An Investigation of Hepatitis B Virus Genome using Markov Models

The human genome encodes a family of editing enzymes known as APOBEC3 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3). Several family members, such as APO-BEC3G, APOBEC3F, and APOBEC3H haplotype II, exhibit activity against viruses such as HIV. These enzymes induce C-to-U mutations in the negative strand of viral genomes, resulting in multiple G-to-A changes, commonly referred to as 'hypermutation.' Mutations catalyzed by these enzymes are sequence context-dependent in the HIV genome; for instance, APOBEC3G preferen-tially mutates G within GG, TGG, and TGGG contexts, while other members mutate G within GA, TGA, and TGAA contexts. However, the same sequence context has not been explored in relation to these enzymes and HBV. In this study, our objective is to identify the mutational footprint of APOBEC3 enzymes in the HBV genome. To achieve this, we employ a multivariable data analytics technique to investigate motif preferences and potential sequence hierarchies of mutation by APOBEC3 enzymes using full genome HBV sequences from a diverse range of naturally infected patients. This approach allows us to distinguish between normal and hypermutated sequences based on the representation of mono- to tetra-nucleotide motifs. Additionally, we aim to identify motifs associated with hypermutation induced by different APOBEC3 enzymes in HBV genomes. Our analyses reveal that either APOBEC3 enzymes are not active against HBV, or the induction of G-to-A mutations by these enzymes is not sequence context-dependent in the HBV genome

The Impact of Host Factors on Retroviral Evolution and the Identification of a Novel Receptor That Was Used by an Ancient Primate Retrovirus

The resurrection of inactive endogenous retroviruses allows us to learn about interactions between extinct pathogens and their hosts that occurred millions of years ago. Two of these paleoviruses, chimpanzee endogenous retrovirus 1 and 2 (CERV1 and CERV2), are relatives of modern murine leukemia viruses that are found in the genomes of a variety of old world primates, but are absent from the human genome. The nonexistence of human CERV1 and CERV2 homologues is peculiar given the numerous apparent cross-species transmissions that occurred between ancestors of old world monkeys, gorillas, and chimpanzees. It is possible that antiviral proteins were able to protect human ancestors from colonization by CERV1 and CERV2. Indeed, sequence analyses of modern primate restriction factors has suggested that these genes have evolved under positive selection, presumably due to their combat with invading pathogens throughout primate history. Here we investigate whether TRIM5 and APOBEC3 antiviral factors were able to restrict the replication of CERV1 and CERV2. Such an interaction would imply a potential involvement of these proteins in the limited host range and, perhaps, even the extinction of CERV1 and CERV2. Reciprocally, activity against CERV1 and CERV2 would suggest that archaic gammaretroviruses contributed to the positive selection observed in TRIM5 and APOBEC3 genes. Our analyses suggest that TRIM5 proteins did not pose a major barrier to the cross-species transmission or contributed to the extinction of CERV1 and CERV2. However, we uncovered extensive evidence for the inactivation of endogenous gammaretroviruses by the action of APOBEC3 cytidine deaminases. Both CERV1 and CERV2, as well as their homologues in the rhesus macaque, bore mutational scars that are characteristic of APOBEC3 activity. A reconstructed CERV2 Gag was used in vitro to confirm that APOBEC3G was capable of restricting CERV2 infection. Therefore, it appears that primate APOBEC3 proteins were capable of targeting ancient primate gammaretroviruses. It remains possible that APOBEC3 proteins were able to limit the cross-species transmission and cause the inactivation of these viruses

Development of a data processing toolkit for the analysis of next-generation sequencing data generated using the primer ID approach

Philosophiae Doctor - PhDSequencing an HIV quasispecies with next generation sequencing technologies yields a dataset with significant amplification bias and errors resulting from both the PCR and sequencing steps. Both the amplification bias and sequencing error can be reduced by labelling each cDNA (generated during the reverse transcription of the viral RNA to DNA prior to PCR) with a random sequence tag called a Primer ID (PID). Processing PID data requires additional computational steps, presenting a barrier to the uptake of this method. MotifBinner is an R package designed to handle PID data with a focus on resolving potential problems in the dataset. MotifBinner groups sequences into bins by their PID tags, identifies and removes false unique bins, produced from sequencing errors in the PID tags, as well as removing outlier sequences from within a bin. MotifBinner produces a consensus sequence for each bin, as well as a detailed report for the dataset, detailing the number of sequences per bin, the number of outlying sequences per bin, rates of chimerism, the number of degenerate letters in the final consensus sequences and the most divergent consensus sequences (potential contaminants). We characterized the ability of the PID approach to reduce the effect of sequencing error, to detect minority variants in viral quasispecies and to reduce the rates of PCR induced recombination. We produced reference samples with known variants at known frequencies to study the effectiveness of increasing PCR elongation time, decreasing the number of PCR cycles, and sample partitioning, by means of dPCR (droplet PCR), on PCR induced recombination. After sequencing these artificial samples with the PID approach, each consensus sequence was compared to the known variants. There are complex relationships between the sample preparation protocol and the characteristics of the resulting dataset. We produce a set of recommendations that can be used to inform sample preparation that is the most useful the particular study. The AMP trial infuses HIV-negative patients with the VRC01 antibody and monitors for HIV infections. Accurately timing the infection event and reconstructing the founder viruses of these infections are critical for relating infection risk to antibody titer and homology between the founder virus and antibody binding sites. Dr. Paul Edlefsen at the Fred Hutch Cancer Research Institute developed a pipeline that performs infection timing and founder reconstruction. Here, we document a portion of the pipeline, produce detailed tests for that portion of the pipeline and investigate the robustness of some of the tools used in the pipeline to violations of their assumptions

Modelling the Evolution of HIV-1 Protein-Coding Sequences with Particular focus on the early stages of Infection

Modelling the Evolution of HIV-1 Protein-Coding Sequences with Particular Focus on the Early Stages of Infection Natasha Thandi Wood, February 2010 The evolution of the viral genome sequence over the course of HIV-1 infection is of interest for vaccine and drug design, and for the development of effective treatment strategies. Characteristics of the transmitted viral genome that could render the virus more sensitive to host immune responses, are of particular interest for vaccine studies. However, sequence samples from the earliest phase of HIV infection are scarce, and inferences about the nature of the infecting virus and its evolution during the course of early infection are often made from samples isolated from later stages, or from chronic infections. To establish in detail the adaptive changes that occur in early infection, an investigation was carried out on a large dataset consisting of sequences isolated from individuals in early infection. The majority of these infections were inferred to have resulted from transmission of a single virion or virally infected cell, which permitted a detailed investigation of HIV-1 diversification in early infection for the first time. Comparing viral diversification across multiple patients, it was possible to identify specific evolutionary patterns in the HIV-1 genome that occur frequently during the earliest stages of infection. The analyses revealed that APOBEC-mediated hypermutation has an important role in early viral diversification and may enable rapid escape from the first wave of host immune responses. Several mutations in early infection that were likely to result in immune escape were identified, some of which have subsequently been confirmed experimentally. In general, experimental verification of model-based inferences is necessary, but can be expensive and time-consuming. To reduce the costs involved, it is essential that the evolutionary methods produce accurate results. Simulation results presented in this thesis show that inferences made about viral evolution can be subject to bias when key aspects of viral biology are not accounted for by the models used. In particular, some previous comparisons between sequence groups that share genealogical histories, positive selection studies that fail to account for recombination, and research on HIV covariation, may need to be revisited, using more accurate evolutionary models. The results presented in this thesis demonstrate the importance of accurate evolutionary models to understand the selection pressures acting on the virus during various stages of infection. Furthermore, using a phylogenetic model it was possible to identify sites in the HIV genome that were evolving adaptively and are implicated in CTL immune escape during early infection. Characterising escape mutations in the transmitted virus may lead to novel approaches to develop vaccines and antiviral drugs

REPLICATION-COMPETENT NON-INDUCED PROVIRUSES IN THE LATENT RESERVOIR INCREASE BARRIER TO HIV-1 CURE

Antiretroviral therapy (ART) fails to cure HIV-1 infection because latent proviruses persist in resting CD4+ T cells. T cell activation reverses latency, but >99% of proviruses are not induced to release infectious virus after maximum in vitro T cell activation under standard viral outgrowth assay conditions. These non-induced proviruses are generally considered defective but have never been characterized. Using limiting dilution near-full length nested PCR and Poisson distribution analysis, we characterized 213 clones of non-induced proviruses from eight aviremic patients on suppressive ART. Most (88.3%) of the non-induced proviruses are defective, including 45.5% large internal deletions, 32.4% APOBEC-mediated G→A hypermutations, 6.6% mutations/deletions in the cis-acting element, and 3.8% insertions/nonsense mutations. Strikingly, 11.7% of the non-induced proviruses have intact genomes. Using direct sequencing and de novo genome synthesis, we reconstructed six full-length non-induced proviral clones and demonstrated growth kinetics comparable to four reconstructed induced proviruses from the same patients. Using luciferase assay to measure non-induced proviral LTR activity, we found that non-induced proviruses have intact promoter function unless they are hypermutated. Using limiting dilution bisulfite sequencing, we found that non-induced proviruses have unmethylated promoters. Using inverse PCR, we found that non-induced proviruses are integrated into active transcription units. We demonstrated that these non-induced proviruses, though not induced after in vitro maximum T cell activation, can be reactivated after repeated stimuli. We propose that maximum T cell activation does not lead to maximum non-induced provirus activation. Rather, activation of non-induced proviruses is stochastic. Thus, it cannot be excluded that non-induced proviruses may become activated in vivo. The discovery of replication-competent non-induced proviruses indicates that the size of the latent reservoir, and hence the barrier to cure, may be ~60-fold greater than previously estimated. Underestimation of intact proviruses by viral outgrowth assays could be reflected in delayed viral rebound after an apparent “cure”, and overestimation of latent reservoir size resulting from detection of defective proviruses by PCR assays could result in prolonged, excessive exposure to toxic latency reversing agents. Thus, the molecular analysis of non-induced proviruses contributes in an important way to HIV-1 eradication efforts

The Interplay between CD27dull and CD27bright B Cells Ensures the Flexibility, Stability, and Resilience of Human B Cell Memory

Summary: Memory B cells (MBCs) epitomize the adaptation of the immune system to the environment. We identify two MBC subsets in peripheral blood, CD27dull and CD27bright MBCs, whose frequency changes with age. Heavy chain variable region (VH) usage, somatic mutation frequency replacement-to-silent ratio, and CDR3 property changes, reflecting consecutive selection of highly antigen-specific, low cross-reactive antibody variants, all demonstrate that CD27dull and CD27bright MBCs represent sequential MBC developmental stages, and stringent antigen-driven pressure selects CD27dull into the CD27bright MBC pool. Dynamics of human MBCs are exploited in pregnancy, when 50% of maternal MBCs are lost and CD27dull MBCs transit to the more differentiated CD27bright stage. In the postpartum period, the maternal MBC pool is replenished by the expansion of persistent CD27dull clones. Thus, the stability and flexibility of human B cell memory is ensured by CD27dull MBCs that expand and differentiate in response to change. : Grimsholm et al. show that CD27dull and CD27bright represent sequential MBC developmental stages. T cell- and germinal center (GC)-independent CD27dull MBCs are the plastic source of strongly selected and GC-dependent CD27bright MBCs. CD27dull MBCs, able to expand and differentiate in response to change, ensure stability and flexibility of human B cell memory. Keywords: memory B cells, pregnancy, immunological memory, CD27, VH repertoire, immunodeficiency, aging, spleen, vaccine, germinal cente

Dynamics of Intact Proviruses in Perinatal HIV-1 Infection

Human Immunodeficiency Virus (HIV) has impacted the lives of nearly 76 million and about a million people become infected each year even with the use of current preventative strategies and antiretroviral therapy (ART). An ART-free remission/cure strategy is needed. The HIV-1 reservoir poses a major hurdle to developing an HIV cure as it fosters HIV-1 persistence through viral quiescence. Measuring the true size of the reservoir is complex owing to dominance of defective HIV-1 genomes, making it harder to specifically target therapies towards the intact proviral reservoir. The assays that are currently used under-or over-estimate the size of the reservoir. A recently established PCR assay, Intact proviral DNA assay (IPDA) was found to differentiate between intact and defective HIV-1 genomes. It has helped in studying the reservoir in adults, but no extensive study using the IPDA have been performed in children. Objective: To apply the IPDA to study the dynamics of the HIV-1 proviral reservoir in children and inform strategies for ART-free remission. Methods: IPDA was optimized for use on the limited number of cells that are generally available from children, by determining the best type of control sample, the optimal DNA isolation technique and genomic DNA input amount to analyze each sample. The IPDA was applied to study the composition and the dynamics of the different DNA species in the HIV-1 reservoir in participants from a previously well characterized cohort (PHACS-AMP) in whom a conventional total HIV-1 DNA assay had been used to quantify HIV-1 reservoirs. Results: 25 PHACS-AMP participants were eligible for this study. The distribution of intact HIV-1 proviruses/million PBMCs was lower in those that achieved virologic suppression at <1 year of age vs the those that achieved virologic suppression (VS) between 1-5 years of age. In contrast the defective HIV- 1 genomes demonstrated an expansion in size regardless of age at VS. Conclusion: Early effective ART decreases the size of the reservoir in children with perinatal HIV-1 infection which is mainly due to the decline in the intact HIV-1 proviral reservoir

Patterns and rates of viral evolution in HIV-1 subtype B infected females and males.

Biological sex differences affect the course of HIV infection, with untreated women having lower viral loads compared to their male counterparts but, for a given viral load, women have a higher rate of progression to AIDS. However, the vast majority of data on viral evolution, a process that is clearly impacted by host immunity and could be impacted by sex differences, has been derived from men. We conducted an intensive analysis of HIV-1 gag and env-gp120 evolution taken over the first 6-11 years of infection from 8 Women's Interagency HIV Study (WIHS) participants who had not received combination antiretroviral therapy (ART). This was compared to similar data previously collected from men, with both groups infected with HIV-1 subtype B. Early virus populations in men and women were generally homogenous with no differences in diversity between sexes. No differences in ensuing nucleotide substitution rates were found between the female and male cohorts studied herein. As previously reported for men, time to peak diversity in env-gp120 in women was positively associated with time to CD4+ cell count below 200 (P = 0.017), and the number of predicted N-linked glycosylation sites generally increased over time, followed by a plateau or decline, with the majority of changes localized to the V1-V2 region. These findings strongly suggest that the sex differences in HIV-1 disease progression attributed to immune system composition and sensitivities are not revealed by, nor do they impact, global patterns of viral evolution, the latter of which proceeds similarly in women and men