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The evolution of HIV-1 reverse transcriptase in route to acquisition of Q151M multi-drug resistance is complex and involves mutations in multiple domains.

Mbisa JL, Gupta RK, Kabamba D, Mulenga V, Kalumbi M, Chintu C, Parry CM, Gibb DM, Walker SA, Cane PA, Pillay D - Retrovirology (2011)

Bottom Line: RT domain-swapping of patient and wild-type RTs showed that patient-derived connection subdomains were not associated with reduced NRTI susceptibility.This was further reduced to ~22% when the Q151M-containing DNA pol domain was expressed with wild-type C-terminal domain, but was then fully compensated by coexpression of the coevolved connection subdomain.The acquisition of the Q151M pathway occurred sequentially over a long period of failing NRTI therapy, and was associated with mutations in multiple RT domains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virus Reference Department, Microbiology Services, Colindale, Health Protection Agency, London, UK.

ABSTRACT

Background: The Q151M multi-drug resistance (MDR) pathway in HIV-1 reverse transcriptase (RT) confers reduced susceptibility to all nucleoside reverse transcriptase inhibitors (NRTIs) excluding tenofovir (TDF). This pathway emerges after long term failure of therapy, and is increasingly observed in the resource poor world, where antiretroviral therapy is rarely accompanied by intensive virological monitoring. In this study we examined the genotypic, phenotypic and fitness correlates associated with the development of Q151M MDR in the absence of viral load monitoring.

Results: Single-genome sequencing (SGS) of full-length RT was carried out on sequential samples from an HIV-infected individual enrolled in ART rollout. The emergence of Q151M MDR occurred in the order A62V, V75I, and finally Q151M on the same genome at 4, 17 and 37 months after initiation of therapy, respectively. This was accompanied by a parallel cumulative acquisition of mutations at 20 other codon positions; seven of which were located in the connection subdomain. We established that fourteen of these mutations are also observed in Q151M-containing sequences submitted to the Stanford University HIV database. Phenotypic drug susceptibility testing demonstrated that the Q151M-containing RT had reduced susceptibility to all NRTIs except for TDF. RT domain-swapping of patient and wild-type RTs showed that patient-derived connection subdomains were not associated with reduced NRTI susceptibility. However, the virus expressing patient-derived Q151M RT at 37 months demonstrated ~44% replicative capacity of that at 4 months. This was further reduced to ~22% when the Q151M-containing DNA pol domain was expressed with wild-type C-terminal domain, but was then fully compensated by coexpression of the coevolved connection subdomain.

Conclusions: We demonstrate a complex interplay between drug susceptibility and replicative fitness in the acquisition Q151M MDR with serious implications for second-line regimen options. The acquisition of the Q151M pathway occurred sequentially over a long period of failing NRTI therapy, and was associated with mutations in multiple RT domains.

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NRTI susceptibilities and replicative capacity associated with RT domains of patient P66. (A) Schematic representation of full-length and chimeras of subtype C wild-type and patient-derived RT gag-pol expressing vectors used for drug susceptibility and replicative capacity testing. The positions of the restriction sites used for cloning of patient-derived PR-RT fragments (ApaI and ClaI) and for RT domain swapping (HpaI and SpeI) are indicated above the vector. The origins of the RT domains are shown as different coloured boxes: black, wild-type virus; dark gray, patient-derived RT at 4 months; light gray, patient-derived RT at 17 months; and white, patient-derived RT at 37 months. The names of the vectors are indicated on the right with a number representing the month when the sample was collected followed by the patient-derived domain(s) being expressed. Mutations present in each domain are shown on the full-length RT constructs as follows: inside the box, NRTI-associated resistance mutations; above the box, NNRTI-associated resistance mutations; and below the box, other mutations. Pol, DNA pol domain; Cn, Connection subdomain; Rh, RNase H domain. (B) Susceptibility to d4T exhibited by patient-derived full-length RTs and RT domains. (C) Susceptibility to second-line NRTI ABC exhibited by patient-derived full-length RTs. (D) Susceptibility to second-line NRTI ddI exhibited by patient-derived full-length RTs. (E) Susceptibility to TDF exhibited by patient-derived full-length RTs. (F) Replicative capacities relative to virus expressing full-length patient-derived RT from 4-months after initiation of therapy, set at 100%, are shown for each virus. The error bars represent standard error of the mean of three or more independent experiments.
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Figure 3: NRTI susceptibilities and replicative capacity associated with RT domains of patient P66. (A) Schematic representation of full-length and chimeras of subtype C wild-type and patient-derived RT gag-pol expressing vectors used for drug susceptibility and replicative capacity testing. The positions of the restriction sites used for cloning of patient-derived PR-RT fragments (ApaI and ClaI) and for RT domain swapping (HpaI and SpeI) are indicated above the vector. The origins of the RT domains are shown as different coloured boxes: black, wild-type virus; dark gray, patient-derived RT at 4 months; light gray, patient-derived RT at 17 months; and white, patient-derived RT at 37 months. The names of the vectors are indicated on the right with a number representing the month when the sample was collected followed by the patient-derived domain(s) being expressed. Mutations present in each domain are shown on the full-length RT constructs as follows: inside the box, NRTI-associated resistance mutations; above the box, NNRTI-associated resistance mutations; and below the box, other mutations. Pol, DNA pol domain; Cn, Connection subdomain; Rh, RNase H domain. (B) Susceptibility to d4T exhibited by patient-derived full-length RTs and RT domains. (C) Susceptibility to second-line NRTI ABC exhibited by patient-derived full-length RTs. (D) Susceptibility to second-line NRTI ddI exhibited by patient-derived full-length RTs. (E) Susceptibility to TDF exhibited by patient-derived full-length RTs. (F) Replicative capacities relative to virus expressing full-length patient-derived RT from 4-months after initiation of therapy, set at 100%, are shown for each virus. The error bars represent standard error of the mean of three or more independent experiments.

Mentions: Consequently, we investigated whether the C-terminal mutations we observed affected susceptibility to NRTIs. Unique restriction sites were introduced in RT and IN genes without changing the amino acid coding, in both the packaging vector and cloned patient fragments in order to facilitate RT domain-swapping (Figure 3A). The patient-derived RTs remained d4T-susceptible until the development of the Q151M mutation at 37 months, when there was a significant increase (~16-fold) in IC50 values compared to wild-type RT (Figure 3B; P < 0.002). At most we observed a 1.3-fold change in susceptibility to d4T at 4 or 17 months leading us to conclude that Q151M is the main contributor to d4T resistance in the Q151M MDR complex. The patient-derived RT exhibited a 23-fold increase in 3TC IC50 values at 4 months which did not increase at 17 and 37 months despite the acquisition of the Q151M MDR mutations (Table 3). The effect on susceptibility to 3TC was probably due to M184I/V mutations which were seen by 4 months. The 23-fold reduction in susceptibility is relatively lower than observed in other studies [30,31]. This could be because our assay uses full-length RT fragments derived from clinical isolates. It has recently been shown that the use of a co-evolved or subtype-specific C-terminal region of RT can influence the magnitude of drug resistance observed in a phenotypic drug susceptibility assay [32].


The evolution of HIV-1 reverse transcriptase in route to acquisition of Q151M multi-drug resistance is complex and involves mutations in multiple domains.

Mbisa JL, Gupta RK, Kabamba D, Mulenga V, Kalumbi M, Chintu C, Parry CM, Gibb DM, Walker SA, Cane PA, Pillay D - Retrovirology (2011)

NRTI susceptibilities and replicative capacity associated with RT domains of patient P66. (A) Schematic representation of full-length and chimeras of subtype C wild-type and patient-derived RT gag-pol expressing vectors used for drug susceptibility and replicative capacity testing. The positions of the restriction sites used for cloning of patient-derived PR-RT fragments (ApaI and ClaI) and for RT domain swapping (HpaI and SpeI) are indicated above the vector. The origins of the RT domains are shown as different coloured boxes: black, wild-type virus; dark gray, patient-derived RT at 4 months; light gray, patient-derived RT at 17 months; and white, patient-derived RT at 37 months. The names of the vectors are indicated on the right with a number representing the month when the sample was collected followed by the patient-derived domain(s) being expressed. Mutations present in each domain are shown on the full-length RT constructs as follows: inside the box, NRTI-associated resistance mutations; above the box, NNRTI-associated resistance mutations; and below the box, other mutations. Pol, DNA pol domain; Cn, Connection subdomain; Rh, RNase H domain. (B) Susceptibility to d4T exhibited by patient-derived full-length RTs and RT domains. (C) Susceptibility to second-line NRTI ABC exhibited by patient-derived full-length RTs. (D) Susceptibility to second-line NRTI ddI exhibited by patient-derived full-length RTs. (E) Susceptibility to TDF exhibited by patient-derived full-length RTs. (F) Replicative capacities relative to virus expressing full-length patient-derived RT from 4-months after initiation of therapy, set at 100%, are shown for each virus. The error bars represent standard error of the mean of three or more independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: NRTI susceptibilities and replicative capacity associated with RT domains of patient P66. (A) Schematic representation of full-length and chimeras of subtype C wild-type and patient-derived RT gag-pol expressing vectors used for drug susceptibility and replicative capacity testing. The positions of the restriction sites used for cloning of patient-derived PR-RT fragments (ApaI and ClaI) and for RT domain swapping (HpaI and SpeI) are indicated above the vector. The origins of the RT domains are shown as different coloured boxes: black, wild-type virus; dark gray, patient-derived RT at 4 months; light gray, patient-derived RT at 17 months; and white, patient-derived RT at 37 months. The names of the vectors are indicated on the right with a number representing the month when the sample was collected followed by the patient-derived domain(s) being expressed. Mutations present in each domain are shown on the full-length RT constructs as follows: inside the box, NRTI-associated resistance mutations; above the box, NNRTI-associated resistance mutations; and below the box, other mutations. Pol, DNA pol domain; Cn, Connection subdomain; Rh, RNase H domain. (B) Susceptibility to d4T exhibited by patient-derived full-length RTs and RT domains. (C) Susceptibility to second-line NRTI ABC exhibited by patient-derived full-length RTs. (D) Susceptibility to second-line NRTI ddI exhibited by patient-derived full-length RTs. (E) Susceptibility to TDF exhibited by patient-derived full-length RTs. (F) Replicative capacities relative to virus expressing full-length patient-derived RT from 4-months after initiation of therapy, set at 100%, are shown for each virus. The error bars represent standard error of the mean of three or more independent experiments.
Mentions: Consequently, we investigated whether the C-terminal mutations we observed affected susceptibility to NRTIs. Unique restriction sites were introduced in RT and IN genes without changing the amino acid coding, in both the packaging vector and cloned patient fragments in order to facilitate RT domain-swapping (Figure 3A). The patient-derived RTs remained d4T-susceptible until the development of the Q151M mutation at 37 months, when there was a significant increase (~16-fold) in IC50 values compared to wild-type RT (Figure 3B; P < 0.002). At most we observed a 1.3-fold change in susceptibility to d4T at 4 or 17 months leading us to conclude that Q151M is the main contributor to d4T resistance in the Q151M MDR complex. The patient-derived RT exhibited a 23-fold increase in 3TC IC50 values at 4 months which did not increase at 17 and 37 months despite the acquisition of the Q151M MDR mutations (Table 3). The effect on susceptibility to 3TC was probably due to M184I/V mutations which were seen by 4 months. The 23-fold reduction in susceptibility is relatively lower than observed in other studies [30,31]. This could be because our assay uses full-length RT fragments derived from clinical isolates. It has recently been shown that the use of a co-evolved or subtype-specific C-terminal region of RT can influence the magnitude of drug resistance observed in a phenotypic drug susceptibility assay [32].

Bottom Line: RT domain-swapping of patient and wild-type RTs showed that patient-derived connection subdomains were not associated with reduced NRTI susceptibility.This was further reduced to ~22% when the Q151M-containing DNA pol domain was expressed with wild-type C-terminal domain, but was then fully compensated by coexpression of the coevolved connection subdomain.The acquisition of the Q151M pathway occurred sequentially over a long period of failing NRTI therapy, and was associated with mutations in multiple RT domains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virus Reference Department, Microbiology Services, Colindale, Health Protection Agency, London, UK.

ABSTRACT

Background: The Q151M multi-drug resistance (MDR) pathway in HIV-1 reverse transcriptase (RT) confers reduced susceptibility to all nucleoside reverse transcriptase inhibitors (NRTIs) excluding tenofovir (TDF). This pathway emerges after long term failure of therapy, and is increasingly observed in the resource poor world, where antiretroviral therapy is rarely accompanied by intensive virological monitoring. In this study we examined the genotypic, phenotypic and fitness correlates associated with the development of Q151M MDR in the absence of viral load monitoring.

Results: Single-genome sequencing (SGS) of full-length RT was carried out on sequential samples from an HIV-infected individual enrolled in ART rollout. The emergence of Q151M MDR occurred in the order A62V, V75I, and finally Q151M on the same genome at 4, 17 and 37 months after initiation of therapy, respectively. This was accompanied by a parallel cumulative acquisition of mutations at 20 other codon positions; seven of which were located in the connection subdomain. We established that fourteen of these mutations are also observed in Q151M-containing sequences submitted to the Stanford University HIV database. Phenotypic drug susceptibility testing demonstrated that the Q151M-containing RT had reduced susceptibility to all NRTIs except for TDF. RT domain-swapping of patient and wild-type RTs showed that patient-derived connection subdomains were not associated with reduced NRTI susceptibility. However, the virus expressing patient-derived Q151M RT at 37 months demonstrated ~44% replicative capacity of that at 4 months. This was further reduced to ~22% when the Q151M-containing DNA pol domain was expressed with wild-type C-terminal domain, but was then fully compensated by coexpression of the coevolved connection subdomain.

Conclusions: We demonstrate a complex interplay between drug susceptibility and replicative fitness in the acquisition Q151M MDR with serious implications for second-line regimen options. The acquisition of the Q151M pathway occurred sequentially over a long period of failing NRTI therapy, and was associated with mutations in multiple RT domains.

Show MeSH
Related in: MedlinePlus