In this study, we demonstrate a role of the commonly selected N348I mutation in the connection domain of HIV-1 RT in both NRTI (AZT) and NNRTI (NVP and EFV) resistance. N348I is more prevalent in clinical samples from patients treated with RTIs compared with samples from treatment-naïve individuals. In this regard, our analysis ranks N348I as the ninth most prevalent resistance mutation from a total of 39 different RT codons that were evaluated in RTI experienced patients and this mutation was observed more frequently in our cohort than mutations at codons 210, 69, 44, 190, 118, and 74, most of which have been the topic of multiple clinical, genetic, virological, biochemical and structural analyses [59
]. As expected with HIV, there was a strong codon bias, with most of the mutant N348I (98.6%) encoded by ATT, while more than 97% of WT N348 was encoded by AAT. Furthermore, the N348I mutation is associated with M184V/I, TAMs, K103N and Y181C and is selected by NVP and AZT treatment. The N348I mutation also appears relatively early in virological failure, generally before the appearance of TAMs and usually at the same time as the acquisition of NNRTI resistance mutations. Notably, N348I was the first mutation observed in two patients in our cohort. The early appearance of N348I after initiation of antiretroviral therapy is consistent with it playing a key role in the development of RTI resistance rather than being an accessory mutation that appears after primary mutations [64
]. This is supported by our findings that its appearance is associated with an increase in viral load, which is at least as large as that observed for any of the other TAMs.
The high prevalence of N348I is not unique to the Centre's database as it was also identified in a separate analysis of mutations beyond RT codon 240 in a large US clinical database (Quest Diagnostics) [65
]. In the latter study, two sets of sequences were analysed. The first comprised 41,122 de-identified clinical samples collected between January 2002 and June 2003 and the second comprised 16,449 samples collected between July 2003 and December 2003. Viruses with at least one resistance mutation in the protease or RT (64.8% in dataset 1 and 57% in dataset 2) were compared with those that contained no key drug resistance mutations (35.2%, dataset 1). The frequency of N348I in the first and second datasets was 13.6% and 13.0%, respectively. A recent study also demonstrates the presence of N348I in RT sequences from over 3,000 treated individuals with subtype B HIV-1 in a UK database [66
]. Thus, the prevalence of N348I in three large databases indicates that this mutation is likely to be present in other clade B cohorts receiving antiretroviral therapy.
To delineate the role of N348I in RTI resistance, this mutation was introduced into molecular HIV-1 clones with defined genetic backbones. In this regard, N348I, alone or in combination with TAMs, conferred 2- to 4-fold AZT resistance. The level of AZT resistance conferred by N348I alone is comparable to the levels of resistance conferred by other individual TAMs. For example, the M41L mutation has been reported to confer between 1.4- to 4-fold AZT resistance, the K70R mutation up to 8-fold resistance, and T215Y up to 16-fold resistance [59
]. Furthermore, other groups have also recently demonstrated that the N348I mutation confers both AZT and NNRTI resistance; with the Monogram group reporting a 2.4-fold change in AZT susceptibility [69
], while another group reported a 23-fold change in AZT susceptibility [70
]. In addition, a recent study demonstrated that reversion of N348I to the WT codon in an RT gene from an isolate derived from an RTI-experienced patient increased AZT sensitivity 5- to 10-fold [13
]. Interestingly, N348I also conferred decreased susceptibility to EFV and NVP, data that are also consistent with studies from other groups [69
]. In our cohort N348I was associated with the appearance of K103N, V108l, Y181C/I, and G190A/S early in virological failure () and was significantly associated with these mutations in analysis of the entire database (). N348I significantly potentiated EFV and NVP resistance when combined with K103N. Consistent with our cell culture–based assays, NNRTI resistance was also observed at the enzyme level with recombinant RT expressing N348I. Furthermore, the phenotypic findings are consistent with our clinical data demonstrating the significant association of N348I with TAMs and NNRTI mutations and its selection in patients on AZT and NVP combination therapy.
N348I was also significantly associated with M184V/I in our cohort. N348I conferred a small increase in 3TC resistance in the context of TAMs but did not counteract the previously observed antagonism between M184V and TAMs. This is in contrast to another connection domain mutation, G333D/E, which is associated with dual resistance to AZT and 3TC [10
]. It is curious that our data demonstrate a strong association of N348I with M184V/I despite it having little effect on drug susceptibility in the context of M184V. However, this can be explained by several possibilities, including that M184V/I and N348I are not genetically linked, that their coincident emergence is due to a high proportion of the Centre's cohort being prescribed 3TC, or that N348I has an effect on M184V/I that is independent of decreasing drug susceptibility such as altering viral fitness. Further studies will be needed to address these possibilities.
Apart from Q145M, which has been reported to confer broad resistance to several NRTIs and NNRTIs [71
], and Y181C/I, which may confer resistance to stavudine in addition to NVP [73
], N348I represents the only other example of an in vivo mutation that confers decreased susceptibility to two classes of RT inhibitors. However, in contrast to N348I, the prevalence of Q145M in an Italian cohort of 3,595 patients was lower (2.36%) and was always found in the presence of key drug resistance mutations [72
]. In our cohort, Q145M was exceedingly rare with a prevalence of less than 0.5%. Additional studies also demonstrate that Q145M significantly decreases viral replication capacity and that the recombinant enzyme displays a large loss in catalytic efficiency [72
]. While the effect of N348I on viral replication fitness has not been established, recombinant enzymes harbouring this mutation facilitate DNA synthesis reactions at least as efficiently as the WT enzyme (unpublished data).
Biochemical analyses designed to elucidate the mechanism by which N348I confers AZT resistance indicate that this mutation, consistent with other TAMs, acts by an excision rather than discrimination phenotype. However, an increase in AZT excision was only observed with a RNA/DNA T/P and not with a DNA/DNA T/P. Recently, it was suggested that mutations in the RT that decrease RNase H activity will enhance the AZT excision phenotype by slowing down the rate at which the RNA template strand is degraded [13
]. In this regard, our analyses confirm that the N348I mutation, alone and in combination with TAMs, decreases the enzyme's RNase H activity. In particular, the mutation significantly reduces the appearance of a 17-nucleotide secondary cleavage product, which corresponds to RNA/DNA duplex length of ten nucleotides. Recent studies that were designed to delineate the relationship between T/P duplex length and efficiency of AZT excision demonstrated that RT could not efficiently unblock chain-terminated T/P if the RNA/DNA duplex length was less than 13 nucleotides [75
]. Thus, the decrease in the formation of this cleavage product is significant and suggests a possible mechanism by which N348I confers AZT resistance. The structural mechanism by which N348I confers AZT (and NNRTI) resistance, however, cannot be inferred from this study. Recent studies have demonstrated that mutations in the RNase H primer grip region can significantly enhance AZT resistance by changing interactions between RT and the T/P complex and/or shifting the balance between the polymerase and RNase H activities [15
]. In contrast, N348I does not make direct contacts with the T/P. Furthermore, N348 in p66 RT is 27 Å away from the polymerase active site and 20 Å from the NNRTI-binding pocket, while N348 in p51 RT is 60 Å from the NNRTI-binding pocket (). Interestingly, N348 in p66 lies in close proximity to I270 and P272 at the base of the thumb domain in some crystal structures, suggesting a possible mechanism by which N348I mediates AZT resistance [77
]. Nevertheless, additional biochemical and structural studies are warranted to define the exact mechanism by which N348I confers RTI resistance.
Position of N348 in the HIV-1 RT Structure
One of the major limitations of this study is that while the data demonstrate that the appearance of N348I is associated with an increase in viral load, which is at least as large as the recognised TAMs, it does not account for the simultaneous appearance of other key PI or RT drug resistance mutations that could also contribute to the observed increase in viral load. Nevertheless, this caveat also applies to our analysis of the individual TAMs. Even with this large study, the number of permutations of mutations is too large to be able to exclude the contribution of other mutations, particularly as these patients are on combination therapy. Nevertheless, this study represents, to our knowledge, the most thorough evaluation of the in vivo relevance of a drug resistance mutation that can contribute to HIV drug resistance, but is not presently included in most genotypic and phenotypic resistance assays.
In conclusion, we have demonstrated that a mutation in the connection domain of the HIV-1 RT, N348I, is prevalent in drug-treated individuals, appears relatively early in drug therapy, and confers decreased susceptibility to AZT and NNRTIs. The mechanism of AZT resistance can be ascribed to increased nucleotide excision, as observed from an RNA/DNA T/P, and is likely to be manifested through decreased RNase H activity conferred by N348I. Decreased susceptibility to NNRTIs can be demonstrated at the enzyme level. Taken together, our data underscore the important role of N348I in conferring drug resistance to AZT and NNRTIs.