HIV-1 subtype F integrase polymorphisms external to the catalytic core domain contribute to severe loss of replication capacity in context of the integrase inhibitor resistance mutation Q148H

Paula C Aulicino; Zoha Momin; Mijael Rozenszajn; Arturo Monzon; Solange Arazi-Caillaud; Rosa Bologna; Andrea Mangano; Jason T Kimata

doi:10.1093/jac/dkac238

. 2022 Jul 28;77(10):2793–2802. doi: 10.1093/jac/dkac238

HIV-1 subtype F integrase polymorphisms external to the catalytic core domain contribute to severe loss of replication capacity in context of the integrase inhibitor resistance mutation Q148H

Paula C Aulicino ^1,^2,^✉, Zoha Momin ³, Mijael Rozenszajn ^4,⁵, Arturo Monzon ⁶, Solange Arazi-Caillaud ⁷, Rosa Bologna ⁸, Andrea Mangano ^9,¹⁰, Jason T Kimata ¹¹

PMCID: PMC9989736 PMID: 35897124

Abstract

Background

In prior studies, HIV-1 BF recombinants with subtype F integrases failed to develop resistance to raltegravir through the Q148H mutational pathway. We aimed to determine the role of subtype-specific polymorphisms in integrase on drug susceptibility, viral replication and integration.

Methods

Integrase sequences were retrieved from the Los Alamos Database or obtained from the Garrahan HIV cohort. HIV-1 infectious molecular clones with or without Q148H (+ G140S) resistance mutations were constructed using integrases of subtype B (NL4-3) or F1(BF) ARMA159 and URTR23. Integrase chimeras were generated by reciprocal exchanges of a 200 bp fragment spanning amino acids 85–150 of the catalytic core domain (CCD) of NL4-3-Q148H and either ARMA159-Q148H or URTR23-Q148H. Viral infections were quantified by p24 ELISA and Alu-gag integration PCR assay.

Results

At least 18 different polymorphisms distinguish subtype B from F1(BF) recombinant integrases. In phenotypic experiments, p24 at Day 15 post-infection was high (10⁵–10⁶ pg/mL) for WT and NL4-3-Q148H; by contrast, it was low (10²–10⁴ pg/mL) for both F1(BF)-Q148H + G140S viruses, and undetectable for the Q148H mutants. Compared with WT viruses, integrated DNA was reduced by 5-fold for NL4-3-Q148H (P = 0.05), 9-fold for URTR23-Q148H (P = 0.01) and 16000-fold for ARMA159-Q148H (P = 0.01). Reciprocal exchange between B and F1(BF) of an integrase CCD region failed to rescue the replicative defect of F1(BF) integrase mutants.

Conclusions

The functional impairment of Q148H in the context of subtype F integrases from BF recombinants explains the lack of selection of this pathway in vivo. Non-B polymorphisms external to the integrase CCD may influence the pathway to integrase strand transfer inhibitor resistance.

Introduction

Integrase strand transfer inhibitors (INSTIs) are a fundamental part of the current anti-HIV armamentarium. However, as with other antiretroviral drugs (ARVs), INSTIs are not immune to development of drug resistance (DR). Of the mutational pathways associated with DR to INSTIs, accumulation of mutations via the Q148H pathway is the most clinically dangerous. Frequently observed in subtype B-infected individuals exposed to first-generation raltegravir or elvitegravir, the Q148H mutational pathway leads to a significant increase in the fold change (FC) in IC₅₀s of all INSTIs¹ and an increased risk of virological failure to dolutegravir.² In many subtype B viruses, Q148H is selected concomitantly with G140S, as it has been shown to compensate for a replication fitness loss of Q148H mutants,^3–7 and to increase DR to the entire drug class. Interestingly, non-B subtypes show an impediment to developing resistance through this pathway, allegedly because of an evolutionary constraint associated with codon usage at position 140 (G140S mutation requires two changes in non-B subtypes but only one in subtype B).⁸

In a previous study evaluating HIV DR patterns in individuals infected with subtype B or BF recombinant strains from Argentina and failing raltegravir-based ART, we confirmed that Q148H (+ G140S) occurs exclusively in the context of subtype B HIV-1 integrase (in) genes, leading to a clinically significant higher level of predicted resistance to raltegravir and also to second-generation INSTIs dolutegravir and bictegravir for individuals carrying HIV-1 with subtype B in.⁹ The observed results were independent of viral load or time under virological failure with raltegravir, reducing the possibility that the Q148H pathway could evolve at a later time or with higher viral loads, and suggesting a possible biological or functional limitation for subtype F integrase (IN) protein to develop these high-level resistance mutations.

HIV-1 IN is composed of three domains: (i) the 46 amino acid N-terminal domain (NTD), which contains a histidine–histidine–cysteine–cysteine (HHCC) motif that coordinates zinc binding; (ii) the 130 amino acid catalytic core domain (CCD), which contains the catalytic triad D64, D116 and E152; and (iii) the 93 amino acid C-terminal domain (CTD), which is involved in host DNA binding. Interaction of IN with lens epithelium-derived growth factor (LEDGF)/p75—a cellular tethering co-factor—through the CCD and CTD domains is crucial for IN activity. Due to functional and structural constraints, IN is the most conserved of the pol-encoded HIV-1 enzymes. However, IN shows a high frequency of polymorphic sites and a complexity not yet fully understood that is important for optimized use and future development of ARVs targeting IN. Recent studies have shown that variations at high-entropy residues in the CCD (particularly S119, T124 and T125) play a fundamental role in protein–DNA recognition,¹⁰ as well as in DR to INSTIs when in combination with Q148H + G140S or N155H mutations.^11,12 In this study, we aimed to assess IN polymorphic diversity in F or BF subtypes—prevalent in Latin American countries—and investigate the effect of viral genetic background on susceptibility to INSTIs, viral replication and IN functionality for mutants carrying Q148H (±G140S) or N155H INSTI-associated drug resistance mutations (DRMs).

Materials and methods

HIV-1 IN sequences

HIV-1 IN sequences from subtypes A, B, C, F, G and Latin American BF recombinants were retrieved from the Los Alamos HIV Sequence Database. Nine novel subtype F IN sequences were obtained from the Garrahan HIV cohort, a well-characterized cohort of HIV-1-infected children and adolescents from Hospital de Pediatría Juan P. Garrahan in Buenos Aires, Argentina. Characterization of HIV-1 subtype was performed using phylogenetic and recombination tools as previously described.⁹ Amino acid sequence alignments of IN were generated to evaluate sequence diversity among subtypes using R. Two Sample Logos was used to identify positions that are enriched or depleted for a given amino acid using the binomial test and a P value cut-off of 0.05 to identify differences between any two alignments.¹³

Cell lines

CEMx174 cells and 293T cells were purchased from the ATCC. ACH-2,^14,15 Jurkat E6-1¹⁶ and TZMbl cells^17,18 were obtained from the NIH HIV Reagent Program.

Generation of infectious HIV-1 clones with subtype F in genes

Infectious molecular clones (IMCs) of HIV-1 bearing subtype F in genes from BF recombinant viruses were generated using HIV-1_NL4-3 as the backbone virus.¹⁹ in genes from two BF recombinant viruses, URTR23 (GenBank #AF385934) and ARMA159 (GenBank #AF385936),^20,21 were synthesized by GenScript with flanking sequences from HIV-1 NL4-3. Because we determined that plasmids with full-length NL4-3 with subtype F in genes were unstable for propagation in Escherichia coli, the in genes were inserted, using HiFi DNA Assembly (New England BioLabs), into a modified 5′ half NL4-3 clone, p83-2,²² which includes the viral sequence through the conserved SalI site (p83-2-ARMA159IN and p83-2-URTR23IN).

To generate infectious virus and determine replication capacity, the 5′ half viral clones were ligated to the 3′ half NL4-3 clone, p83-10²² at the SalI site and the combined DNA of each virus was transfected into triplicate cultures of 293T cells using GeneJuice (MilliporeSigma). One day post-transfection, the 293T cells were co-cultured with CEMx174 cells for 3 days in complete RPMI. CEMx174 cells were removed from the co-cultures and cultured for 2–3 additional weeks. HIV-1 p24 was quantified by ELISA (Advanced Bioscience Laboratories). For viral replication experiments, statistical significance was determined using two-way ANOVA using Tukey’s correction (Prism 9, GraphPad Software).

The 50% tissue culture infectious dose (TCID₅₀/mL) was determined by limiting dilution using the reporter cell line TZMbl as we previously described.²³

in mutagenesis

INSTI-associated DRMs were introduced into the NL4-3, ARMA159, or URTR23 in genes by overlap extension PCR site-specific mutagenesis using Phusion Green-high fidelity DNA polymerase (ThermoFisher) and the modified p83-2 plasmid with the NL4-3, ARMA159, or URTR23 in genes as templates. Primer design and Phusion DNA polymerase PCR to introduce mutations G140S, Q148H, N155H and G/K163R were based on the methods of Xia et al.²⁴

For reciprocal exchanges of the CCD between in genes with Q148H mutations (NL4-3-Q148H and either ARMA159-Q148H or URTR23-Q148H), 200 bp fragments of each in gene that includes the coding region from amino acids 85 to 150 were PCR amplified with Phusion DNA polymerase and cloned by HiFi DNA Assembler into the complementary sites of the p83-2 clones to form p83-2-NL4-3(ARMA or URTR), p83-2-ARMA159(NL), or p83-2-URTR23(NL). Infectious viruses were produced and viral replication analysed as described in the previous section.

INSTI susceptibility assay

The susceptibility of each virus to raltegravir and dolutegravir was determined using a TZMbl cell-based drug susceptibility assay²⁵ and 2-fold serial dilutions of raltegravir or dolutegravir starting at a concentration of 640 nM. The 50% effective concentrations (EC₅₀) were calculated by non-linear regression using Prism 9 (GraphPad Software).

Integration assay

Integrated HIV DNA was quantified by the Alu-gag PCR method described by Yu et al.²⁶ For this assay, the ARMA159 and URTR23 in genes were introduced into the HIV-based vector NL4-3 (pNL4-3.Luc.R-E-) with or without the Q148H mutation.^27,28 The vector constructs were pseudotyped with VSV-G by co-transfection of 293T cells. Viral transductions of Jurkat E6-1 cells were performed in triplicate with equal amounts of HIV-1 p24, as determined by ELISA. Two days post-infection, the number of integrants was quantified by qPCR, using an input of 500 ng of cellular DNA and a DNA standard curve of ACH2 cells: peripheral blood lymphocytes in ratios of 1:10 to 1:100 000. Differences in relative integration were statistically analysed by two-tailed Welch’s t-test using Prism 9 (GraphPad).

Results

Genotypic differences between HIV subtype B and Latin American F1 IN sequences are distributed along the CCD and NTD regions

IN sequences from the prototypic subtype B HIV viruses (n = 3026) were compared with those of Latin American F1/BF recombinants (F1/BFrec) (n = 42). Because HIV F1 IN sequences with a Latin American origin are phylogenetically distinct from most of the HIV F1 strains isolated elsewhere (Figure S1, available as Supplementary data at JAC Online), the F1/BFrec dataset included 20 HIV F1 sequences from pure HIV F1 (19 from Brazil, 1 from Argentina); 13 CRF_BF recombinants from Brazil, Argentina, Uruguay or Peru; and 9 novel subtype F1 IN sequences belonging to BF recombinant strains of the Garrahan HIV cohort.

As shown in Figure 1, Latin American F1/BFrec IN dataset showed 18 significant amino acid changes with respect to the subtype B dataset. Of them, 1 was located in the NTD region of the enzyme (S17N), 10 were located in the CCD region (I72V, I84L, L101I, S119T/P, T/N124A, T125A, K136Q, N156K, G163S/K/R and V165I) and 7 were located in the CTD region (V201I, A205S, K211R, T218I, L234V, D256E and S283G). Among amino acids typical of IN from Latin American F1/BFrec, G163K/R has previously been associated with INSTI resistance in subtype B strains, although it showed little effect on INSTI susceptibility in the absence of major INSTI-associated resistance mutations.

Polymorphisms in CCD are common between BF recombinants and non-B group M subtypes

Curated alignments of IN sequences belonging to subtypes C (n = 1035), A (n = 163), G (n = 79), F1 (n = 52) and BFrec (n = 42) were compared at each amino acid position to subtype B. Non-B amino acids were considered polymorphisms. At each position, the frequency of polymorphisms above 10% was compared between subtypes (Figure 2).

Figure 2. — Predominant amino acid changes differing from the most prevalent in subtype B in HIV-1 IN, according to subtype. Polymorphisms in amino acids 1–283 of the HIV-1 IN are shown for a given subtype if 10% or more of the sequences in the dataset were polymorphic at that site; the percentage of sequences that were polymorphic appear within the coloured bars. The subtype is shown in the key. This figure appears in colour in the online version of *JAC* and in black and white in the print version of *JAC*.

A total of 54 IN sites (18.7%) were polymorphic. Of them, 8 were highly frequent (>50%) in BFrec and at least 3 other non-B subtypes analysed: L101I, T124A/N/S, T125A/V, K136Q/T in the CCD, V201I, L234I/V, D256E and S283G in the CTD. While a number of subtype B sequences were also polymorphic at positions 101, 124, 125 and 201, positions 136, 234 and 283 were typically polymorphic in non-B strains.

Q148H disrupts replication of HIV-1 with subtype F INs and reduces integration

To evaluate the effect of Q148H mutations and subtype-specific polymorphisms on HIV-1 replication capacity and susceptibility to INSTIs, we constructed HIV-1 IMCs carrying WT and Q148H ± G140S mutant in genes using an NL4-3 subtype B backbone and in gene fragments from NL4-3, or from either of two CRF12_BF recombinants: ARMA159 or URTR23 from Argentina and Uruguay, respectively. Both BF recombinants carry subtype F1 in genes. As shown in Figure 3, NL4-3(B) differs from BF recombinants at 17 sites along the IN protein, representing most of the inter-subtype diversity evidenced in the previous sections. Also, seven amino acids differentiate ARMA159 and URTR23 at positions 72, 101, 119, 122, 124, 163 and 254.

Figure 3. — HIV-1 IN sequence alignment from IMCs NL4-3, URTR23 and ARMA159. Polymorphic sites are marked in yellow over each of the IN domains. Shaded region shows the CCD fragment reciprocally exchanged between B and BF for phenotypic studies. Conserved sites for IN function are marked with symbols. *HHCC motif relevant to co-ordinate zinc binding; #catalytic triad D64, D116 and E152. This figure appears in colour in the online version of *JAC* and in black and white in the print version of *JAC*.

To determine if polymorphisms had any effect on susceptibility to INSTIs, we first assessed in vitro susceptibility of the WT IMCs to raltegravir and dolutegravir (Table 1). Mean EC₅₀s to raltegravir were similar between viruses encoding the subtype F1 IN and NL4-3 (5.9 nM for NL4-3, 5.0 nM for ARMA159 and 4.9 nM for URTR23). Furthermore, the EC₅₀s to dolutegravir were 2.4 nM for NL4-3, 1.8 nM for ARMA159 and 2.8 nM for URTR23 and not significantly different. These data indicate that the BF-specific polymorphisms do not affect susceptibility to INSTIs.

Table 1.

Drug susceptibilities for HIV-1 WT and site-specific integrase mutant viruses^a

Viruses	WT		148H		140S+148H		155H		155H+163R		163R
Raltegravir (EC₅₀± SD (nM)/fold change)
NL4-3	5.9 ± 0.9	–	64.9 ± 3.9	11	132.7 ± 3.0	23	47.6 ± 5.2	8.1	72.7 ± 4.8	12	NT	–
URTR23	4.9 ± 2.6	–	NT	–	120.7 ± 29.7	25	71.7 ± 7.1	15	65.5 ± 4.0	13	6.2 ± 0.3	1.3
ARMA159	5.0 ± 1.0	–	NT	–	NT	–	NT	–	NT	–	NT	–
Dolutegravir (EC₅₀± SD (nM)/fold change)
NL4-3	2.4 ± 0.7	–	2.8 ± 0.9	1.2	6.8 ± 0.6	2.8	4.0 ± 0.4	1.7	3.0 ± 0.2	1.3	NT	–
URTR23	2.8 ± 1.0	–	NT	–	8.9 ± 1.1	3.2	3.9 ± 1.6	1.4	3.6 ± 1.4	1.3	3.7 ± 1.4	1.3
ARMA159	1.8 ± 1.1	–	NT	–	NT	–	NT	–	NT	–	NT	–

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NT, not tested.

Data shown represent the means and SD of at least two independent experiments performed in triplicate. The fold changes in EC₅₀s of the IN mutants are relative to the WT virus.

EC₅₀s were determined by TZMbl infection assay as described in the Materials and methods section.

As we previously observed the emergence of N155H in subtype F1 in encoding HIV-1 during virological failures to raltegravir, we introduced 155H into URTR23 IN, which has a 163K polymorphism. The 155H mutation reduced susceptibility to raltegravir by 15-fold, which was not significantly different from NL4-3 155H (Table 1). These data confirm the importance of the N155H DRM for resistance to raltegravir. On the other hand, although 163R was also associated with resistance to raltegravir, its introduction into URTR23-155H did not further increase the EC₅₀ to raltegravir, and alone this mutation did not impact susceptibility to raltegravir. Additionally, these mutations did not affect URTR23 IN virus susceptibility to dolutegravir.

As expected, introduction of Q148H INSTI-associated mutation into the NL4-3 in gene resulted in an 11-fold reduction in susceptibility to raltegravir and a 1.2-fold reduction in susceptibility to dolutegravir. Addition of G140S to the Q148H NL4-3 mutant further increased the resistance to 23-fold for raltegravir and 2.8-fold for dolutegravir. While Q148H is known to reduce IN activity and viral replication capacity of subtype B viruses, the introduction of this mutation into ARMA159 or URTR23 abolished the ability of the viruses to replicate in vitro. As shown in Figure 4, Q148H delayed replication of NL4-3, but the mutant achieved similar peak p24 levels as the WT virus within 15 days post-infection (Figure 4a). By contrast, unlike the WT viruses, which achieved p24 levels, neither ARMA159 nor URTR23 with Q148H alone demonstrated increasing p24 levels, indicating the absence of viral replication (Figure 4b and c). Addition of G140S to the URTR23 Q148H mutant partially rescued viral replication and increased EC₅₀s to raltegravir and dolutegravir that are similar to those observed for NL4-3 G140S/Q148H double mutant. However, viral replication kinetics were delayed and the levels of p24 failed to reach those of the WT virus during the time frame analysed. ARMA159 was also only partially rescued by the G140S/Q148H double mutant.

Figure 4. — Replication dynamics of HIV-1 (a) NL4-3(B); (b) URTR23(BF); and (c) ARMA159(BF) WT, Q148H and Q148H+G140S mutant viruses. *P < 0.05; **P < 0.01; and ****P < 0.0001 (two-way ANOVA using Tukey’s correction). This figure appears in colour in the online version of *JAC* and in black and white in the print version of *JAC*.

As integration is vital for HIV-1 replication, we assessed the level of integrated DNA for Q148H mutants relative to the WT viruses. Figure 5 shows that integration was reduced by 6-fold for NL4-3-Q148H (P = 0.05), 9-fold for URTR23-Q148H (P = 0.01) and 16 000-fold for ARMA159-Q148H (P = 0.01) compared with their respective WT viruses.

IN CCD chimeric HIV-1 mutants fail to rescue the severe loss of replication capacity of Q148H subtype F mutants

To further explore the role of inter-subtype genetic diversity in the dramatic loss of replication capacity observed for Q148H subtype F mutants, we constructed four chimeric HIV-1 viruses. Using the parental mutant IMCs NL4-3, ARMA159 and URTR23, we exchanged a fragment spanning amino acids 85–150 of the in CCD between viruses of different subtypes. This region contains 7 of the 9 subtype-specific polymorphisms common to BF recombinants. The Q148H mutation was included in all chimeras. NL(ARMA) and NL(URTR) had a NL4-3 backbone and ARMA159 or URTR23 in CCD fragment, while the reverse was constructed for ARMA(NL) and URTR(NL). In vitro HIV-1 replication experiments of the chimeras and Q148H parental viruses showed that all chimeric viruses with NL4-3 IN NTD and CTD replicated at similar levels as the Q148H parental NL4-3 virus, whereas chimeric viruses with ARMA159 or URTR23 IN NTD and CTD continued to show a dramatic loss of viral replicative capacity (Figure 6). These results show that subtype-specific differences in the CCD do not play a role in the loss of replication capacity only observed for Q148H mutants in the context of subtype F INs and suggest that this effect is associated with polymorphisms located external to it.

Figure 6. — Replication dynamics of HIV-1 chimeric and parental NL4-3(B), URTR23(BF) and ARMA159(BF) Q148H mutant viruses. Chimeric viruses with NL4-3 backbone and F(BF) CCD region are named NL(URTR) and NL(ARMA). Reciprocally, chimeric viruses with F(BF) backbones and NL4-3 CCD region are named URTR(NL) and ARMA(NL). HIV-1 p24 was measured by ELISA at 0, 5, 8, 11 and 14 days post-infection. ****P < 0.0001 (two-way ANOVA using Tukey’s correction); ns, statistically not significant. This figure appears in colour in the online version of *JAC* and in black and white in the print version of *JAC*.

Discussion

Amino acid polymorphisms typical of F1 HIV-1 INs from BF recombinants did not affect in vitro susceptibility to INSTIs significantly. However, introduction of Q148H in subtype F1 INs resulted in a complete inhibition of viral replication, providing a plausible explanation for its lack of selection in vivo. The effect of Q148H in F1 INs was independent of polymorphisms in the CCD, consistent with a subtype-specific disruption of one or more of the pleiotropic activities of IN during the viral life cycle.

Resistance to INSTIs is generally associated with mutations at one of three IN codons: Y143, Q148, or N155. While mutations at positions 143 and 155 confer moderate- to high-level resistance to first-generation INSTIs only, the Q148 pathway is associated with at least 3-fold resistance to dolutegravir.²⁹ In vitro, raltegravir, elvitegravir and cabotegravir have shown to select for Q148R or Q148K,³⁰ whereas Q148H is more frequently observed in raltegravir-exposed individuals undergoing virological failure.³¹ In most cases, a secondary mutation, G140S/A/C, accompanies Q148 mutations in the same viral genomes,³² with Q148H + G140S being the most frequent combination found in vivo. G140S contributes both to resistance to INSTIs and to recovery of viral replicative capacity of 148 single mutants.^32,33 Importantly these mutations are very rarely found in non-B subtypes,⁸ suggesting a higher susceptibility of non-B HIV strains to INSTIs, even after treatment with non-suppressive raltegravir regimens. By comparing the replication capacity of IN Q148H mutants of subtypes B and F1 from BF recombinants, we showed that the loss of function due to Q148H is more drastic for subtype F1 INs. Viruses with this subtype F1 IN mutation failed to replicate and would therefore be less likely to select for compensatory mutations. Thus, in addition to the high genetic barrier to G140S mutation for non-B subtypes,⁸ the severe loss of viral replication fitness that results from Q148H may be a primary reason for absence of its selection in subtype F1 INs during INSTI treatment failures. Furthermore, we demonstrated a reduction in integration for both viruses encoding F1 IN Q148H. However, proviral integration was not abolished and therefore the reduction in integration activity does not fully explain the drastic loss of viral replication capacity.

The epidemiological importance of HIV F is linked to its increasing frequency as part of a variety of recombinant forms circulating in South America. Since the identification of CRF12_BF in the early 1990s as the first autochthonous BF recombinant strain circulating in Argentina and Uruguay,²¹ already 17 other CRF_BF recombinants have been described in Latin American countries, representing 19% of the total number of CRFs. Besides CRF12_BF, an HIV F in gene can be found in 6 of them: CRF17_BF from Argentina, CRF29_BF from Brazil, CRF38_BF from Uruguay, CRF44_BF and CRF46_BF from Chile, and CRF89_BF from Bolivia. Also, HIV F in can be found as part of CRF81cpx, a newly identified B/C/F recombinant circulating in Brazil. In previous studies from our group, we showed that South American subtype F1 env sequences present either in pure or recombinant genomes share a common ancestor, responsible for initiating the F1 epidemic in South America during late 1970s.³⁴ Therefore, restricting our study to F1 INs from BF recombinants was important for inter-subtype comparison of polymorphisms, and allowed us to show for the first known time a difference in HIV replication capacity caused by mutations associated with high-level resistance to INSTIs between the prototypic HIV B and a non-B subtype typical of South America.

At least 18 amino acid positions in the CCD and CTD regions of IN were found to differ significantly between subtypes B strains, and F1 or BF recombinants circulating in Latin America. Among them, G163K/R are secondary mutations that confer low-level DR to INSTIs when present with major mutations in HIV B strains. Despite being a common polymorphism for BF recombinants, G163R has been observed to emerge during virological failure to INSTIs in children from our cohort (P. C. Aulicino, unpublished data), as well as in other non-B subtypes,^30,35 suggesting a possible role of this mutation in DR. However, the G163R polymorphism did not have a significant effect on in vitro susceptibility to raltegravir or dolutegravir either alone or in combination with the major resistance mutation N155H in a BF recombinant genetic background.

A number of polymorphisms were found at high frequency in BF recombinants, F1 strains and also in subtypes A, C and AG (L101I, T124A/N/S, T125A/V, K136Q/T and V201I). We hypothesized that one or more non-B polymorphisms were responsible for the phenotypic effects of Q148H mutants in the context of F1 INs, in agreement with previous observations in subtypes A1/A6^36,37 and subtype C.³⁸ Our studies of chimeric INs with exchanges of CCD coding sequences (i.e. codons 84–150) between NL4-3 and ARMA159 or URTR23 showed that polymorphisms in the central part of the CCD (namely L101I, V113I, S119P/T, T122S/I, T124A, T125A/M and K136Q) do not contribute to the loss of viral replication capacity of subtype F1 Q148H mutant IN-encoding viruses. Rather, polymorphisms C-terminal to this CCD segment restrict subtype F1 IN function when Q148H is present. One possible explanation is that Q148H may also significantly affect IN function in other parts of the viral life cycle, and that this effect is more pronounced in context of subtype F1 IN polymorphisms. Potentially, these polymorphisms may behave like class II IN mutations and disrupt virion assembly, morphogenesis, or reverse transcription.^39–42 Alternatively, the interaction with LEDGF may be partially compromised. Further studies will be necessary to elucidate the specific amino acids and possible defects in function that arise in association with Q148H for subtype F1 INs.

A shortcoming of our studies is that we only examined the effect of Q148H in two subtype F1 in genes. However, we observed a consistent severe disruption of viral replication capacity for viruses encoding either IN variant and a role for the genetic background outside the CCD region of resistant INs that is yet to be unveiled. Whether HIV subtype or specific polymorphisms in IN commonly affect the resistance pathways for INSTIs will be important to further investigate. Additional studies using a larger number of viruses of different non-B subtypes should be conducted to validate our findings.

This study provides an explanation for the lack of selection of Q148H pathway in individuals failing a raltegravir-based ART and carrying BF recombinant strains in Argentina, and is a proof-of-concept demonstrating that HIV-1 subtype in in gene affects the resistance pathways for INSTIs. The evidence presented adds to the equal or higher potency of dolutegravir, bictegravir and cabotegravir on non-B HIV subtypes⁴³ and further supports their use in low- and middle-income countries where non-B subtypes predominate and sheds light on molecular impairments associated with the genomic background of HIV IN that can be exploited for future drug developments targeting this important step of the viral life cycle. Our results reinforce the importance of characterizing HIV subtype as well as DRMs during standard HIV genotyping.

Supplementary Material

dkac238_Supplementary_Data

Click here for additional data file.^{(36.7KB, docx)}

Acknowledgements

The following reagents were obtained through the NIH HIV reagent program, Division of AIDS, NIAID, NIH: Human Immunodeficiency Virus 1 (HIV-1), Strain NL4-3 Infectious Molecular Clone (pNL4-3), ARP-2852, contributed by Dr M. Martin; pNL4-3.Luc.R-E-, ARP-3418, was contributed by Nathaniel Landau; TZM-bl Cells, ARP-8129, contributed by Dr J. C. Kappes and Dr X. Wu; ACH-2 cells, ARP-349, contributed by Thomas Folks; and Jurkat E6-1, ARP-177, contributed by Arthur Weiss (via the ATCC). P.C.A., M.R. and A.M. gratefully thank Mrs Silvina Juarez and Miss Romina Guelho for technical assistance.

Contributor Information

Paula C Aulicino, Laboratory of Cellular Biology and Retroviruses, Unit of Virology and Molecular Epidemiology, Hospital de Pediatría “Prof. Dr. Juan P. Garrahan”, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.

Zoha Momin, Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX, USA.

Mijael Rozenszajn, Laboratory of Cellular Biology and Retroviruses, Unit of Virology and Molecular Epidemiology, Hospital de Pediatría “Prof. Dr. Juan P. Garrahan”, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.

Arturo Monzon, Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX, USA.

Solange Arazi-Caillaud, Unit of Epidemiology and Infectology, Hospital de Pediatría “Prof. Dr. Juan P. Garrahan”, Buenos Aires, Argentina.

Rosa Bologna, Unit of Epidemiology and Infectology, Hospital de Pediatría “Prof. Dr. Juan P. Garrahan”, Buenos Aires, Argentina.

Andrea Mangano, Laboratory of Cellular Biology and Retroviruses, Unit of Virology and Molecular Epidemiology, Hospital de Pediatría “Prof. Dr. Juan P. Garrahan”, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.

Jason T Kimata, Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX, USA.

Funding

This work was in part supported by a Fulbright-CONICET Scholar Award to P.C.A; AI116167 to J.T.K.; and the Basic Science Core of the Texas D-CFAR (AI161943).

Transparency declarations

None to declare.

Supplementary data

Figure S1 is available as Supplementary data at JAC Online.

References

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2. Castagna A, Maggiolo F, Penco G et al. Dolutegravir in antiretroviral-experienced patients with raltegravir- and/or elvitegravir-resistant HIV-1: 24-week results of the phase III VIKING-3 study. J Infect Dis 2014; 210: 354–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
3. Nguyen HL, Charpentier C, Nguyen N et al. Longitudinal analysis of integrase N155H variants in heavily treated patients failing raltegravir-based regimens. HIV Med 2013; 14: 85–91. [DOI] [PubMed] [Google Scholar]
4. Malet I, Delelis O, Soulie C et al. Quasispecies variant dynamics during emergence of resistance to raltegravir in HIV-1-infected patients. J Antimicrob Chemother 2009; 63: 795–804. [DOI] [PubMed] [Google Scholar]
5. Fransen S, Gupta S, Danovich R et al. Loss of raltegravir susceptibility by human immunodeficiency virus type 1 is conferred via multiple nonoverlapping genetic pathways. J Virol 2009; 83: 11440–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Delelis O, Malet I, Li N et al. The G140S mutation in HIV integrases from raltegravir-resistant patients rescues catalytic defect due to the resistance Q148H mutation. Nucleic Acids Res 2009; 37: 1193–201. [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Cheung PK, Shahid A, Dong W et al. Impact of combinations of clinically observed HIV integrase mutations on phenotypic resistance to integrase strand transfer inhibitors (INSTIs): a molecular study. J Antimicrob Chemother 2022; 77: 979–88. [DOI] [PubMed] [Google Scholar]
8. Doyle T, Dunn DT, Ceccherini-Silberstein F et al. Integrase inhibitor (INI) genotypic resistance in treatment-naive and raltegravir-experienced patients infected with diverse HIV-1 clades. J Antimicrob Chemother 2015; 70: 3080–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Sánchez D, Caillaud SA, Zapiola I et al. Impact of genotypic diversity on selection of subtype-specific drug resistance profiles during raltegravir-based therapy in individuals infected with B and BF recombinant HIV-1 strains. J Antimicrob Chemother 2020; 75: 1567–74. [DOI] [PubMed] [Google Scholar]
10. Machado LA, Gomes MF da C, Guimarães ACR. Raltegravir-induced adaptations of the HIV-1 integrase: Analysis of structure, variability, and mutation Co-occurrence. Front Microbiol 2019; 10: 1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Hachiya A, Ode H, Matsuda M et al. Natural polymorphism S119R of HIV-1 integrase enhances primary INSTI resistance. Antiviral Res 2015; 119: 84–8. [DOI] [PubMed] [Google Scholar]
12. Mbhele N, Gordon M. Structural effects of HIV-1 subtype C integrase mutations on the activity of integrase strand transfer inhibitors in South African patients. J Biomol Struct Dyn 2021: 1–11. [DOI] [PubMed] [Google Scholar]
13. Vacic V, Iakoucheva LM, Radivojac P. Two Sample Logo: a graphical representation of the differences between two sets of sequence alignments. Bioinformatics 2006; 22: 1536–7. [DOI] [PubMed] [Google Scholar]
14. Folks TM, Clouse KA, Justement J et al. Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc Natl Acad Sci USA 1989; 86: 2365–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Clouse K, Powell D, Washington I et al. Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J Immunol 1989; 142: 431–8. [PubMed] [Google Scholar]
16. Weiss A, Wiskocil R, Stobo JD. The role of T3 surface molecules in the activation of human T cells: a two-stimulus requirement for IL 2 production reflects events occurring at a pre-translational level. J Immunol 1984; 133: 123–8. [PubMed] [Google Scholar]
17. Derdeyn CA, Decker JM, Sfakianos JN et al. Sensitivity of Human Immunodeficiency Virus Type 1 to the Fusion Inhibitor T-20 Is Modulated by Coreceptor Specificity Defined by the V3 Loop of gp120. J Virol 2000; 74: 8358–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Wei X, Decker JM, Liu H et al. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 2002; 46: 1896–905. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Adachi A, Gendelman HE, Koenig S et al. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 1986; 59: 284–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Carr JK, Avila M, Gomez Carrillo M et al. Diverse BF recombinants have spread widely since the introduction of HIV-1 into South America. AIDS 2001; 15: F41–7. [DOI] [PubMed] [Google Scholar]
21. Quarleri JF, Rubio A, Carobene M et al. HIV type 1 BF recombinant strains exhibit different pol gene mosaic patterns: descriptive analysis from 284 patients under treatment failure. AIDS Res Hum Retroviruses 2004; 20: 1100–7. [DOI] [PubMed] [Google Scholar]
22. Gibbs JS, Regier DA, Desrosiers RC. Construction and in vitro properties of HIV-1 mutants with deletions in ‘nonessential’ genes. AIDS Res Hum Retroviruses 1994; 10: 343–50. [DOI] [PubMed] [Google Scholar]
23. Misra A, Gleeson E, Wang W et al. Glycosyl-phosphatidylinositol-anchored anti-HIV Env single-chain variable fragments interfere with HIV-1 Env processing and viral infectivity. J Virol 2018; 92: e02080-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Xia Y, Chu W, Qi Q et al. New insights into the QuikChangeTM process guide the use of Phusion DNA polymerase for site-directed mutagenesis. Nucleic Acids Res 2015; 43: e12. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Thippeshappa R, Polacino P, Chandrasekar SS et al. In vivo serial passaging of human-simian immunodeficiency virus clones identifies characteristics for persistent viral replication. Front Microbiol 2021; 12: 779460. [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Yu JJ, Wu TL, Liszewski MK et al. A more precise HIV integration assay designed to detect small differences finds lower levels of integrated DNA in HAART treated patients. Virology 2008; 379: 78–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Connor RI, Chen BK, Choe S et al. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 1995; 206: 935–44. [DOI] [PubMed] [Google Scholar]
28. He J, Choe S, Walker R et al. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol 1995; 69: 6705–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Kobayashi M, Nakahara K, Seki T et al. Selection of diverse and clinically relevant integrase inhibitor-resistant human immunodeficiency virus type 1 mutants. Antiviral Res 2008; 80: 213–22. [DOI] [PubMed] [Google Scholar]
30. Oliveira M, Ibanescu RI, Anstett K et al. Selective resistance profiles emerging in patient-derived clinical isolates with cabotegravir, bictegravir, dolutegravir, and elvitegravir. Retrovirology 2018; 15: 56. [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Cooper DA, Steigbigel RT, Gatell JM et al. Subgroup and resistance analyses of raltegravir for resistant HIV-1 infection. N Engl J Med 2008; 359: 355–65. [DOI] [PubMed] [Google Scholar]
32. Fransen S, Gupta S, Frantzell A et al. Substitutions at amino acid positions 143, 148, and 155 of HIV-1 integrase define distinct genetic barriers to raltegravir resistance in vivo. J Virol 2012; 86: 7249–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Hu Z, Kuritzkes DR. Altered viral fitness and drug susceptibility in HIV-1 carrying mutations that confer resistance to nonnucleoside reverse transcriptase and integrase strand transfer inhibitors. J Virol 2014; 88: 9268–76. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Aulicino PC, Bello G, Rocco C et al. Description of the first full-length HIV type 1 subtype F1 strain in Argentina: implications for the origin and dispersion of this subtype in South America. AIDS Res Hum Retroviruses 2007; 23: 1176–82. [DOI] [PubMed] [Google Scholar]
35. Ndashimye E, Li Y, Reyes PS et al. High-level resistance to bictegravir and cabotegravir in subtype A- and D-infected HIV-1 patients failing raltegravir with multiple resistance mutations. J Antimicrob Chemother 2021; 76: 2965–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Jeffrey JL, Clair M, Wang P et al. Impact of integrase sequences from HIV-1 subtypes A6/A1 on the In Vitro potency of cabotegravir or rilpivirine. Antimicrob Agents Chemother 2022; 66: e0170221. [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Charpentier C, Storto A, Soulié C et al. Prevalence of genotypic baseline risk factors for cabotegravir + rilpivirine failure among ARV-naive patients. J Antimicrob Chemother 2021; 76: 2983–7. [DOI] [PubMed] [Google Scholar]
38. Arimide DA, Szojka ZI, Zealiyas K et al. Pre-treatment integrase inhibitor resistance and natural polymorphisms among HIV-1 subtype C infected patients in Ethiopia. Viruses 2022; 14: 729. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Engelman A, Englund G, Orenstein JM et al. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J Virol 1995; 69: 2729–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Elliott J, Eschbach JE, Koneru PC et al. Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis. Elife 2020; 9: 1–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Elliott JL, Kutluay SB. Going beyond integration: The emerging role of HIV-1 integrase in virion morphogenesis. Viruses 2020; 12: 1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Bukovsky A, Göttlinger H. Lack of integrase can markedly affect human immunodeficiency virus type 1 particle production in the presence of an active viral protease. J Virol 1996; 70: 6820–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Neogi U, Singh K, Aralaguppe SG et al. Ex-vivo antiretroviral potency of newer integrase strand transfer inhibitors cabotegravir and bictegravir in HIV type 1 non-B subtypes. AIDS 2018; 32: 469–76. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

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[dkac238-B1] 1. Kobayashi M, Yoshinaga T, Seki T et al. In Vitro antiretroviral properties of S/GSK1349572, a next-generation HIV integrase inhibitor. Antimicrob Agents Chemother 2011; 55: 813–21. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B2] 2. Castagna A, Maggiolo F, Penco G et al. Dolutegravir in antiretroviral-experienced patients with raltegravir- and/or elvitegravir-resistant HIV-1: 24-week results of the phase III VIKING-3 study. J Infect Dis 2014; 210: 354–62. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B3] 3. Nguyen HL, Charpentier C, Nguyen N et al. Longitudinal analysis of integrase N155H variants in heavily treated patients failing raltegravir-based regimens. HIV Med 2013; 14: 85–91. [DOI] [PubMed] [Google Scholar]

[dkac238-B4] 4. Malet I, Delelis O, Soulie C et al. Quasispecies variant dynamics during emergence of resistance to raltegravir in HIV-1-infected patients. J Antimicrob Chemother 2009; 63: 795–804. [DOI] [PubMed] [Google Scholar]

[dkac238-B5] 5. Fransen S, Gupta S, Danovich R et al. Loss of raltegravir susceptibility by human immunodeficiency virus type 1 is conferred via multiple nonoverlapping genetic pathways. J Virol 2009; 83: 11440–6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B6] 6. Delelis O, Malet I, Li N et al. The G140S mutation in HIV integrases from raltegravir-resistant patients rescues catalytic defect due to the resistance Q148H mutation. Nucleic Acids Res 2009; 37: 1193–201. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B7] 7. Cheung PK, Shahid A, Dong W et al. Impact of combinations of clinically observed HIV integrase mutations on phenotypic resistance to integrase strand transfer inhibitors (INSTIs): a molecular study. J Antimicrob Chemother 2022; 77: 979–88. [DOI] [PubMed] [Google Scholar]

[dkac238-B8] 8. Doyle T, Dunn DT, Ceccherini-Silberstein F et al. Integrase inhibitor (INI) genotypic resistance in treatment-naive and raltegravir-experienced patients infected with diverse HIV-1 clades. J Antimicrob Chemother 2015; 70: 3080–6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B9] 9. Sánchez D, Caillaud SA, Zapiola I et al. Impact of genotypic diversity on selection of subtype-specific drug resistance profiles during raltegravir-based therapy in individuals infected with B and BF recombinant HIV-1 strains. J Antimicrob Chemother 2020; 75: 1567–74. [DOI] [PubMed] [Google Scholar]

[dkac238-B10] 10. Machado LA, Gomes MF da C, Guimarães ACR. Raltegravir-induced adaptations of the HIV-1 integrase: Analysis of structure, variability, and mutation Co-occurrence. Front Microbiol 2019; 10: 1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B11] 11. Hachiya A, Ode H, Matsuda M et al. Natural polymorphism S119R of HIV-1 integrase enhances primary INSTI resistance. Antiviral Res 2015; 119: 84–8. [DOI] [PubMed] [Google Scholar]

[dkac238-B12] 12. Mbhele N, Gordon M. Structural effects of HIV-1 subtype C integrase mutations on the activity of integrase strand transfer inhibitors in South African patients. J Biomol Struct Dyn 2021: 1–11. [DOI] [PubMed] [Google Scholar]

[dkac238-B13] 13. Vacic V, Iakoucheva LM, Radivojac P. Two Sample Logo: a graphical representation of the differences between two sets of sequence alignments. Bioinformatics 2006; 22: 1536–7. [DOI] [PubMed] [Google Scholar]

[dkac238-B14] 14. Folks TM, Clouse KA, Justement J et al. Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc Natl Acad Sci USA 1989; 86: 2365–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B15] 15. Clouse K, Powell D, Washington I et al. Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J Immunol 1989; 142: 431–8. [PubMed] [Google Scholar]

[dkac238-B16] 16. Weiss A, Wiskocil R, Stobo JD. The role of T3 surface molecules in the activation of human T cells: a two-stimulus requirement for IL 2 production reflects events occurring at a pre-translational level. J Immunol 1984; 133: 123–8. [PubMed] [Google Scholar]

[dkac238-B17] 17. Derdeyn CA, Decker JM, Sfakianos JN et al. Sensitivity of Human Immunodeficiency Virus Type 1 to the Fusion Inhibitor T-20 Is Modulated by Coreceptor Specificity Defined by the V3 Loop of gp120. J Virol 2000; 74: 8358–67. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B18] 18. Wei X, Decker JM, Liu H et al. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 2002; 46: 1896–905. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B19] 19. Adachi A, Gendelman HE, Koenig S et al. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 1986; 59: 284–91. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B20] 20. Carr JK, Avila M, Gomez Carrillo M et al. Diverse BF recombinants have spread widely since the introduction of HIV-1 into South America. AIDS 2001; 15: F41–7. [DOI] [PubMed] [Google Scholar]

[dkac238-B21] 21. Quarleri JF, Rubio A, Carobene M et al. HIV type 1 BF recombinant strains exhibit different pol gene mosaic patterns: descriptive analysis from 284 patients under treatment failure. AIDS Res Hum Retroviruses 2004; 20: 1100–7. [DOI] [PubMed] [Google Scholar]

[dkac238-B22] 22. Gibbs JS, Regier DA, Desrosiers RC. Construction and in vitro properties of HIV-1 mutants with deletions in ‘nonessential’ genes. AIDS Res Hum Retroviruses 1994; 10: 343–50. [DOI] [PubMed] [Google Scholar]

[dkac238-B23] 23. Misra A, Gleeson E, Wang W et al. Glycosyl-phosphatidylinositol-anchored anti-HIV Env single-chain variable fragments interfere with HIV-1 Env processing and viral infectivity. J Virol 2018; 92: e02080-17. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B24] 24. Xia Y, Chu W, Qi Q et al. New insights into the QuikChangeTM process guide the use of Phusion DNA polymerase for site-directed mutagenesis. Nucleic Acids Res 2015; 43: e12. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B25] 25. Thippeshappa R, Polacino P, Chandrasekar SS et al. In vivo serial passaging of human-simian immunodeficiency virus clones identifies characteristics for persistent viral replication. Front Microbiol 2021; 12: 779460. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B26] 26. Yu JJ, Wu TL, Liszewski MK et al. A more precise HIV integration assay designed to detect small differences finds lower levels of integrated DNA in HAART treated patients. Virology 2008; 379: 78–86. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B27] 27. Connor RI, Chen BK, Choe S et al. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 1995; 206: 935–44. [DOI] [PubMed] [Google Scholar]

[dkac238-B28] 28. He J, Choe S, Walker R et al. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol 1995; 69: 6705–11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B29] 29. Kobayashi M, Nakahara K, Seki T et al. Selection of diverse and clinically relevant integrase inhibitor-resistant human immunodeficiency virus type 1 mutants. Antiviral Res 2008; 80: 213–22. [DOI] [PubMed] [Google Scholar]

[dkac238-B30] 30. Oliveira M, Ibanescu RI, Anstett K et al. Selective resistance profiles emerging in patient-derived clinical isolates with cabotegravir, bictegravir, dolutegravir, and elvitegravir. Retrovirology 2018; 15: 56. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B31] 31. Cooper DA, Steigbigel RT, Gatell JM et al. Subgroup and resistance analyses of raltegravir for resistant HIV-1 infection. N Engl J Med 2008; 359: 355–65. [DOI] [PubMed] [Google Scholar]

[dkac238-B32] 32. Fransen S, Gupta S, Frantzell A et al. Substitutions at amino acid positions 143, 148, and 155 of HIV-1 integrase define distinct genetic barriers to raltegravir resistance in vivo. J Virol 2012; 86: 7249–55. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B33] 33. Hu Z, Kuritzkes DR. Altered viral fitness and drug susceptibility in HIV-1 carrying mutations that confer resistance to nonnucleoside reverse transcriptase and integrase strand transfer inhibitors. J Virol 2014; 88: 9268–76. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B34] 34. Aulicino PC, Bello G, Rocco C et al. Description of the first full-length HIV type 1 subtype F1 strain in Argentina: implications for the origin and dispersion of this subtype in South America. AIDS Res Hum Retroviruses 2007; 23: 1176–82. [DOI] [PubMed] [Google Scholar]

[dkac238-B35] 35. Ndashimye E, Li Y, Reyes PS et al. High-level resistance to bictegravir and cabotegravir in subtype A- and D-infected HIV-1 patients failing raltegravir with multiple resistance mutations. J Antimicrob Chemother 2021; 76: 2965–74. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B36] 36. Jeffrey JL, Clair M, Wang P et al. Impact of integrase sequences from HIV-1 subtypes A6/A1 on the In Vitro potency of cabotegravir or rilpivirine. Antimicrob Agents Chemother 2022; 66: e0170221. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B37] 37. Charpentier C, Storto A, Soulié C et al. Prevalence of genotypic baseline risk factors for cabotegravir + rilpivirine failure among ARV-naive patients. J Antimicrob Chemother 2021; 76: 2983–7. [DOI] [PubMed] [Google Scholar]

[dkac238-B38] 38. Arimide DA, Szojka ZI, Zealiyas K et al. Pre-treatment integrase inhibitor resistance and natural polymorphisms among HIV-1 subtype C infected patients in Ethiopia. Viruses 2022; 14: 729. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B39] 39. Engelman A, Englund G, Orenstein JM et al. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J Virol 1995; 69: 2729–36. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B40] 40. Elliott J, Eschbach JE, Koneru PC et al. Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis. Elife 2020; 9: 1–56. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B41] 41. Elliott JL, Kutluay SB. Going beyond integration: The emerging role of HIV-1 integrase in virion morphogenesis. Viruses 2020; 12: 1005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B42] 42. Bukovsky A, Göttlinger H. Lack of integrase can markedly affect human immunodeficiency virus type 1 particle production in the presence of an active viral protease. J Virol 1996; 70: 6820–5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dkac238-B43] 43. Neogi U, Singh K, Aralaguppe SG et al. Ex-vivo antiretroviral potency of newer integrase strand transfer inhibitors cabotegravir and bictegravir in HIV type 1 non-B subtypes. AIDS 2018; 32: 469–76. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

HIV-1 subtype F integrase polymorphisms external to the catalytic core domain contribute to severe loss of replication capacity in context of the integrase inhibitor resistance mutation Q148H

Paula C Aulicino

Zoha Momin

Mijael Rozenszajn

Arturo Monzon

Solange Arazi-Caillaud

Rosa Bologna

Andrea Mangano

Jason T Kimata

Abstract

Background

Methods

Results

Conclusions

Introduction

Materials and methods

HIV-1 IN sequences

Cell lines

Generation of infectious HIV-1 clones with subtype F in genes

in mutagenesis

INSTI susceptibility assay

Integration assay

Results

Genotypic differences between HIV subtype B and Latin American F1 IN sequences are distributed along the CCD and NTD regions

Figure 1.

Polymorphisms in CCD are common between BF recombinants and non-B group M subtypes

Figure 2.

Q148H disrupts replication of HIV-1 with subtype F INs and reduces integration

Figure 3.

Table 1.

Figure 4.

Figure 5.

IN CCD chimeric HIV-1 mutants fail to rescue the severe loss of replication capacity of Q148H subtype F mutants

Figure 6.

Discussion

Supplementary Material

Acknowledgements

Contributor Information

Funding

Transparency declarations

Supplementary data

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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