The Evolutionary Analysis of Emerging Low Frequency HIV-1 CXCR4 Using Variants through Time—An Ultra-Deep Approach

Large-scale parallel pyrosequencing produces unprecedented quantities of sequence data. However, when generated from viral populations current mapping software is inadequate for dealing with the high levels of variation present, resulting in the potential for biased data loss. In order to apply the 454 Life Sciences' pyrosequencing system to the study of viral populations, we have developed software for the processing of highly variable sequence data. Here we demonstrate our software by analyzing two temporally sampled HIV-1 intra-patient datasets from a clinical study of maraviroc. This drug binds the CCR5 coreceptor, thus preventing HIV-1 infection of the cell. The objective is to determine viral tropism (CCR5 versus CXCR4 usage) and track the evolution of minority CXCR4-using variants that may limit the response to a maraviroc-containing treatment regimen. Five time points (two prior to treatment) were available from each patient. We first quantify the effects of divergence on initial read k-mer mapping and demonstrate the importance of utilizing population-specific template sequences in relation to the analysis of next-generation sequence data. Then, in conjunction with coreceptor prediction algorithms that infer HIV tropism, our software was used to quantify the viral population structure pre- and post-treatment. In both cases, low frequency CXCR4-using variants (2.5–15%) were detected prior to treatment. Following phylogenetic inference, these variants were observed to exist as distinct lineages that were maintained through time. Our analysis, thus confirms the role of pre-existing CXCR4-using virus in the emergence of maraviroc-insensitive HIV. The software will have utility for the study of intra-host viral diversity and evolution of other fast evolving viruses, and is available from http://www.bioinf.manchester.ac.uk/segminator/

Simultaneous gene expression analysis of steady-state and actively translated mRNA populations from osteosarcoma MG-63 cells in response to IL-1α via an open expression analysis platform

Pro-inflammatory cytokines play a key role in various forms of metabolic bone diseases, including osteopenia and osteoporosis. Human MG-63 cells treated with IL-1α were used as a model system to identify potential marker genes that are differentially expressed. This study is designed to quantitate gene expression of actively translated mRNAs as compared to the steady-state mRNA population. Both steady-state mRNAs and actively translated mRNAs from control MG-63 cells and MG-63 cells treated with IL-1α were isolated and converted to cDNA. The gene expression analysis from these samples was then quantitated with an open expression analysis platform with no requirement for a priori knowledge of sequence information. As a result, many differentially regulated genes were discovered via IL-1α treatment. Some of the genes have been described previously as playing important roles in the regulation of inflammation and cell adhesion. These comparisons provided a panoramic overview of gene expression at both the total transcript and post-transcriptional levels. In addition, the quantitation of actively translated mRNAs associated with polysomes also provided a better estimation of protein expression levels. This methodology allows for the identification of genes acutely regulated during translation. Furthermore, the process may aid in the identification of new drug targets or biomarkers

“One plate/three-reporter” assay format for the detection and validation of yeast two-hybrid interactions

We describe a novel assay format for the Gal4-based yeast two-hybrid-system, in which the readout from three different reporter genes is measured sequentially in a single microplate. Activation of the URA3, MEL1, and lacZ reporters in response to a protein-protein interaction is monitored by measuring sequentially: (i) growth in medium lacking uracil, (ii) α-galactosidase activity, and (iii) β-galactosidase. The data thus generated permit elimination of many false positive signals and provide a preliminary measurement of reporter activation-strength that may be confirmed by further analysis. The assay procedure is inexpensive and requires few liquid-handling steps. It is appropriate for automated high-throughput interaction mating assays, validation of putative interactor strains and hybrid-protein self-activator tests

Detection of low-frequency pretherapy chemokine (CXC motif) receptor 4 (CXCR4)-using HIV-1 with ultra-deep pyrosequencing

Objective: Identification of low-frequency variants is of clinical importance in the identification of preexisting drug resistance. Using ‘ultra-deep’ sequencing, we address the detection of potential resistance to the chemokine (C–C motif) receptor 5 (CCR5) antagonist, maraviroc, due to the pretreatment presence of low levels of chemokine (CXC motif) receptor 4 (CXCR4)-using virus. Methods: We present a novel protocol for the phenotyping of HIV based on ‘454’ pyrosequence data and apply this to two large data sets comprised of 104 628 (before treatment, day 1) and 191 637 (after treatment, day 11) reads from the envelope region. We study resistance in the context of the evolutionary history of the intrapatient viral population. Variation was also investigated both within and outside the V3 region, the region associated with the receptor switch. Results: CXCR4-using virus can be detected at low frequency prior to maraviroc treatment (~0.5%) and at high frequency after failure of monotherapy (~81%). Inferring an evolutionary tree from the 1674 unique reads that span the V3 region confirms that the CXCR4-using population emerged from low-frequency CXCR4-using variants present before treatment. Changes in the frequency of amino acid residues used at individual sites were found in regions outside the V3 region, indicative of other potential sites associated with receptor usage. Conclusion: We have provided a high-resolution snapshot of intrapatient viral variation, prior and after treatment with maraviroc, and detected preexisting CXCR4-using variants present at an extremely low frequency. The evolutionary analysis demonstrates the extent of diversity present at a single time point within an infected individual and the rapid effect of drug pressure on the structure of a viral population

Deoptimization of FMDV P1 Region Results in Robust Serotype-Independent Viral Attenuation

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious disease of cloven-hoofed livestock that can have severe economic impacts. Control and prevention strategies, including the development of improved vaccines, are urgently needed to effectively control FMD outbreaks in endemic settings. Previously, we employed two distinct strategies (codon pair bias deoptimization (CPD) and codon bias deoptimization (CD)) to deoptimize various regions of the FMDV serotype A subtype A12 genome, which resulted in the development of an attenuated virus in vitro and in vivo, inducing varying levels of humoral responses. In the current study, we examined the versatility of the system by using CPD applied to the P1 capsid coding region of FMDV serotype A subtype, A24, and another serotype, Asia1. Viruses carrying recoded P1 (A24-P1Deopt or Asia1-P1Deopt) exhibited different degrees of attenuation (i.e., delayed viral growth kinetics and replication) in cultured cells. Studies in vivo using a mouse model of FMD demonstrated that inoculation with the A24-P1Deopt and Asia1-P1Deopt strains elicited a strong humoral immune response capable of offering protection against challenge with homologous wildtype (WT) viruses. However, different results were obtained in pigs. While clear attenuation was detected for both the A24-P1Deopt and Asia1-P1Deopt strains, only a limited induction of adaptive immunity and protection against challenge was detected, depending on the inoculated dose and serotype deoptimized. Our work demonstrates that while CPD of the P1 coding region attenuates viral strains of multiple FMDV serotypes/subtypes, a thorough assessment of virulence and induction of adaptive immunity in the natural host is required in each case in order to finely adjust the degree of deoptimization required for attenuation without affecting the induction of protective adaptive immune responses

Second generation sequencing of the mesothelioma tumor genome.

The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM) tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.6X depth of coverage. Read density analysis uncovered significant aneuploidy and numerous rearrangements. Method-dependent informatics rules, which combined the results of different sequencing platforms, were developed to identify and validate candidate mutations of multiple types. Many more tumor-specific rearrangements than point mutations were uncovered at this depth of sequencing, resulting in novel, large-scale, inter- and intra-chromosomal deletions, inversions, and translocations. Nearly all candidate point mutations appeared to be previously unknown SNPs. Thirty tumor-specific fusions/translocations were independently validated with PCR and Sanger sequencing. Of these, 15 represented disrupted gene-encoding regions, including kinases, transcription factors, and growth factors. One large deletion in DPP10 resulted in altered transcription and expression of DPP10 transcripts in a set of 53 additional MPM tumors correlated with survival. Additionally, three point mutations were observed in the coding regions of NKX6-2, a transcription regulator, and NFRKB, a DNA-binding protein involved in modulating NFKB1. Several regions containing genes such as PCBD2 and DHFR, which are involved in growth factor signaling and nucleotide synthesis, respectively, were selectively amplified in the tumor. Second-generation sequencing uncovered all types of mutations in this MPM tumor, with DNA rearrangements representing the dominant type