Polypurine Tract Research Articles

Post-transcriptional reprogramming by thousands of mRNA untranslated regions in trypanosomes

Although genome-wide polycistronic transcription places major emphasis on post-transcriptional controls in trypanosomatids, messenger RNA cis-regulatory untranslated regions (UTRs) have remained largely uncharacterised. Here, we describe a genome-scale massive parallel reporter assay coupled with 3’-UTR-seq profiling in the African trypanosome and identify thousands of regulatory UTRs. Increased translation efficiency was associated with dosage of adenine-rich poly-purine tracts (pPuTs). An independent assessment of native UTRs using machine learning based predictions confirmed the robust correspondence between pPuTs and positive control, as did an assessment of synthetic UTRs. Those 3’-UTRs associated with upregulated expression in bloodstream-stage cells were also enriched in uracil-rich poly-pyrimidine tracts, suggesting a mechanism for developmental activation through pPuT ‘unmasking’. Thus, we describe a cis-regulatory UTR sequence ‘code’ that underpins gene expression control in the context of a constitutively transcribed genome. We conclude that thousands of UTRs post-transcriptionally reprogram gene expression profiles in trypanosomes.

Characterizing HIV drug resistance in cases of vertical transmission in the VESTED randomized antiretroviral treatment trial.

VESTED (NCT03048422) compared the safety and efficacy of three antiretroviral treatment (ART) regimens in pregnant and postpartum women: dolutegravir+emtricitabine/tenofovir alafenamide fumarate; dolutegravir+emtricitabine/tenofovir disoproxil fumarate (TDF); efavirenz/emtricitabine/TDF. Vertical HIV transmission (VT) occurred to 4/617 (0.60%) live-born infants, who were evaluated for HIV drug resistance (HIVDR) and other risk factors. In 2018-2020, pregnant (weeks-14-28) women living with HIV and ≤14 days of ART were enrolled at 22 international sites and followed with their infants through 50 weeks postpartum. HIV sequences derived by single genome amplification (SGA) from longitudinally collected specimens were assessed from VT Cases for HIVDR in protease, reverse transcriptase, integrase, and the nef 3'polypurine tract (3'PPT). The four Case mothers were prescribed efavirenz-based-ART for 1-7 days prior to randomization to study ART. Their infants received postnatal nevirapine+/-zidovudine prophylaxis and were breastfed. A total of 833 SGA sequences were derived. The "major" (Stanford HIVDR Score ≥60) non-nucleoside reverse transcriptase inhibitor (NNRTI) mutation (K103N) was detected persistently in one viremic mother, and likely contributed to VT of HIVDR. Major NNRTI HIVDR mutations were detected in all three surviving infants. No integrase, nor high frequencies of 3'PPT mutations conferring dolutegravir HIVDR were detected. The timing of HIV infant diagnosis, plasma HIV RNA levels and HIVDR suggests one in utero, one peripartum, one early, and one late breastfeeding transmission. VT was rare. New-onset NNRTI HIVDR in Case mothers was likely from efavirenz-ART prescribed prior to study dolutegravir-ART, and in one case appeared transmitted to the infant despite nevirapine prophylaxis.

Comprehensive Assessment of Force-Field Performance in Molecular Dynamics Simulations of DNA/RNA Hybrid Duplexes.

Mixed double helices formed by RNA and DNA strands, commonly referred to as hybrid duplexes or hybrids, are essential in biological processes like transcription and reverse transcription. They are also important for their applications in CRISPR gene editing and nanotechnology. Yet, despite their significance, the hybrid duplexes have been seldom modeled by atomistic molecular dynamics methodology, and there is no benchmark study systematically assessing the force-field performance. Here, we present an extensive benchmark study of polypurine tract (PPT) and Dickerson-Drew dodecamer hybrid duplexes using contemporary and commonly utilized pairwise additive and polarizable nucleic acid force fields. Our findings indicate that none of the available force-field choices accurately reproduces all the characteristic structural details of the hybrid duplexes. The AMBER force fields are unable to populate the C3'-endo (north) pucker of the DNA strand and underestimate inclination. The CHARMM force field accurately describes the C3'-endo pucker and inclination but shows base pair instability. The polarizable force fields struggle with accurately reproducing the helical parameters. Some force-field combinations even demonstrate a discernible conflict between the RNA and DNA parameters. In this work, we offer a candid assessment of the force-field performance for mixed DNA/RNA duplexes. We provide guidance on selecting utilizable force-field combinations and also highlight potential pitfalls and best practices for obtaining optimal performance.

Potent anti-coronaviral activity of pateamines and new insights into their mode of action

Pateamines, derived from the sponge Mycale hentscheli, function as inhibitors of the RNA helicase eIF4A and exhibit promising antiviral and anticancer properties. eIF4A plays a pivotal role in unwinding stable RNA structures within the 5′-UTR of selected mRNAs, facilitating the binding of the 43S preinitiation complex during translation initiation. Pateamines function by clamping RNA substrates onto the eIF4A surface, effectively preventing eIF4A from carrying out the unwinding step. Rocaglates, a compound class isolated from plants of the genus Aglaia, target the same binding pocket on eIF4A, and based on structural data, a similar mode of action has been proposed for pateamines and rocaglates. In this study, we conducted a detailed characterization of pateamines' binding mode and assessed their antiviral activity against human pathogenic coronaviruses (human coronavirus 229E (HCoV-229E), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)). Our findings reveal significant differences in the binding behavior of pateamines compared to rocaglates when interacting with an eIF4A-RNA complex. We also observed that pateamines do not depend on the presence of a polypurine tract in the RNA substrate for efficient RNA clamping, as it is the case for rocaglates. Most notably, pateamines demonstrate potent antiviral activity against coronaviruses in the low nanomolar range. Consequently, pateamines broaden our toolbox for combating viruses that rely on the host enzyme eIF4A to conduct their viral protein synthesis, indicating a possible future treatment strategy against new or re-emerging pathogenic viruses.

Abstract IA021: Novel genomic features of POLE-mutant tumors: the utility for tumor classification and identification of new driver alleles

Abstract Alterations in the exonuclease domain of POLE drive the development of ultramutated endometrial tumors with better prognosis. A hallmark of these tumors is the accumulation of G>T mutations in AGA sequences; however, understanding of their genomic features beyond the trinucleotide motifs is limited. We applied a novel computational framework to the whole-exome sequencing data of 524 endometrial tumors reported by The Cancer Genome Atlas network to analyze the extended DNA sequence context of ultramutation. We found that the presence of POLE driver alleles is associated with an increased frequency of G>T mutations in polypurine tracts. Sequences containing three consecutive purines, including the classic AGA context, are only moderately mutable, but the mutability increased abruptly as the tract length reached six or more purines. Moreover, mutations showed a strong preference for certain positions within the tracts, creating a characteristic quantifiable pattern. Using this signature, we developed a machine learning classifier to identify tumors with hitherto unknown POLE drivers and validated two new drivers, POLE-E978G and POLE-S461L, by functional assays in yeast. Unlike previously known pathogenic variants, the E978G substitution affects the DNA polymerase rather than exonuclease domain of POLE. We further show that the extended genomic signature of POLE ultramutation is shared by tumors with POLD1 drivers. These findings uncover new mechanisms of ultramutation and highlight the limitations of the current focus on POLE exonuclease domain variants when stratifying patients. Citation Format: Daria Ostroverkhova, Kathryn Tyryshkin, Annette K. Beach, Elizabeth A. Moore, Yosef Masoudi-Sobhanzadeh, Stephanie R. Barbari, Igor B Rogozin, Konstantin V Shaitan, Anna R Panchenko, Polina V. Shcherbakova. Novel genomic features of POLE-mutant tumors: the utility for tumor classification and identification of new driver alleles [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA021.

Helenus and Ajax, Two Groups of Non-Autonomous LTR Retrotransposons, Represent a New Type of Small RNA Gene-Derived Mobile Elements.

Terminal repeat retrotransposons in miniature (TRIMs) are short non-autonomous long terminal repeat (LTR) retrotransposons found from various eukaryotes. Cassandra is a unique TRIM lineage which contains a 5S rRNA-derived sequence in its LTRs. Here, two new groups of TRIMs, designated Helenus and Ajax, are reported based on bioinformatics analysis and the usage of Repbase. Helenus is found from fungi, animals, and plants, and its LTRs contain a tRNA-like sequence. It includes two LTRs and between them, a primer-binding site (PBS) and polypurine tract (PPT) exist. Fungal and plant Helenus generate 5 bp target site duplications (TSDs) upon integration, while animal Helenus generates 4 bp TSDs. Ajax includes a 5S rRNA-derived sequence in its LTR and is found from two nemertean genomes. Ajax generates 5 bp TSDs upon integration. These results suggest that despite their unique promoters, Helenus and Ajax are TRIMs whose transposition is dependent on autonomous LTR retrotransposon. These TRIMs can originate through an insertion of SINE in an LTR of TRIM. The discovery of Helenus and Ajax suggests the presence of TRIMs with a promoter for RNA polymerase III derived from a small RNA gene, which is here collectively termed TRIMp3.

DNA polymerase ε and δ variants drive mutagenesis in polypurine tracts in human tumors

Alterations in the exonuclease domain of DNA polymerase ε cause ultramutated cancers. These cancers accumulate AGA>ATA transversions; however, their genomic features beyond the trinucleotide motifs are obscure. We analyze the extended DNA context of ultramutation using whole-exome sequencing data from 524 endometrial and 395 colorectal tumors. We find that G>T transversions in POLE-mutant tumors predominantly affect sequences containing at least six consecutive purines, with a striking preference for certain positions within polypurine tracts. Using this signature, we develop a machine-learning classifier to identify tumors with hitherto unknown POLE drivers and validate two drivers, POLE-E978G and POLE-S461L, by functional assays in yeast. Unlike other pathogenic variants, the E978G substitution affects the polymerase domain of Pol ε. We further show that tumors with POLD1 drivers share the extended signature of POLE ultramutation. These findings expand the understanding of ultramutation mechanisms and highlight peculiar mutagenic properties of polypurine tracts in the human genome.

Nucleobase and Linker Modification for Triple-Helical Recognition of Pyrimidines in RNA Using Peptide Nucleic Acids.

Triple-helical recognition of any sequence of double-stranded RNA requires high affinity Hoogsteen hydrogen binding to pyrimidine interruptions of polypurine tracts. Because pyrimidines have only one hydrogen bond donor/acceptor on Hoogsteen face, their triple-helical recognition is a formidable problem. The present study explored various five-membered heterocycles and linkers that connect nucleobases to backbone of peptide nucleic acid (PNA) to optimize formation of X•C-G and Y•U-A triplets. Molecular modeling and biophysical (UV melting and isothermal titration calorimetry) results revealed a complex interplay between the heterocyclic nucleobase and linker to PNA backbone. While the five-membered heterocycles did not improve pyrimidine recognition, increasing the linker length by four atoms provided promising gains in binding affinity and selectivity. The results suggest that further optimization of heterocyclic bases with extended linkers to PNA backbone may be a promising approach to triple-helical recognition of RNA.

HIV-1 3'-Polypurine Tract Mutations Confer Dolutegravir Resistance by Switching to an Integration-Independent Replication Mechanism via 1-LTR Circles.

Several recent studies indicate that mutations in the human immunodeficiency virus type 1 (HIV-1) 3'polypurine tract (3'PPT) motif can reduce sensitivity to the integrase inhibitor dolutegravir (DTG). Using an in vivo systematic evolution of ligands by exponential enrichment (SELEX) approach, we discovered that multiple different mutations in this viral RNA element can confer DTG resistance, suggesting that the inactivation of this critical reverse transcription element causes resistance. An analysis of the viral DNA products formed upon infection by these 3'PPT mutants revealed that they replicate without integration into the host cell genome, concomitant with an increased production of 1-LTR circles. As the replication of these virus variants is activated by the human T-lymphotropic virus 1 (HTLV-1) Tax protein, a factor that reverses epigenetic silencing of episomal HIV DNA, these data indicate that the 3'PPT-mutated viruses escape from the integrase inhibitor DTG by switching to an integration-independent replication mechanism. IMPORTANCE The integrase inhibitor DTG is a potent inhibitor of HIV replication and is currently recommended in drug regimens for people living with HIV. Whereas HIV normally escapes from antiviral drugs by the acquisition of specific mutations in the gene that encodes the targeted enzyme, mutational inactivation of the viral 3'PPT sequence, an RNA element that has a crucial role in the viral reverse transcription process, was found to allow HIV replication in the presence of DTG in cell culture experiments. While the integration of the viral DNA into the cellular genome is considered one of the hallmarks of retroviruses, including HIV, 3'PPT inactivation caused integration-independent replication, which can explain the reduced DTG sensitivity. Whether this exotic escape route can also contribute to viral escape in HIV-infected persons remains to be determined, but our results indicate that screening for 3'PPT mutations in patients that fail on DTG therapy should be considered.

2-Guanidyl pyridine PNA nucleobase for triple-helical Hoogsteen recognition of cytosine in double-stranded RNA.

In triplex-forming peptide nucleic acid, a novel 2-guanidyl pyridine nucleobase (V) enables recognition of up to two cytosine interruptions in polypurine tracts of dsRNA by engaging the entire Hoogsteen face of C-G base pair. Ab initio and molecular dynamics simulations provided insights into H-bonding interactions that stabilized V·C-G triplets. Our results provided insights for future design of improved nucleobases, which is an important step towards the ultimate goal of recognition of any sequence of dsRNA.

INSTIs and NNRTIs Potently Inhibit HIV-1 Polypurine Tract Mutants in a Single Round Infection Assay.

Integrase strand transfer inhibitors (INSTIs) are a class of antiretroviral compounds that prevent the insertion of a DNA copy of the viral genome into the host genome by targeting the viral enzyme integrase (IN). Dolutegravir (DTG) is a leading INSTI that is given, usually in combination with nucleoside reverse transcriptase inhibitors (NRTIs), to treat HIV-1 infections. The emergence of resistance to DTG and other leading INSTIs is rare. However, there are recent reports suggesting that drug resistance mutations can occur at positions outside the integrase gene either in the HIV-1 polypurine tract (PPT) or in the envelope gene (env). Here, we used single round infectivity assays to measure the antiviral potencies of several FDA-approved INSTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs) against a panel of HIV-1 PPT mutants. We also tested several of our promising INSTIs and NNRTIs in these assays. No measurable loss in potency was observed for either INSTIs or NNRTIs against the HIV-1 PPT mutants. This suggests that HIV-1 PPT mutants are not able, by themselves, to confer resistance to INSTIs or NNRTIs.

Specific mutations in the HIV-1 G-tract of the 3'-polypurine tract cause resistance to integrase strand transfer inhibitors.

In vitro selection experiments identified viruses resistant to integrase strand transfer inhibitors (INSTIs) carrying mutations in the G-tract (six guanosines) of the 3'-polypurine tract (3'-PPT). A clinical study also reported that mutations in the 3'-PPT were observed in a patient receiving dolutegravir monotherapy. However, recombinant viruses with the 3'-PPT mutations that were found in the clinical study were recently shown to be susceptible to INSTIs. To identify the specific mutation(s) in the G-tract of the 3'-PPT for acquiring INSTI resistance, we constructed infectious clones bearing single or multiple mutations and systematically characterized the susceptibility of these clones to both first- and second-generation INSTIs. The infectious clones were tested for their infectivity and susceptibility to INSTIs in a single-cycle assay using TZM-bl cells. A single mutation of thymidine (T) at the fifth position (GGG GTG) in the G-tract of the 3'-PPT had no effect on INSTI resistance. A double mutation, cytidine (C) or 'T' at the second position and 'T' at the fifth position (GCG GTG and GTG GTG), increased resistance to INSTIs, with the appearance of a plateau in the maximal percentage inhibition (MPI) of the dose-response curves, consistent with a non-competitive mechanism of inhibition. Mutations at the second and fifth positions in the G-tract of the 3'-PPT may result in complex resistance mechanism(s), rather than simply affecting INSTI binding at the IN active site.

Mutations in the HIV-1 3′-Polypurine Tract Can Confer Dolutegravir Resistance

With interest we read the Letter to the Editor of Wei and Sluis-Cremer about the role of human immunodeficiency virus 1 (HIV-1) 3’polypurine tract (3’PPT) mutations in dolutegravir (DTG) resistance 1.…

Safety and efficiency modifications of SIV-based integrase-defective lentiviral vectors for immunization

Integrase-defective lentiviral vectors (IDLVs) represent an attractive platform for vaccine development as a result of the ability to induce persistent humoral- and cellular-mediated immune responses against the encoded transgene. Compared with the parental integrating vector, the main advantages for using IDLV are the reduced hazard of insertional mutagenesis and the decreased risk for vector mobilization by wild-type viruses. Here we report on the development and use in the mouse immunogenicity model of simian immunodeficiency virus (SIV)-based IDLV containing a long deletion in the U3 region and with the 3′ polypurine tract (PPT) removed from the transfer vector for improving safety and/or efficacy. Results show that a safer extended deletion of U3 sequences did not modify integrase-mediated or -independent integration efficiency. Interestingly, 3′ PPT deletion impaired integrase-mediated integration but did not reduce illegitimate, integrase-independent integration efficiency, contrary to what was previously reported in the HIV system. Importantly, although the extended deletion in the U3 did not affect expression or immunogenicity from IDLV, deletion of 3′ PPT considerably reduced both expression and immunogenicity of IDLV.

Limited HIV-1 Subtype C nef 3'PPT Variation in Combination Antiretroviral Therapy Naïve and Experienced People Living with HIV in Botswana.

Dolutegravir (DTG) is a potent anti-HIV drug that is used to treat HIV globally. There have been reports of mutations in the HIV-1 3′-polypurine tract (3′PPT) of the nef gene, contributing to DTG failure; however, there are limited ‘real-world’ data on this. In addition, there is a knowledge gap on the variability of 3′PPT residues in patients receiving combination antiretroviral therapy (cART) with and without viral load (VL) suppression. HIV-1 subtype C (HIV-1C) whole-genome sequences from cART naïve and experienced individuals were generated using next-generation sequencing. The nef gene sequences were trimmed from the generated whole-genome sequences using standard bioinformatics tools. In addition, we generated separate integrase and nef gene sequences by Sanger sequencing of plasma samples from individuals with virologic failure (VF) while on a DTG/raltegravir (RAL)-based cART. Analysis of 3′PPT residues was performed, and comparison of proportions computed using Pearson’s chi-square test with p-values < 0.05 was considered statistically significant. A total of 6009 HIV-1C full genome sequences were generated and had a median log10 HIV-1 VL (Q1, Q3) copies/mL of 1.60 (1.60, 2.60). A total of 12 matching integrase and nef gene sequences from therapy-experienced participants failing DTG/ RAL-based cART were generated. HIV-1C 3′PPT nef gene sequences from therapy-experienced patients failing DTG cART (n = 12), cART naïve individuals (n = 1263), and individuals on cART with and without virological suppression (n = 4696) all had a highly conserved 3′PPT motif with no statistically significant differences identified. Our study confirms the high conservation of the HIV-1 nef gene 3′PPT motif in ‘real-world’ patients and showed no differences in the motif according to VL suppression or INSTI-based cART failure. Future studies should explore other HIV-1 regions outside of the pol gene for associations with DTG failure.

Pyridazine Nucleobase in Triplex-Forming PNA Improves Recognition of Cytosine Interruptions of Polypurine Tracts in RNA.

Sequence specific recognition of regulatory noncoding RNAs would open new possibilities for fundamental science and medicine. However, molecular recognition of such complex double-stranded RNA (dsRNA) structures remains a formidable problem. Recently, we discovered that peptide nucleic acids (PNAs) form an unusually stable and sequence-specific triple helix with dsRNA. Triplex-forming PNAs could become universal tools for recognition of noncoding dsRNAs but are limited by the requirement of polypurine tracts in target RNAs as only purines form stable Hoogsteen hydrogen bonded base triplets. Herein, we systematically surveyed simple nitrogen heterocycles PN as modified nucleobases for recognition of cytosine in PN*C-G triplets. We found that a 3-pyridazinyl nucleobase formed significantly more stable PN*C-G triplets than other heterocycles including the pyrimidin-2-one previously used by us and others for recognition of cytosine interruptions in polypurine tracts of PNA-dsRNA triplexes. Our results improve triple helical recognition of dsRNA and provide insights for future development of new nucleobases to expand the sequence scope of noncoding dsRNAs that can be targeted by triplex-forming PNAs.

Analysis and Molecular Determinants of HIV RNase H Cleavage Specificity at the PPT/U3 Junction.

HIV reverse transcriptases (RTs) convert viral genomic RNA into double-stranded DNA. During reverse transcription, polypurine tracts (PPTs) resilient to RNase H cleavage are used as primers for plus-strand DNA synthesis. Nonnucleoside RT inhibitors (NNRTIs) can interfere with the initiation of plus-strand DNA synthesis by enhancing PPT removal, while HIV RT connection subdomain mutations N348I and N348I/T369I mitigate this effect by altering RNase H cleavage specificity. Now, we demonstrate that among approved nonnucleoside RT inhibitors (NNRTIs), nevirapine and doravirine show the largest effects. The combination N348I/T369I in HIV-1BH10 RT has a dominant effect on the RNase H cleavage specificity at the PPT/U3 site. Biochemical studies showed that wild-type HIV-1 and HIV-2 RTs were able to process efficiently and accurately all tested HIV PPT sequences. However, the cleavage accuracy at the PPT/U3 junction shown by the HIV-2EHO RT was further improved after substituting the sequence YQEPFKNLKT of HIV-1BH10 RT (positions 342–351) for the equivalent residues of the HIV-2 enzyme (HQGDKILKV). Our results highlight the role of β-sheets 17 and 18 and their connecting loop (residues 342–350) in the connection subdomain of the large subunit, in determining the RNase H cleavage window of HIV RTs.

Arabidopsis retrotransposon virus-like particles and their regulation by epigenetically activated small RNA.

In Arabidopsis, LTR retrotransposons are activated by mutations in the chromatin gene DECREASE in DNA METHYLATION 1 (DDM1), giving rise to 21- to 22-nt epigenetically activated siRNA (easiRNA) that depend on RNA DEPENDENT RNA POLYMERASE 6 (RDR6). We purified virus-like particles (VLPs) from ddm1 and ddm1rdr6 mutants in which genomic RNA is reverse transcribed into complementary DNA. High-throughput short-read and long-read sequencing of VLP DNA (VLP DNA-seq) revealed a comprehensive catalog of active LTR retrotransposons without the need for mapping transposition, as well as independent of genomic copy number. Linear replication intermediates of the functionally intact COPIA element EVADE revealed multiple central polypurine tracts (cPPTs), a feature shared with HIV in which cPPTs promote nuclear localization. For one member of the ATCOPIA52 subfamily (SISYPHUS), cPPT intermediates were not observed, but abundant circular DNA indicated transposon “suicide” by auto-integration within the VLP. easiRNA targeted EVADE genomic RNA, polysome association of GYPSY (ATHILA) subgenomic RNA, and transcription via histone H3 lysine-9 dimethylation. VLP DNA-seq provides a comprehensive landscape of LTR retrotransposons and their control at transcriptional, post-transcriptional, and reverse transcriptional levels.

Variability in HIV-1 Integrase Gene and 3'-Polypurine Tract Sequences in Cameroon Clinical Isolates, and Implications for Integrase Inhibitors Efficacy.

Integrase strand-transfer inhibitors (INSTIs) are now included in preferred first-line antiretroviral therapy (ART) for HIV-infected adults. Studies of Western clade-B HIV-1 show increased resistance to INSTIs following mutations in integrase and nef 3′polypurine tract (3′-PPT). With anticipated shifts in Africa (where 25.6-million HIV-infected people resides) to INSTIs-based ART, it is critical to monitor patients in African countries for resistance-associated mutations (RAMs) affecting INSTIs efficacy. We analyzed HIV-1 integrase and 3′-PPT sequences in 345 clinical samples from INSTIs-naïve HIV-infected Cameroonians for polymorphisms and RAMs that affect INSTIs. Phylogeny showed high genetic diversity, with the predominance of HIV-1 CRF02_AG. Major INSTIs RAMs T66A and N155K were found in two (0.6%) samples. Integrase polymorphic and accessory RAMs found included T97A, E157Q, A128T, M50I, S119R, L74M, L74I, S230N, and E138D (0.3′23.5% of samples). Ten (3.2%) samples had both I72V+L74M, L74M+T97A, or I72V+T97A mutations; thirty-one (9.8%) had 3′-PPT mutations. The low frequency of major INSTIs RAMs shows that INSTIs-based ART can be successfully used in Cameroon. Several samples had ≥1 INSTIs accessory RAMs known to reduce INSTIs efficacy; thus, INSTIs-based ART would require genetic surveillance. The 3′-PPT mutations could also affect INSTIs. For patients failing INSTIs-based ART with no INSTIs RAMs, monitoring 3′-PPT sequences could reveal treatment failure etiology.

A scale-free analysis of the HIV-1 genome demonstrates multiple conserved regions of structural and functional importance.

HIV-1 replicates via a low-fidelity polymerase with a high mutation rate; strong conservation of individual nucleotides is highly indicative of the presence of critical structural or functional properties. Identifying such conservation can reveal novel insights into viral behaviour. We analysed 3651 publicly available sequences for the presence of nucleic acid conservation beyond that required by amino acid constraints, using a novel scale-free method that identifies regions of outlying score together with a codon scoring algorithm. Sequences with outlying score were further analysed using an algorithm for producing local RNA folds whilst accounting for alignment properties. 11 different conserved regions were identified, some corresponding to well-known cis-acting functions of the HIV-1 genome but also others whose conservation has not previously been noted. We identify rational causes for many of these, including cis functions, possible additional reading frame usage, a plausible mechanism by which the central polypurine tract primes second-strand DNA synthesis and a conformational stabilising function of a region at the 5′ end of env.