Mapping of positive selection sites in the HIV-1 genome in the context of RNA and protein structural constraints

<p>Abstract</p> <p>Background</p> <p>The HIV-1 genome is subject to pressures that target the virus resulting in escape and adaptation. On the other hand, there is a requirement for sequence conservation because of functional and structural constraints. Mapping the sites of selective pressure and conservation on the viral genome generates a reference for understanding the limits to viral escape, and can serve as a template for the discovery of sites of genetic conflict with known or unknown host proteins.</p> <p>Results</p> <p>To build a thorough evolutionary, functional and structural map of the HIV-1 genome, complete subtype B sequences were obtained from the Los Alamos database. We mapped sites under positive selective pressure, amino acid conservation, protein and RNA structure, overlapping coding frames, CD8 T cell, CD4 T cell and antibody epitopes, and sites enriched in AG and AA dinucleotide motives. Globally, 33% of amino acid positions were found to be variable and 12% of the genome was under positive selection. Because interrelated constraining and diversifying forces shape the viral genome, we included the variables from both classes of pressure in a multivariate model to predict conservation or positive selection: structured RNA and α-helix domains independently predicted conservation while CD4 T cell and antibody epitopes were associated with positive selection.</p> <p>Conclusions</p> <p>The global map of the viral genome contains positive selected sites that are not in canonical CD8 T cell, CD4 T cell or antibody epitopes; thus, it identifies a class of residues that may be targeted by other host selective pressures. Overall, RNA structure represents the strongest determinant of HIV-1 conservation. These data can inform the combined analysis of host and viral genetic information.</p

Activity of ancestral restriction factors against ancient retroviruses

Analysis of TRIM5α and APOBEC3G genes suggests that these two restriction factors underwent strong positive selection throughout primate evolution. This pressure was possibly imposed by ancient exogenous retroviruses, of which endogenous retroviruses are remnants. Our study aims to assess in vitro the activity of these factors against ancient retroviruses by reconstructing their ancestral gag sequences, as well as the ancestral TRIM5α and APOBEC3G for primates. Based on evolutionary genomics approach, we reconstructed ancestors of the two largest families of human endogenous retroviruses (HERV), namely HERV-K and HERV-H, as well as primate ancestral TRIM5α and APOBEC3G variants. The oldest TRIM5α sequence was the catarhinne TRIM5α, common ancestor of Old World monkeys and hominoids, dated from 25 million years ago (mya). From the oldest, to the youngest, ancestral TRIM5α variants showed less restriction of HIV-1 in vitro [1]. Likewise three ancestral APOBEC3Gs sequences common to hominoids (18 mya), Old World monkeys, and catarhinnes (25 mya) were reconstructed. All ancestral APOBEC3G variants inhibited efficiently HIV-1Δvif in vitro, compared to modern APOBEC3Gs. The ability of Vif proteins (HIV-1, HIV-2, SIVmac and SIVagm) to counteract their activity tallied with the residue 128 on ancestral APOBEC3Gs. Moreover we are attempting to reconstruct older ancestral sequences of both restriction factors by using prosimian orthologue sequences. An infectious onemillion- years-old HERV-KCON previously reconstituted was shown to be resistant to modern TRIM5α and APOBEC3G [2]. Our ancestral TRIM5α and APOBEC3G variants were inactive against HERV-KCON. Besides we reconstructed chimeric HERV-K bearing ancestral capsids (up to 7 mya) that resulted in infectious viruses resistant to modern and ancestral TRIM5α. Likewise HERV-K viruses bearing ancestral nucleocapsids will be tested for ancestral and modern APOBEC3G restriction. In silico reconstruction and structural modeling of ancestral HERV-H capsids resulted in structures homologous to that of the gammaretrovirus MLV. Thus we are attempting to construct chimeric MLV virus bearing HERV-H ancestral capsids. These chimeric ancestral HERVs will be tested for infectivity and restriction by ancestral TRIM5α. Similarly chimeric MLV viruses bearing ancestral HERV-H nucleocapsids will be reconstructed and tested for APOBEC3G restriction

HIV-1 protease mutation 82M contributes to phenotypic resistance to protease inhibitors in subtype G

Objectives The purpose of this study was the qualitative and quantitative assessment of the in vitro effect of HIV-1 protease (PR) mutation 82M on replication capacity and susceptibility to the eight clinically available PR inhibitors (PIs). Methods The 82M substitution was introduced by site-directed mutagenesis in wild-type subtype B and G strains, as well as reverted back to wild-type in a therapy-failing strain. The recombinant viruses were evaluated for their replication capacity and susceptibility to PIs. Results The single 82M mutation within a wild-type subtype B or G background did not result in drug resistance. However, the in vitro effect of single PR mutations on PI susceptibility is not always distinguishable from wild-type virus, and particular background mutations and polymorphisms are required to detect significant differences in the drug susceptibility profile. Consequently, reverting the 82M mutation back to wild-type (82I) in a subtype G isolate from a patient that failed therapy with multiple other PR mutations did result in significant increases in susceptibility towards indinavir and lopinavir and minor increases in susceptibility towards amprenavir and atazanavir. The presence of the 82M mutation also slightly decreased viral replication, whether it was in the genetic background of subtype B or subtype G. Conclusions Our results suggest that 82M has an impact on PI susceptibility and that this effect is not due to a compensatory effect on the replication capacity. Because 82M is not observed as a polymorphism in any subtype, these observations support the inclusion of 82M in drug resistance interpretation systems and PI mutation list

DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication by Preventing a Cyclophilin A Induced Cis-Trans Isomerisation in Domain II of NS5A

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance

An exploratory survey of professionals on the use of stored tissue samples from minors for genetic research

The ethical aspects of the use of stored tissue samples collected from minors are of topical interest. However, the views of professionals working in the field of genetics have not been investigated in depth anywhere. We conducted a survey among 194 such professionals in Belgium. This list was composed of the members of the High Council for Anthropogenetics, supplemented with all professionals working in the field of genetics that we found on the websites of the eight Belgian centers of human genetics and of the associated university registries. We achieved a response rate of 35.5%. The vast majority (92%) think that research on stored tissue samples is useful. Most respondents stated that parental consent is valid (82.5%), and 76.5% thought that children should also be given the right to assent when they are able to comprehend the implications of the storage of biological samples and of genetic research. Slightly more than half put the age at which young people can understand storage or research rather high: 16-18 years (51 and 53.1%, respectively). Although there is some consensus in the literature that donors should be allowed to give broad consent for future research on their biological samples, only 47.6% in our survey thought that parents should be allowed to consent to any future research on their children’s samples. The aim of our study was to give some basis for future ethical reflections and policies on the subject of stored tissue samples from minors for genetic research. We concluded that a large majority of Belgian researchers and clinicians in the field of genetic research think research on stored tissue samples from minors is useful. They also think that parental consent for such research is valid, but that children should be allowed to assent as they grow older.status: publishe

Analysis of NGS data from heterogeneous HCV1b populations

Background: The analysis of next-generation sequencing (NGS) data from heterogeneous viral populations such as hepatitis C virus (HCV) remains a non-trivial exercise. In order to accommodate the specific characteristics of short read fragment data derived from such diverse populations, we studied the impact of tailored analysis methodologies and of reference sequence divergence on the recovery of minority variants. Materials and methods: Four samples from treatment-naïve HCV1b infected patients were amplified by nested PCR and sequenced with Illumina’s Genome Analyzer IIx. The performance of five software packages (MAQ, Bowtie, BWA, Velvet and Segminator II) for aligning the quality trimmed Illumina reads against a reference sequence was tested. To assess the impact of using a related reference sequence, all reads of each sample were mapped to a published HCV1b reference sequence (GenBank: AB049087), to a contig of the same sample obtained by Sanger sequencing and to an in silico reconstructed data-specific reference sequence (VICUNA and V-FAT). Concordance between the viral population obtained after Sanger sequencing and after read mapping with Segminator II using the three mentioned reference sequences, was compared. The observed differences in minority variants between the analysis with a sample-specific and a more distantly related sequence were compared. Positions with high difference (>5% threshold) were compared with the location of high divergent regions, defined as areas with Shannon entropy above the 75th percentile. Results: The number of mapped reads was consistently higher when a sample-specific sequence was used instead of the standardly used but more divergent HCV1b reference sequence. For the different software packages, recovery of reads varied between 20% and 82% with Segminator II performing best: 80.6% (± 3.0) against the sample-specific Sanger sequence, 77.3% (± 2.4) against the Genbank reference sequence (using the automatic cyclic remapping), and 82.1% (± 4.0) against the in silico reconstructed reference sequence. Simple scoring of aligned positions as matched/unmatched revealed a high degree of concordance between the NGS consensus sequences obtained by Segminator II (all three reference sequences) and the sample-specific sequence obtained with Sanger sequencing. After neglecting the minor discordances (predominantly attributable to ambiguity characters in the NGS consensus sequences), overall concordance increased to a nearly perfect match (>99%). However, the use of the standard HCV1b reference sequence resulted into slightly higher discordance values (0.30%) as for the Sanger (0.026%) and in silico reconstructed reference sequence (0.05%). Although the main differences in minority variants were located below the 0.5% threshold, in total 184 differences at frequencies above 5% were observed. All these positions were located at (70.34%) or within 5 nucleotides of a divergent area. Conclusions: Only a few software packages can accommodate the diversity present in heterogeneous viral populations, and using a distantly related reference sequence leads to substantial data losses, even with the best among the software packages, Segminator II. Although the viral population of the three reference sequences did not differ substantially, discordances were enriched in highly divergent regions. So we recommend the use of a sample-/data-specific reference sequence for read mapping.status: accepte

Evaluation of software to map NGS reads from heterogeneous HCV1b populations

We studied the impact of analysis software and of reference sequence divergence on the recovery of minority variants with Illumina deep sequencing applied to highly variable HCV populations. Four HCV1b full genomes were sequenced by Sanger population and Illumina deep sequencing, starting from the same PCR fragments. Reads were mapped to a published HCV1b reference sequence, to the sample-specific Sanger sequence and to an in silico reconstructed data-specific reference sequence (VICUNA). Of the four tested software packages (MAQ, Bowtie, BWA and Segminator II), Segminator II consistently mapped the largest number of reads, recovering respectively 77.3% (± 2.4), 80.6% (± 3.0) and 82.1% (± 4.0) of reads whereas this was between 29% and 77% for the other methods. For all packages, the number of mapped reads increased when a sample-specific sequence was used instead of a more distantly related HCV1b reference sequence. For Segminator II, the concordance between the three NGS consensus sequences (representing the three mapping strategies) and the obtained Sanger sequence, was >99% when neglecting ambiguities. When using a 5% threshold, for one sample 184 differences in ambiguities between the NGS analysis with a data-specific and HCV1b published reference sequence were all located in or near a divergent area, and therefore most probably represented true minority variants. To conclude, for deep sequencing of highly variable viral sequences, we recommend the use of a sample-/data-specific reference sequence and of tailored mapping software in order to map the maximum number of reads and recover as much as possible minority variants.status: accepte