Alessandro Bruselles

Use of Massively Parallel Ultradeep Pyrosequencing To Characterize the Genetic Diversity of Hepatitis B Virus in Drug-Resistant and Drug-Naive Patients and To Detect Minor Variants in Reverse Transcriptase and Hepatitis B S Antigen

ABSTRACT Direct population sequencing and reverse hybridization (line probe assay [LiPA])-based methods are the most common methods for detecting hepatitis B virus (HBV) drug resistance mutations, although only mutations present in viral quasispecies with a prevalence of ≥20% can be detected by sequencing, and only known mutations are detected by LiPA. Massively parallel ultradeep pyrosequencing (UDPS; GS FLX platform) was used to analyze HBV quasispecies in reverse transcriptase (RT) and hepatitis B S antigen (HBsAg) from five drug-naive patients and eight drug-resistant patients. Eight primer pairs were used to obtain partially overlapping amplicons, covering the RT gene from codons 1 to 288 and the complete overlapping HBsAg sequence. A 1% mutation frequency was selected as the cutoff based on an error rate estimated on plasmid DNA. This technology enabled simultaneous analysis of between 2,852 and 18,016 clonally amplified fragments from each patient. The results indicate that UDPS has a relative sensitivity much higher than both direct sequencing and LiPA. In addition, the UDPS results are quantitative, allowing establishment of the relative frequency of both known mutations and novel substitutions. Some of the detected RT substitutions led to changes also in HBsAg. On the whole, genotype D presented a higher heterogeneity than genotype A. Considering the high quantity of information that can be provided by a single test from one patient, the short turnaround time, the information on substitution frequency, and the detection of rare variants, there are strong advantages conferred by UDPS, and the new method could play a relevant role in the clinical management of HBV infection and therapy.

Massively parallel pyrosequencing highlights minority variants in the HIV-1 env quasispecies deriving from lymphomonocyte sub-populations

BackgroundVirus-associated cell membrane proteins acquired by HIV-1 during budding may give information on the cellular source of circulating virions. In the present study, by applying immunosorting of the virus and of the cells with antibodies targeting monocyte (CD36) and lymphocyte (CD26) markers, it was possible to directly compare HIV-1 quasispecies archived in circulating monocytes and T lymphocytes with that present in plasma virions originated from the same cell types. Five chronically HIV-1 infected patients who underwent therapy interruption after prolonged HAART were enrolled in the study. The analysis was performed by the powerful technology of ultra-deep pyrosequencing after PCR amplification of part of the env gene, coding for the viral glycoprotein (gp) 120, encompassing the tropism-related V3 loop region. V3 amino acid sequences were used to establish heterogeneity parameters, to build phylogenetic trees and to predict co-receptor usage.ResultsThe heterogeneity of proviral and viral genomes derived from monocytes was higher than that of T-lymphocyte origin. Both monocytes and T lymphocytes might contribute to virus rebounding in the circulation after therapy interruptions, but other virus sources might also be involved. In addition, both proviral and circulating viral sequences from monocytes and T lymphocytes were predictive of a predominant R5 coreceptor usage. However, minor variants, segregating from the most frequent quasispecies variants, were present. In particular, in proviral genomes harboured by monocytes, minority variant clusters with a predicted X4 phenotype were found.ConclusionThis study provided the first direct comparison between the HIV-1 quasispecies archived as provirus in circulating monocytes and T lymphocytes with that of plasma virions replicating in the same cell types. Ultra-deep pyrosequencing generated data with some order of magnitude higher than any previously obtained with conventional approaches. Next generation sequencing allowed the analysis of previously inaccessible aspects of HIV-1 quasispecies, such as co-receptor usage of minority variants present in archived proviral sequences and in actually replicating virions, which may have clinical and therapeutic relevance.

Single Mutation in the Linker Domain Confers Protein Flexibility and Camptothecin Resistance to Human Topoisomerase I

DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin specifically targets DNA topoisomerase I, and several mutations have been isolated that render the enzyme camptothecin-resistant. The catalytic and structural dynamical properties of a human DNA topoisomerase I mutant in which Ala-653 in the linker domain was mutated into Pro have been investigated. The mutant is resistant to camptothecin and in the absence of the drug displays a cleavage-religation equilibrium strongly shifted toward religation. The shift is mainly because of an increase in the religation rate relative to the wild type enzyme, indicating that the unperturbed linker is involved in slowing religation. Molecular dynamics simulation indicates that the Ala to Pro mutation increases the linker flexibility allowing it to sample a wider conformational space. The increase in religation rate of the mutant, explained by means of the enhanced linker flexibility, provides an explanation for the mutant camptothecin resistance.

Combinatorial analysis and algorithms for quasispecies reconstruction using next-generation sequencing

BackgroundNext-generation sequencing (NGS) offers a unique opportunity for high-throughput genomics and has potential to replace Sanger sequencing in many fields, including de-novo sequencing, re-sequencing, meta-genomics, and characterisation of infectious pathogens, such as viral quasispecies. Although methodologies and software for whole genome assembly and genome variation analysis have been developed and refined for NGS data, reconstructing a viral quasispecies using NGS data remains a challenge. This application would be useful for analysing intra-host evolutionary pathways in relation to immune responses and antiretroviral therapy exposures. Here we introduce a set of formulae for the combinatorial analysis of a quasispecies, given a NGS re-sequencing experiment and an algorithm for quasispecies reconstruction. We require that sequenced fragments are aligned against a reference genome, and that the reference genome is partitioned into a set of sliding windows (amplicons). The reconstruction algorithm is based on combinations of multinomial distributions and is designed to minimise the reconstruction of false variants, called in-silico recombinants.ResultsThe reconstruction algorithm was applied to error-free simulated data and reconstructed a high percentage of true variants, even at a low genetic diversity, where the chance to obtain in-silico recombinants is high. Results on empirical NGS data from patients infected with hepatitis B virus, confirmed its ability to characterise different viral variants from distinct patients.ConclusionsThe combinatorial analysis provided a description of the difficulty to reconstruct a quasispecies, given a determined amplicon partition and a measure of population diversity. The reconstruction algorithm showed good performance both considering simulated data and real data, even in presence of sequencing errors.

Detection of quasispecies variants predicted to use CXCR4 by ultra-deep pyrosequencing during early HIV infection.

Objectives:HIV-1 V3 quasispecies was analyzed by ultra-deep pyrosequencing, in early HIV-infected patients, to assess possible correlations between quasispecies diversity, frequency of variants predicted to use CXCR4 and need for early antiretroviral treatment. Methods:Twenty patients were retrospectively enrolled: 10 patients (group A) required HAART within 6 months from seroconversion and 10 (group B) remained free of therapy during this period. V3 quasispecies was assessed on plasma viral RNA and in peripheral blood mononuclear cell-associated proviral DNA. Prediction of coreceptor usage was performed by position-specific score matrix analysis. Results:Variants predicted to use CXCR4 were detected (frequency ≥0.3%) in the plasma of 50% of early infected patients (60% from group A and 40% from group B). Intrapatient frequency of these variants was highly variable (0.3–56.3%). A positive correlation was observed between the proportion of X4 variants and intrapatient quasispecies diversity. Quasispecies diversity and absolute numbers of X4 variants were significantly higher in patients from group A. The analysis of proviral DNA quasispecies, performed in a subgroup of five patients, showed that X4 variants were not detected in patients with RNA frequency below 0.3%, and detected at 3.6% in the patient with 56.3% of X4 plasma variants. Conclusion:Our findings show that X4 variants may be frequently found, at variable intrapatient frequency, in early infected patients, and that quasispecies diversity and absolute numbers of X4 variants are significantly higher in patients undergoing early antiretroviral treatment. Further studies are mandatory to explore the clinical relevance of X4 variants present during early infection with respect to clinical progression and possible therapeutic implications.

Analysis of co‐receptor usage of circulating viral and proviral HIV genome quasispecies by ultra‐deep pyrosequencing in patients who are candidates for CCR5 antagonist treatment

UDPS combined with genotypic algorithms for prediction of HIV-1 co-receptor usage may provide quantitative data about the tropism of each variant present in the viral quasispecies. The aim of the present study was to assess co-receptor usage by ultra-deep pyrosequencing (UDPS), in comparison with the reference phenotypic test (Trofile), in patients who are candidates for CCR5 antagonist treatment, in both circulating and proviral HIV-1. Seventeen patients who were tested by Trofile were enrolled. UDPS of the V3 loop region was carried out on both plasma RNA and proviral DNA. Genotypic prediction of co-receptor usage was established by position-specific score matrices (PSSM) and confirmed, in discordant cases, with geno2pheno. Genetic heterogeneity of the RNA and DNA quasispecies was assessed as well. A total of 196,729 V3 sequences were considered (mean coverage per site, 6346). Concordance between phenotypic test and UDPS with PSSM was 0.82. Geno2pheno results were in line with those obtained with PSSM. Proviral quasispecies were more heterogeneous than those found in circulating HIV. In most patients eligible for CCR5 antagonist treatment, X4 variants were detected in proviral DNA, ranging from 1.0% to 52.7%. UDPS combined with genotypic algorithms for co-receptor usage prediction highlighted the presence of minority variants, with a discordant tropism with respect to the predominant population, in both circulating viral and proviral HIV. In most patients treated with Maraviroc the virological response was independent of the presence of X4 in proviral DNA. The clinical impact of minority X4 variants present in patients who are candidates for anti-CCR5 antagonists remains a crucial point to be addressed.

Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study

The functional and dynamical properties of the human topoisomerase I Thr718Ala mutant have been compared to that of the wild-type enzyme using functional assays and molecular dynamics (MD) simulations. At physiological ionic strength, the cleavage and religation rates, evaluated on oligonucleotides containing the preferred topoisomerase I DNA sequence, are almost identical for the wild-type and the mutated enzymes, as is the cleavage/religation equilibrium. On the other hand, the Thr718Ala mutant shows a decreased efficiency in a DNA plasmid relaxation assay. The MD simulation, carried out on the enzyme complexed with its preferred DNA substrate, indicates that the mutant has a different dynamic behavior compared to the wild-type enzyme. Interestingly, no changes are observed in the proximity of the mutation site, whilst a different flexibility is detected in regions contacting the DNA scissile strand, such as the linker and the V-shaped α helices. Taken together, the functional and simulation results indicate a direct communication between the mutation site and regions located relatively far away, such as the linker domain, that with their altered flexibility confer a reduced DNA relaxation efficiency. These results provide evidence that the comprehension of the topoisomerase I dynamical properties are an important element in the understanding of its complex catalytic cycle.

Use of Massive Parallel Pyrosequencing for Near Full-Length Characterization of a Unique HIV Type 1 BF Recombinant Associated with a Fatal Primary Infection

Near full length genome characterization of a BF recombinant from a patient who died from multiorgan failure during HIV-1 seroconversion is reported. Massive parallel pyrosequencing was used with the shotgun approach. Intrahost genetic variability along the whole genome was calculated and coreceptor usage of viral quasispecies was predicted. A consensus sequence was established to perform subtype assignment, phylogenetic analysis, and recombination tests. The sequence clustered with two recently described BF unique recombinant forms from Brazil, consistent with the recombination pattern, yielding breakpoints located at the same positions, with the exception of the second env breakpoint. The actual prevalence of recombinant forms is probably underestimated if partial genomic regions are considered. Here the first full length BF recombinant from Italy is described, together with an evaluation of quasispecies heterogeneity. Our data provide evidence that next generation sequencing may provide a major contribution to HIV-1 molecular epidemiology and to the comprehension of intrapatient heterogeneity.

The open state of human topoisomerase I as probed by molecular dynamics simulation

The open state of human topoisomerase I has been probed by molecular dynamics simulation, starting from the coordinates of the closed structure of the protein complexed with DNA, after elimination of the 22-bp DNA duplex oligonucleotide. A repulsion force between the two lips of the protein has been introduced for a short time to induce destabilization of the local minimum, after which an unperturbed simulation has been carried out for 10 ns. The simulation shows that the protein undergoes a large conformational change due to rearrangements in the orientation of the protein domains, which however move as a coherent unit, fully maintaining their secondary and tertiary structures. Despite movements between the domains as large as 80–90 Å, the catalytic pentad remains preassembled, the largest deviation of the active site backbone atoms from the starting crystallographic structure being only 1.7 Å. Electrostatic calculation of the open protein structure shows that the protein displays a vast positive region with the active site residues located nearly at its center, in a conformation perfectly suited to interact with the negatively charged supercoiled DNA substrate.

Ultra-deep sequencing reveals hidden HIV-1 minority lineages and shifts of viral population between the main cellular reservoirs of the infection after therapy interruption

Viral quasispecies population dynamics between monocytes and T‐lymphocytes were analyzed in patients after highly active antiretroviral therapy (HAART) interruption, during a follow‐up of 3–6 months. V3 env region underwent ultra‐deep pyrosequencing. Co‐receptor usage prediction was performed by Position Specific Score Matrix Analysis. Phylogenetic trees were constructed to evaluate the relationships between the variants. Gene flow was also investigated. Even though at the moment of therapy interruption monocyte‐derived HIV‐1 genomes presented higher genetic heterogeneity than that of T‐lymphocytes, at subsequent times, this difference in genetic heterogeneity disappeared, due to different waves of expansion and reduction of quasispecies variability associated with monocytes and T‐lymphocytes. Phylogenetic analysis and gene flow evaluation supported the hypothesis of extensive interchange of variants between cellular compartments of the infection. A spread of proviral X4 lineages hidden in monocytes to T cells was observed, but this was not associated with an overall shift towards CXCR4 using variants during the observation period. J. Med. Virol. 84:839–844, 2012.