucleotides present in the cell, thereby decreasing the incorporation of the NRTI-triphosphate into the elongation HIV DNA strand. Excision mutations enhance the phosphorolytic excision of the NRTI-triphosphate that had been added to the elongation HIV RNA DNA, resulting in unblocking the primer. Examples of mutations that cause decreased NRTI incorporation include M184I/V, K65R, L74V, and the Q151M complex (Q151M followed by the accessory mutations A62V, V75I, F77L, and F116Y).[,,] Characteristic mutations that occur via the primer unblocking pathway include the M41L, D67N, K70R, L210W, T215Y/F, and K219Q/E.[,,]
The M184I/V mutation is the signature mutations that develops with resistance to the medications lamivudine (Epivir) and emtricitabine (Emtriva). The M184I mutation typically develops first and usually is rapidly replaced by the M184V, primarily because the M184I mutation causes a greater impairment in viral fitness than does the M184V. Accordingly, the M184V is identified much more frequently in genotypic resistance testing than the M184I mutation and overall the M184V mutation is the most frequently identified NRTI mutation. The M184I/V mutations develop via the discriminatory pathway.[,] One early studiy has shown that patients treated with lamivudine monotherapy develop virologic failure within 4 weeks of starting lamivudine and the increase in HIV RNA levels correlates with the emergence of the M184V mutation; even with development of the M184V and high-level resistance, lamivudine continues to exert an approximately 0.5 log10 decrease in HIV level (Figure 4). In clinical trials involving combination antiretroviral therapy, the M184V mutation was the most common mutation to develop with initial virologic failure. In vitro data demonstrates the the M184V mutation cause high-level resistance to emtricitabine and lamivudine, low-level resistance to abacavir and didanosine, and enhanced susceptibility to stavudine, tenofovir, and zidovudine. The development of the M184V mutation results in some loss of of virologic fitnees. Several studies have suggested that treatment with lamivudine in the presence of an M184V mutation may confer clinical benefit, potentially through residual antiviral activity, resultant decreased viral fitness, and hypersensitivity to some other NRTIs, and perhaps delaying development of mutations in other NRTIs.[,] In the absence of drug pressure from either emtricitabine or lamivudine, the M184V mutation rapidly disappears, reflecting the overall negative impact of the 184V on viral fitness. Once the M184V mutation develops, there are no further cascading mutations that develop that would negatively impact other antiretroviral medications. The M184V mutation is not known to impact mediations outside of the NRTI class, but the M184I mutation augments resistance to rilpivirine (Edurant) resistance in conjunction with a E138K mutation.[,]
Thymidine Analog Mutations
The TAM mutations (Figure 5), which include M41L, D67N, K70R, L210W, T215Y/F, and K219Q/E, develop in the setting of virologic failure with a regimen that include the thymidine analog medications stavudine or zidovudine.[,] Although these medications are infrequently used in current clinical practice, patients with long-standing HIV may have acquired TAM mutation in the past. In addition, patients from resource limited regions who have immigrated to the United States recently may have received stavudine or zidovudine in recent years, or may be currently taking these medications. In the United States, thymidine analog mutations infrequently develop in patients on modern antiretroviral regimens that have tenofovir-emtricitabine (Truvada) or abacavir-lamivudine (Epzicom) as the NRTI backbone of the regimen. Although the TAM mutations are selected by stavudine and zidovdine, the accumulation of multiple TAMs can also have significant impact on HIV susceptibility to abacavir, didanosine, and tenofovir.[,,,] The TAM mutations tend to accumulate in one of two characteristic, but overlapping patterns: (a) the type I pattern that has M41L, L210W, and T215Y or (b) type II pattern consisting of D67N, K70R, T215F, and K219Q/E (Figure 6).[,,,,] In general, type I TAM mutations result in higher levels of phenotypic resistance to stavudine and zidovdine, as well as greater cross resistance to abacavir, didanosine, and tenofovir.[,] Indeed, if all three type I TAM mutations are detected, clinical response to abacavir, didanosine, and tenofovir is markedly reduced. Some patients develop the D67N mutation will the type I cluster. The presence of a M184V mutation reduces the impact of the TAM mutations to some degree, but the favorable impact of the M184V is negligible with high numbers of TAM mutations.
Selection of the K65R mutation can occur with exposure to abacavir, didanosine, stavudine, and tenofovir. In clinical trials, the development of the K65R mutation has primarily occurred in patients who were taking an antiretroviral regimen that did not include a thymidine analog (stavudine or zidovudine). In early trials of abacavir monotherapy, approximately 10% of patients developed the K5R mutation. Even higher rates (greater than 50%) of K65R mutation were observed in patients treated with the triple nucleoside regimen of abacavir plus lamivudine plus tenofovir. Addition of a drug from a class other than NRTI appears to markedly reduce the likelihood of developing the K65R mutation in patients receiving tenofovir-emtricitabine. Development of the K65R can have variable impact on NRTI medications, including intermediate-level resistance (abacavir, didanosine, emtricitabine, lamivudine, and tenofovir), low-level resistance (stavudine), and hypersusceptibility (zidovudine). The mechanism of resistance with the K65R is decrease incorporation and the K65R mutation shows bilateral antagonism with the primer unblocking (excision) activity of the reverse transcriptase enzyme that contains TAMS.[,,] In clinical trials and clinical practice, it is very uncommon to observe the K65R mutation in conjunction with multiple TAMs.[,] The 65R and M184V double mutation causes higher-level resistance to abacavir than either mutation alone and the K65R mutation reverses the hypersusceptibility effect of the M184V on stavudine, tenofovir, and zidovudine.[,] With abacavir resistance, the M184V typically precedes the K65R.
The L74V mutation was first identified with didanosine and abacavir monotherapy; this mutation alone causes high-level resistance to didanosine and intermediate-level resistance to abacavir. Similar to the M184V mutation, the L74V mutation causes in in vitro hypersusceptibility to tenofovir, zidovudine, and possibly stavudine. The L74V in combination with M184V has been seen in patients treated with an abacavir plus lamivudine or didanosine plus lamivudine NRTI backbone. Overall, the L74V mutation is an uncommon NRTI mutation, but is identified in approximately 25% of samples that contain HIV with K101E plus G190S mutations and in approximately 50% of samples L100I plus K103N mutations.
Multi-Nucleoside Resistance Mutations
The multi-nucleoside resistance mutations occur relatively infrequently, but may have a major impact on the NRTIs. The T69-insertion mutation consists of double amino acid (diserine) insertion between codons 69 and 70 in the reverse transcriptase enzyme.[,] The T69 inoccurs only in the setting of existing TAM-1 mutations and together the T69-insertion and TAM-1 generate high-level resistance to all of the NRTI medications, except for lamivudine and emtricitabine, which have intermediate resistance.[,] The Q151M mutation complex usually occurs with several accessory mutations (A62V, V75I, F77L, and F116Y) and these mutations in tandem cause high-level resistance to abacavir, didanosine, and zidovudine, as well as intermediate resistance to emtricitabine, lamivudine, and tenofovir. The Q15M mutation complex develops only in the setting of prolonged viremia while on therapy.