Maureen Oliveira

Persistence and Fitness of Multidrug-Resistant Human Immunodeficiency Virus Type 1 Acquired in Primary Infection

ABSTRACT This study examines the persistence and fitness of multidrug-resistant (MDR) viruses acquired during primary human immunodeficiency virus infection (PHI). In four individuals, MDR infections persisted over the entire study period, ranging from 36 weeks to 5 years, in the absence of antiretroviral therapy. In stark contrast, identified source partners in two cases showed expected outgrowth of wild-type (WT) virus within 12 weeks of treatment interruption. In the first PHI case, triple-class MDR resulted in low plasma viremia (1.6 to 3 log copies/ml) over time compared with mean values obtained for an untreated PHI group harboring WT infections (4.1 to 4.3 log copies/ml). Increasing viremia in PHI patient 1 at week 52 was associated with the de novo emergence of a protease inhibitor-resistant variant through a recombination event involving the original MDR virus. MDR infections in two other untreated PHI patients yielded viremia levels typical of the untreated WT group. A fourth patients MDR infection yielded low viremia (<50 to 500 copies/ml) for 5 years despite his having phenotypic resistance to all antiretroviral drugs in his treatment regimen. In two of these PHI cases, a rebound to higher levels of plasma viremia only occurred when the M184V mutation in reverse transcriptase could no longer be detected and, in a third case, nondetection of M184V was associated with an inability to isolate virus. To further evaluate the fitness of MDR variants acquired in PHI, MDR and corresponding WT viruses were isolated from index and source partners, respectively. Although MDR viral infectivity (50% tissue culture infective dose) was comparable to that observed for WT viruses, MDR infections in each case demonstrated 2-fold and 13- to 23-fold reductions in p24 antigen and reverse transcriptase enzymatic activity, respectively. In dual-infection competition assays, MDR viruses consistently demonstrated a marked replicative disadvantage compared with WT virus. These results indicate that MDR viruses that are generated following PHI can establish persistent infections as dominant quasispecies despite their impaired replicative competence.

HIV-1 subtype C viruses rapidly develop K65R resistance to tenofovir in cell culture

Background:Genotypic diversity among HIV-1 subtypes and circulating recombinant forms (CRF) may lead to distinct pathways to drug resistance. This study evaluated subtype-related differences in the development of resistance in culture to tenofovir. Methods:Genotyping determined nucleotide diversity among subtypes. Representative subtype B, C, CRF1_AE, CRF2_AG, G, and HIV-2 isolates were selected for resistance to tenofovir, lamivudine and didanosine in cell culture. Phenotypic assays determined the effects of the K65R substitution in reverse transcriptase (RT) on drug susceptibility. Results:Subtype C isolates show unique polymorphisms in RT codons 64 (AAG→AAA), 65 (AAA→AAG), and 66 (AAA→AAG), absent in other subtypes. The K65R mutation (AAG→AGG) arose with tenofovir by week 12 in four subtype C selections. In contrast, no tenofovir resistance arose in four subtype B (> 34–74 weeks), one each of CRF2_AG and G (> 30–33 weeks), and three HIV-2 (> 27–28 weeks) selections. K65R appeared after 55 and 73 weeks in two CRF1_AE selections with tenofovir. In contrast, times to the appearance of M184V with lamivudine pressure (weeks 8–14) did not vary among subtypes. Selective didanosine pressure resulted in the appearance of M184V and L74V after 38 weeks in two of four subtype C selections. The K65R transitions in subtype C and other subtypes (AGG and AGA) conferred similar 6.5–10-fold resistance to tenofovir and five to 25-fold crossresistance to each of abacavir, lamivudine, and didanosine, while not affecting zidovudine susceptibility. Conclusion:Tenofovir -based regimens will need to be carefully monitored in subtype C infections for the possible selection of K65R.

Characterization of the R263K Mutation in HIV-1 Integrase That Confers Low-Level Resistance to the Second-Generation Integrase Strand Transfer Inhibitor Dolutegravir

ABSTRACT Integrase (IN) strand transfer inhibitors (INSTIs) have been developed to inhibit the ability of HIV-1 integrase to irreversibly link the reverse-transcribed viral DNA to the host genome. INSTIs have proven their high efficiency in inhibiting viral replication in vitro and in patients. However, first-generation INSTIs have only a modest genetic barrier to resistance, allowing the virus to escape these powerful drugs through several resistance pathways. Second-generation INSTIs, such as dolutegravir (DTG, S/GSK1349572), have been reported to have a higher resistance barrier, and no novel drug resistance mutation has yet been described for this drug. Therefore, we performed in vitro selection experiments with DTG using viruses of subtypes B, C, and A/G and showed that the most common mutation to emerge was R263K. Further analysis by site-directed mutagenesis showed that R263K does confer low-level resistance to DTG and decreased integration in cell culture without altering reverse transcription. Biochemical cell-free assays performed with purified IN enzyme containing R263K confirmed the absence of major resistance against DTG and showed a slight decrease in 3′ processing and strand transfer activities compared to the wild type. Structural modeling suggested and in vitro IN-DNA binding assays show that the R263K mutation affects IN-DNA interactions.

Genetic Divergence of Human Immunodeficiency Virus Type 1 Ethiopian Clade C Reverse Transcriptase (RT) and Rapid Development of Resistance against Nonnucleoside Inhibitors of RT

ABSTRACT We sequenced and phylogenetically analyzed the reverse transcriptase (RT) region of five human immunodeficiency virus type 1 isolates from treatment-naive Ethiopian émigrés to Israel. Heteroduplex mobility assays were performed to confirm the clade C status of env genomic regions. The RT sequences showed that the strains clustered phylogenetically with clade C viruses, and a KVEQ-specific motif of silent mutations (amino acids 65, 106, 138, and 161, respectively) at resistance sites was present in the polymerase region of all studied Ethiopian isolates and subtype C reference strains. In addition, many other silent mutations were observed in the clade C viruses at various resistance sites. In general, the Ethiopian isolates were more closely related genotypically to a clade C reference strain from Botswana (southern Africa) than to previously sequenced Ethiopian reference strains. Genotypic analysis showed that two Ethiopian isolates naturally harbored the mutations K70R and G190A associated with resistance to ZDV and nonnucleoside reverse transcriptase inhibitors, respectively. Phenotypic assays revealed that the K70R substitution in this context did not reduce susceptibility to ZDV, whereas the G190A substitution resulted in high-level resistance to nevirapine (NVP). Moreover, variants resistant to NVP, delavirdine (DLV), and efavirenz (EFV) were more rapidly selected at lower drug doses culture with clade C than with clade B wild-type isolates. In the case of subtype C, selection with NVP and/or EFV led to the appearance of several previously unseen mutations in RT, i.e., V106M and S98I, as well as other mutations that have been previously reported (e.g., K103N, V106A, V108I, and Y181C). After selection with DLV, a polymorphism, A62A, initially observed in the Ethiopian isolate 4762, mutated to A62V; the latter is a secondary substitution associated with multidrug resistance against nucleoside RT inhibitors. Phenotypic analysis of clade C mutants selected against NVP, DLV, and EFV revealed broad cross-resistance, particularly in regard to NVP and DLV. These findings suggest that RT genotypic diversity may influence the emergence of drug resistance.

Transmission networks of drug resistance acquired in primary/early stage HIV infection

Objectives:Population-based sequencing of primary/recent HIV infections (PHIs) can provide a framework for understanding transmission dynamics of local epidemics. In Quebec, half of PHIs represent clustered transmission events. This study ascertained the cumulative implications of clustering on onward transmission of drug resistance. Methods:HIV-1 pol sequence datasets were available for all genotyped PHI (<6 months postseroconversion; n = 848 subtype B infections, 1997–2007). Phylogenetic analysis established clustered transmission events, based on maximum likelihood topologies having high bootstrap values (>98%) and short genetic distances. The distributions of resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors and protease inhibitors in unique and clustered transmissions were ascertained. Results:Episodic clustering was observed in half of recent/early stage infections from 1997–2008. Overall, 29 and 28% of new infections segregated into small (<5 PHI/cluster, n = 242/848) and large transmission chains (≥5 PHI/cluster, n = 239/848), averaging 2.8 ± 0.1 and 10.3 ± 1.0 PHI/cluster, respectively. The transmission of nucleoside analogue mutations and 215 resistant variants (T215C/D/I/F/N/S/Y) declined with clustering (7.9 vs. 3.4 vs. 1.2 and 5.8 vs. 1.7 vs. 1.1% for unique, small, and large clustered transmissions, respectively). In contrast, clustering was associated with the increased transmission of viruses harbouring resistance to nonnucleoside reverse transcriptase inhibitors (6.6 vs. 6.0 vs. 15.5%, respectively). Conclusion:Clustering in early/PHI stage infection differentially affects transmission of drug resistance to different drug classes. Public health, prevention and diagnostic strategies, targeting PHI, afford a unique opportunity to curb the spread of transmitted drug resistance.

High Prevalence of the K65R Mutation in Human Immunodeficiency Virus Type 1 Subtype C Isolates from Infected Patients in Botswana Treated with Didanosine-Based Regimens

ABSTRACT We analyzed the reverse transcriptase genotypes of human immunodeficiency virus type 1 subtype C viruses isolated from 23 patients in Botswana treated with didanosine-based regimens. The K65R mutation was selected either alone or together with the Q151M, S68G, or F116Y substitution in viruses from seven such individuals. The results of in vitro passage experiments were consistent with an apparent increased propensity of subtype C viruses to develop the K65R substitution.

Compensation by the E138K Mutation in HIV-1 Reverse Transcriptase for Deficits in Viral Replication Capacity and Enzyme Processivity Associated with the M184I/V Mutations

ABSTRACT Recently, several phase 3 clinical trials (ECHO and THRIVE) showed that E138K and M184I were the most frequent mutations to emerge in patients who failed therapy with rilpivirine (RPV) together with two nucleos(t)ide reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir (TDF). To investigate the basis for the copresence of E138K and M184I, we generated recombinant mutated and wild-type (WT) reverse transcriptase (RT) enzymes and HIV-1NL4-3 infectious clones. Drug susceptibilities were determined in cord blood mononuclear cells (CBMCs). Structural modeling was performed to analyze any impact on deoxynucleoside triphosphate (dNTP) binding. The results of phenotyping showed that viruses containing both the E138K and M184V mutations were more resistant to each of FTC, 3TC, and ETR than viruses containing E138K and M184I. Viruses with E138K displayed only modest resistance to ETR, little resistance to efavirenz (EFV), and no resistance to either FTC or 3TC. E138K restored viral replication capacity (RC) in the presence of M184I/V, and this was confirmed in cell-free RT processivity assays. RT enzymes containing E138K, E138K/184I, or E138K/184V exhibited higher processivity than WT RT at low dNTP concentrations. Steady-state kinetic analysis demonstrated that the E138K mutation resulted in decreased K m s for dNTPs. In contrast, M184I/V resulted in an increased K m for dNTPs compared to those for WT RT. These results indicate that the E138K mutation compensates for both the deficit in dNTP usage and impairment in replication capacity by M184I/V. Structural modeling shows that the addition of E138K to M184I/V promotes tighter dNTP binding.

Template Usage Is Responsible for the Preferential Acquisition of the K65R Reverse Transcriptase Mutation in Subtype C Variants of Human Immunodeficiency Virus Type 1

ABSTRACT We propose that a nucleotide template-based mechanism facilitates the acquisition of the K65R mutation in subtype C human immunodeficiency virus type 1 (HIV-1). Different patterns of DNA synthesis were observed using DNA templates from viruses of subtype B or C origin. When subtype C reverse transcriptase (RT) was employed to synthesize DNA from subtype C DNA templates, preferential pausing was seen at the nucleotide position responsible for the AAG-to-AGG K65R mutation. This did not occur when the subtype B RT and template were used. Template factors can therefore increase the probability of K65R development in subtype C HIV-1.

Identification of Novel Mutations Responsible for Resistance to MK-2048, a Second-Generation HIV-1 Integrase Inhibitor

ABSTRACT MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer “off” rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg2+ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.

Signature Nucleotide Polymorphisms at Positions 64 and 65 in Reverse Transcriptase Favor the Selection of the K65R Resistance Mutation in HIV-1 Subtype C

Recently, we described a novel nucleotide template-based mechanism that may be the basis for the facilitated acquisition of the K65R resistance mutation in subtype C versus subtype B human immunodeficiency virus type 1 (HIV-1). In this article, we evaluated the effects of subtype C-specific silent polymorphisms in cell culture drug-selection experiments using nucleoside and nucleotide reverse-transcriptase inhibitors. The K65R pathway was selected more frequently in a subtype B virus that contained subtype C nucleotide polymorphisms at both positions 64 and 65 than in a wild-type NL4-3 subtype B virus. This is the first demonstration of the significance of silent nucleotide polymorphisms in the development of drug resistance.