Selective ablation of 3' RNA ends and processive RTs facilitate direct cDNA sequencing of full-length host cell and viral transcripts.

Alternative splicing (AS) is necessary for viral proliferation in host cells and a critical regulatory component of viral gene expression. Conventional RNA-seq approaches provide incomplete coverage of AS due to their short read lengths and are susceptible to biases and artifacts introduced in prevailing library preparation methodologies. Moreover, viral splicing studies are often conducted separately from host cell transcriptome analysis, precluding an assessment of the viral manipulation of host splicing machinery. To address current limitations, we developed a quantitative full-length direct cDNA sequencing strategy to simultaneously profile viral and host cell transcripts. This nanopore-based approach couples processive reverse transcriptases with a novel one-step chemical ablation of 3' RNA ends (termed CASPR), which decreases ribosomal RNA reads and enriches polyadenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show that CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4 kb), while being functionally equivalent to PolyA+ selection for transcript quantification. We used our approach to interrogate host cell and HIV-1 transcript dynamics during viral reactivation and identified novel putative HIV-1 host factors containing exon skipping or novel intron retentions and delineated the HIV-1 transcriptional state associated with these differentially regulated host factors.

Covariation of viral recombination with single nucleotide variants during virus evolution revealed by CoVaMa.

Adaptation of viruses to their environments occurs through the acquisition of both novel single-nucleotide variants (SNV) and recombination events including insertions, deletions, and duplications. The co-occurrence of SNVs in individual viral genomes during their evolution has been well-described. However, unlike covariation of SNVs, studying the correlation between recombination events with each other or with SNVs has been hampered by their inherent genetic complexity and a lack of bioinformatic tools. Here, we expanded our previously reported CoVaMa pipeline (v0.1) to measure linkage disequilibrium between recombination events and SNVs within both short-read and long-read sequencing datasets. We demonstrate this approach using long-read nanopore sequencing data acquired from Flock House virus (FHV) serially passaged in vitro. We found SNVs that were either correlated or anti-correlated with large genomic deletions generated by nonhomologous recombination that give rise to Defective-RNAs. We also analyzed NGS data from longitudinal HIV samples derived from a patient undergoing antiretroviral therapy who proceeded to virological failure. We found correlations between insertions in the p6Gag and mutations in Gag cleavage sites. This report confirms previous findings and provides insights on novel associations between SNVs and specific recombination events within the viral genome and their role in viral evolution.

HSPA8 Chaperone Complex Drives Chaperone-Mediated Autophagy Regulation in Acute Promyelocytic Leukemia Cell Differentiation.

INTRODUCTION: Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapies are focused toward tumor debulking, all-trans retinoic acid (ATRA) induces neutrophil differentiation in the AML subtype acute promyelocytic leukemia (APL). Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML remains elusive. METHODS: We took advantage of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) APL cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cyclic adenosine monophosphate. RESULTS: Here, we report that CMA-related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA-resistant NB4-R1 cells to differentiate upon ATRA treatment but reduced the association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. CONCLUSION: Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones.

MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution.

Technical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolved genetic interactions given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of these genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s of viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure and enables the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

Correction: Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na+/K+ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses.

[This corrects the article DOI: 10.1371/journal.ppat.1006892.].

Engineered Zinc Finger Protein Targeting 2LTR Inhibits HIV Integration in Hematopoietic Stem and Progenitor Cell-Derived Macrophages: In Vitro Study.

Human hematopoietic stem/progenitor cell (HSPC)-based gene therapy is a promising direction for curing HIV-1-infected individuals. The zinc finger protein (2LTRZFP) designed to target the 2-LTR-circle junction of HIV-1 cDNA was previously reported as an intracellular antiviral molecular scaffold that prevents HIV integration. Here, we elucidate the efficacy and safety of using 2LTRZFP in human CD34+ HSPCs. We transduced 2LTRZFP which has the mCherry tag (2LTRZFPmCherry) into human CD34+ HSPCs using a lentiviral vector. The 2LTRZFPmCherry-transduced HSPCs were subsequently differentiated into macrophages. The expression levels of pro-apoptotic proteins of the 2LTRZFPmCherry-transduced HSPCs showed no significant difference from those of the non-transduced control. Furthermore, the 2LTRZFPmCherry-transduced HSPCs were successfully differentiated into mature macrophages, which had normal phagocytic function. The cytokine secretion assay demonstrated that 2LTRZFPmCherry-transduced CD34+ derived macrophages promoted the polarization towards classically activated (M1) subtypes. More importantly, the 2LTRZFPmCherry transduced cells significantly exhibited resistance to HIV-1 integration in vitro. Our findings demonstrate that the 2LTRZFPmCherry-transduced macrophages were found to be functionally and phenotypically normal, with no adverse effects of the anti-HIV-1 scaffold. Our data suggest that the anti-HIV-1 integrase scaffold is a promising antiviral molecule that could be applied to human CD34+ HSPC-based gene therapy for AIDS patients.

Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes.

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.

mTOR inhibitors lower an intrinsic barrier to virus infection mediated by IFITM3.

Rapamycin and its derivatives are specific inhibitors of mammalian target of rapamycin (mTOR) kinase and, as a result, are well-established immunosuppressants and antitumorigenic agents. Additionally, this class of drug promotes gene delivery by facilitating lentiviral vector entry into cells, revealing its potential to improve gene therapy efforts. However, the precise mechanism was unknown. Here, we report that mTOR inhibitor treatment results in down-regulation of the IFN-induced transmembrane (IFITM) proteins. IFITM proteins, especially IFITM3, are potent inhibitors of virus-cell fusion and are broadly active against a range of pathogenic viruses. We found that the effect of rapamycin treatment on lentiviral transduction is diminished upon IFITM silencing or knockout in primary and transformed cells, and the extent of transduction enhancement depends on basal expression of IFITM proteins, with a major contribution from IFITM3. The effect of rapamycin treatment on IFITM3 manifests at the level of protein, but not mRNA, and is selective, as many other endosome-associated transmembrane proteins are unaffected. Rapamycin-mediated degradation of IFITM3 requires endosomal trafficking, ubiquitination, endosomal sorting complex required for transport (ESCRT) machinery, and lysosomal acidification. Since IFITM proteins exhibit broad antiviral activity, we show that mTOR inhibition also promotes infection by another IFITM-sensitive virus, Influenza A virus, but not infection by Sendai virus, which is IFITM-resistant. Our results identify the molecular basis by which mTOR inhibitors enhance virus entry into cells and reveal a previously unrecognized immunosuppressive feature of these clinically important drugs. In addition, this study uncovers a functional convergence between the mTOR pathway and IFITM proteins at endolysosomal membranes.

Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na+/K+ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses.

Several mammalian arenaviruses (mammarenaviruses) cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein (NP) containing an affinity tag (rLCMV/Strep-NP) and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.

Prostaglandin E2 Increases Lentiviral Vector Transduction Efficiency of Adult Human Hematopoietic Stem and Progenitor Cells.

Gene therapy currently in development for hemoglobinopathies utilizes ex vivo lentiviral transduction of CD34+ hematopoietic stem and progenitor cells (HSPCs). A small-molecule screen identified prostaglandin E2 (PGE2) as a positive mediator of lentiviral transduction of CD34+ cells. Supplementation with PGE2 increased lentiviral vector (LVV) transduction of CD34+ cells approximately 2-fold compared to control transduction methods with no effect on cell viability. Transduction efficiency was consistently increased in primary CD34+ cells from multiple normal human donors and from patients with ß-thalassemia or sickle cell disease. Notably, PGE2 increased transduction of repopulating human HSPCs in an immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 gamma receptor null [NSG]) xenotransplantation mouse model without evidence of in vivo toxicity, lineage bias, or a de novo bias of lentiviral integration sites. These data suggest that PGE2 improves lentiviral transduction and increases vector copy number, therefore resulting in increased transgene expression. As a result, PGE2 may be useful in clinical gene therapy applications using lentivirally modified HSPCs.

Inference of Epistatic Effects Leading to Entrenchment and Drug Resistance in HIV-1 Protease.

Understanding the complex mutation patterns that give rise to drug resistant viral strains provides a foundation for developing more effective treatment strategies for HIV/AIDS. Multiple sequence alignments of drug-experienced HIV-1 protease sequences contain networks of many pair correlations which can be used to build a (Potts) Hamiltonian model of these mutation patterns. Using this Hamiltonian model, we translate HIV-1 protease sequence covariation data into quantitative predictions for the probability of observing specific mutation patterns which are in agreement with the observed sequence statistics. We find that the statistical energies of the Potts model are correlated with the fitness of individual proteins containing therapy-associated mutations as estimated by in vitro measurements of protein stability and viral infectivity. We show that the penalty for acquiring primary resistance mutations depends on the epistatic interactions with the sequence background. Primary mutations which lead to drug resistance can become highly advantageous (or entrenched) by the complex mutation patterns which arise in response to drug therapy despite being destabilizing in the wildtype background. Anticipating epistatic effects is important for the design of future protease inhibitor therapies.

Human DMTF1ß antagonizes DMTF1α regulation of the p14(ARF) tumor suppressor and promotes cellular proliferation.

The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, ß and γ, arise from the human gene. We now show that DMP1α, ß and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1ß and γ did not activate the ARF promoter, whereas only ß resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1ß reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1ß- and γ-isoforms share domains necessary for the inhibitory function of the ß-isoform. That DMP1ß may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/ß in the nucleus. Cells stably expressing DMP1ß, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and ß-ratios are tightly regulated in hematopoietic cells and DMP1ß antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.

The Response to Burn Injury in Mice With Human Hematolymphoid Systems.

OBJECTIVE: To develop an animal model of injury that more closely represents the human inflammatory cell response to injury. BACKGROUND: Because the mouse inflammatory response to burn injury cannot account for the contribution of human-specific genes, animal models are needed to more closely recapitulate the human inflammatory response and improve the translational impact of injury research. To this end, we hypothesized that the human inflammatory cell response to injury could be selectively assessed after severe burn injury using humanized mice. METHODS: NOD-Scid-IL2Rγ null mice were transplanted with human hematopoietic CD34+ progenitor cells; their engraftment confirmed and then subjected to 30% total body surface area steam burn injury. Blood, bone marrow, and lung tissue were collected 4 hours after injury and human inflammatory cell mobilization analyzed using flow cytometry and immunohistochemistry. RESULTS: Burn injury caused mobilization of human inflammatory cells into the systemic circulation. Next, burn injury was accompanied by evidence of histologic lung injury and concomitant mobilization of human CD45+ immune cells into the lung that were associated with increased trafficking of human CD11b+ myeloid cells. CONCLUSIONS: These experiments are the first to demonstrate the suitability of humanized mice for injury research. They offer the possibility to address very specific research questions that are not amenable to traditional mouse models of injury, for example, the emerging role of certain human-specific genes that are either unrepresented or totally absent, from the mouse genome.

Rapamycin relieves lentiviral vector transduction resistance in human and mouse hematopoietic stem cells.

Transplantation of genetically modified hematopoietic stem cells (HSCs) is a promising therapeutic strategy for genetic diseases, HIV, and cancer. However, a barrier for clinical HSC gene therapy is the limited efficiency of gene delivery via lentiviral vectors (LVs) into HSCs. We show here that rapamycin, an allosteric inhibitor of the mammalian target of rapamycin complexes, facilitates highly efficient lentiviral transduction of mouse and human HSCs and dramatically enhances marking frequency in long-term engrafting cells in mice. Mechanistically, rapamycin enhanced postbinding endocytic events, leading to increased levels of LV cytoplasmic entry, reverse transcription, and genomic integration. Despite increasing LV copy number, rapamycin did not significantly alter LV integration site profile or chromosomal distribution in mouse HSCs. Rapamycin also enhanced in situ transduction of mouse HSCs via direct intraosseous infusion. Collectively, rapamycin strongly augments LV transduction of HSCs in vitro and in vivo and may prove useful for therapeutic gene delivery.

Nucleocapsid Protein: A Desirable Target for Future Therapies Against HIV-1.

The currently available anti-HIV-1 therapeutics is highly beneficial to infected patients. However, clinical failures occur as a result of the ability of HIV-1 to rapidly mutate. One approach to overcome drug resistance is to target HIV-1 proteins that are highly conserved among phylogenetically distant viral strains and currently not targeted by available therapies. In this respect, the nucleocapsid (NC) protein, a zinc finger protein, is particularly attractive, as it is highly conserved and plays a central role in virus replication, mainly by interacting with nucleic acids. The compelling rationale for considering NC as a viable drug target is illustrated by the fact that point mutants of this protein lead to noninfectious viruses and by the inability to select viruses resistant to a first generation of anti-NC drugs. In our review, we discuss the most relevant properties and functions of NC, as well as recent developments of small molecules targeting NC. Zinc ejectors show strong antiviral activity, but are endowed with a low therapeutic index due to their lack of specificity, which has resulted in toxicity. Currently, they are mainly being investigated for use as topical microbicides. Greater specificity may be achieved by using non-covalent NC inhibitors (NCIs) targeting the hydrophobic platform at the top of the zinc fingers or key nucleic acid partners of NC. Within the last few years, innovative methodologies have been developed to identify NCIs. Though the antiviral activity of the identified NCIs needs still to be improved, these compounds strongly support the druggability of NC and pave the way for future structure-based design and optimization of efficient NCIs.

Safety and efficacy of a tCD25 preselective combination anti-HIV lentiviral vector in human hematopoietic stem and progenitor cells.

The successful suppression of human immunodeficiency virus (HIV) in the "Berlin Patient" has highlighted the ability of HIV-resistant hematopoietic stem cells to offer a potential functional cure for HIV-infected patients. HIV stem cell gene therapy can mimic this result by genetically modifying a patient's own cells with anti-HIV genes. Previous attempts of HIV gene therapy have been hampered by a low percentage of transplanted HIV-resistant cells which has led to minimal clinical efficacy. In our current study, we have evaluated the in vitro and in vivo safety and efficacy of a truncated/mutated form of human CD25 preselective anti-HIV lentiviral vector in human hematopoietic stem cells. This preselective vector allows us to purify vector-transduced cells prior to transplantation so an increased percentage of gene-modified cells can be delivered. Here, we demonstrate the safety of this strategy with successful engraftment and multilineage hematopoiesis of transduced cells in a humanized NOD-RAG1-/-IL-2rγ-/- knockout mouse model. Efficacy was also demonstrated with significant protection from HIV-1 infection including maintenance of human CD4+ cell levels and a decrease in HIV-1 plasma viremia. Collectively, these results establish the utility of this HIV stem cell gene therapy strategy and bring it closer to providing a functional cure for HIV-infected patients.

Deep sequencing of protease inhibitor resistant HIV patient isolates reveals patterns of correlated mutations in Gag and protease.

While the role of drug resistance mutations in HIV protease has been studied comprehensively, mutations in its substrate, Gag, have not been extensively cataloged. Using deep sequencing, we analyzed a unique collection of longitudinal viral samples from 93 patients who have been treated with therapies containing protease inhibitors (PIs). Due to the high sequence coverage within each sample, the frequencies of mutations at individual positions were calculated with high precision. We used this information to characterize the variability in the Gag polyprotein and its effects on PI-therapy outcomes. To examine covariation of mutations between two different sites using deep sequencing data, we developed an approach to estimate the tight bounds on the two-site bivariate probabilities in each viral sample, and the mutual information between pairs of positions based on all the bounds. Utilizing the new methodology we found that mutations in the matrix and p6 proteins contribute to continued therapy failure and have a major role in the network of strongly correlated mutations in the Gag polyprotein, as well as between Gag and protease. Although covariation is not direct evidence of structural propensities, we found the strongest correlations between residues on capsid and matrix of the same Gag protein were often due to structural proximity. This suggests that some of the strongest inter-protein Gag correlations are the result of structural proximity. Moreover, the strong covariation between residues in matrix and capsid at the N-terminus with p1 and p6 at the C-terminus is consistent with residue-residue contacts between these proteins at some point in the viral life cycle.

Mice engrafted with human hematopoietic stem cells support a human myeloid cell inflammatory response in vivo.

Mice engrafted with human CD34+ hematopoietic stem and progenitor cells (CD34+ -HSPCs) have been used to study human infection, diabetes, sepsis, and burn, suggesting that they could be highly amenable to characterizing the human inflammatory response to injury. To this end, human leukocytes infiltrating subcutaneous implants of polyvinyl alcohol (PVA) sponges were analyzed in immunodeficient NSG mice reconstituted with CD34+ -HSPCs. It was reported that human CD45+ (hCD45+ ) leukocytes were present in PVA sponges 3 and 7 days postimplantation and could be localized within the sponges by immunohistochemistry. The different CD45+ subtypes were characterized by flow cytometry and the profile of human cytokines they secreted into PVA wound fluid was assessed using a human-specific multiplex bead analyses of human IL-12p70, TNFα, IL-10, IL-6, IL1ß, and IL-8. This enabled tracking the functional contributions of HLA-DR+ , CD33+ , CD19+ , CD62L+ , CD11b+ , or CX3CR1+ hCD45+ infiltrating inflammatory leukocytes. PCR of cDNA prepared from these cells enabled the assessment and differentiation of human, mouse, and uniquely human genes. These findings support the hypothesis that mice engrafted with CD34+ -HSPCs can be deployed as precision avatars to study the human inflammatory response to injury.

CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data.

Next-Generation Sequencing (NGS) has transformed our understanding of the dynamics and diversity of virus populations for human pathogens and model systems alike. Due to the sensitivity and depth of coverage in NGS, it is possible to measure the frequency of mutations that may be present even at vanishingly low frequencies within the viral population. Here, we describe a simple bioinformatic pipeline called CoVaMa (Co-Variation Mapper) scripted in Python that detects correlated patterns of mutations in a viral sample. Our algorithm takes NGS alignment data and populates large matrices of contingency tables that correspond to every possible pairwise interaction of nucleotides in the viral genome or amino acids in the chosen open reading frame. These tables are then analysed using classical linkage disequilibrium to detect and report evidence of epistasis. We test our analysis with simulated data and then apply the approach to find epistatically linked loci in Flock House Virus genomic RNA grown under controlled cell culture conditions. We also reanalyze NGS data from a large cohort of HIV infected patients and find correlated amino acid substitution events in the protease gene that have arisen in response to anti-viral therapy. This both confirms previous findings and suggests new pairs of interactions within HIV protease. The script is publically available at http://sourceforge.net/projects/covama.

Role of the mammalian target of rapamycin pathway in lentiviral vector transduction of hematopoietic stem cells.

PURPOSE OF REVIEW: A major goal in repopulating hematopoietic stem cell (HSC) gene therapies is achieving high-efficacy gene transfer, while maintaining robust HSC engraftment and differentiation in vivo. Recent studies have documented that rapamycin treatment of HSC during lentiviral vector transduction enhances gene transfer to human and mouse HSCs and maintains engraftment capacity. In this review, we place into context the role of mammalian target of rapamycin (mTOR) pathways in HSC quiescence and function, endocytic regulation, and lentiviral gene delivery. RECENT FINDINGS: Lentiviral vector transduction of human and mouse HSCs is considerably enhanced by rapamycin treatment. Furthermore, rapamycin preserves long-term engraftment of human and mouse HSCs. Investigations of cellular mechanisms that contribute to increased transduction in HSCs uncover a role for mTOR inhibition-dependent activation of endocytosis. SUMMARY: Rapamycin enhances lentiviral vector transduction of HSCs through regulation of endocytic activity via mTOR inhibition. An important attribute of rapamycin treatment during transduction is the preservation of HSC function, allowing reconstitution of long-term hematopoiesis in vivo in murine models.