Designer Oncolytic Adenovirus: Coming of Age

The licensing of talimogene laherparepvec (T-Vec) represented a landmark moment for oncolytic virotherapy, since it provided unequivocal evidence for the long-touted potential of genetically modified replicating viruses as anti-cancer agents. Whilst T-Vec is promising as a locally delivered virotherapy, especially in combination with immune-checkpoint inhibitors, the quest continues for a virus capable of specific tumour cell killing via systemic administration. One candidate is oncolytic adenovirus (Ad); it’s double stranded DNA genome is easily manipulated and a wide range of strategies and technologies have been employed to empower the vector with improved pharmacokinetics and tumour targeting ability. As well characterised clinical and experimental agents, we have detailed knowledge of adenoviruses’ mechanisms of pathogenicity, supported by detailed virological studies and in vivo interactions. In this review we highlight the strides made in the engineering of bespoke adenoviral vectors to specifically infect, replicate within, and destroy tumour cells. We discuss how mutations in genes regulating adenoviral replication after cell entry can be used to restrict replication to the tumour, and summarise how detailed knowledge of viral capsid interactions enable rational modification to eliminate native tropisms, and simultaneously promote active uptake by cancerous tissues. We argue that these designer-viruses, exploiting the viruses natural mechanisms and regulated at every level of replication, represent the ideal platforms for local overexpression of therapeutic transgenes such as immunomodulatory agents. Where T-Vec has paved the way, Ad-based vectors now follow. The era of designer oncolytic virotherapies looks decidedly as though it will soon become a reality

Molecular mechanisms of human innate immune restriction factors APOBEC3G and APOBEC3H for inhibiting the retrovirus HIV-1 and retrotransposon LINE1

The APOBEC3 restriction factors belong to a family of cytidine deaminases that are able to suppress the replication of viruses with a single-stranded DNA intermediate by inducing mutagenesis and functional inactivation of the virus. Of the seven common human APOBEC3 enzymes, only APOBEC3-D, -F, -G and -H appear to be relevant to HIV-1 restriction in CD4+ T cells. The restriction of HIV-1 by APOBEC3 enzymes occurs most potently in the absence of HIV-1 Vif, which counteracts APOBEC3s by inducing polyubiquitination and subsequent degradation of APOBEC3 enzymes. Virion-encapsidated APOBEC3s can deaminate cytosines to uracils in viral (-)DNA. Upon replication of (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite to the uracils hereby inducing C/G to T/A transition mutations. Among all APOBEC3 enzymes that are relevant to HIV-1 restriction, APOBEC3G is the most studied APOBEC3 enzyme. APOBEC3G has been shown to processively catalyze deamination reactions on single-stranded (-)DNA using a mechanism called facilitated diffusion, which involves sliding and jumping movements in search of target cytosine-containing motifs. This single-stranded DNA scanning mechanism allows APOBEC3G to efficiently deaminate multiple cytosines within one enzyme-DNA encounter and it is important for the mutational inactivation of HIV-1 in vivo. Vif attempts to neutralize APOBEC3G’s function not only by inducing proteasomal degradation, but also by several degradation-independent mechanisms, such as inhibiting APOBEC3G virion encapsidation, mRNA translation, and for those APOBEC3G molecules that still become virion encapsidated, Vif has been shown to inhibit APOBEC3G’s deamination activity. My Ph.D. thesis work investigated the molecular mechanism of degradation-independent Vif-mediated inhibition of APOBEC3G and APOBEC3H deamination activity. This research led to the development of the hypothesis that Vif has developed a unique interaction with each APOBEC3 enzyme due to the different selection pressures they impose on HIV-1. Thus, we investigated how the interaction of Vif differs between APOBEC3G and APOBEC3H and characterized the activity of APOBEC3H as a restriction factor. This research allowed us to have a better understanding of the molecular determinants that govern an efficient APOBEC3-degradatation by HIV-1 Vif and provide insights for APOBEC3-based HIV-1 therapeutics. Two Vif variants obtained from HIV-1 laboratory isolates, VifHXB2 and VifIIIB, were used to determine the degradation-independent effects of Vif on APOBEC3G. Biochemical assays using a model HIV-1 replication assay and synthetic single-stranded or partially double-stranded DNA substrates demonstrated that APOBEC3G has an altered processive mechanism in the presence of Vif, and this caused APOBEC3G to be less effective at inducing mutagenesis in a model HIV-1 replication assay. APOBEC3H is unique in that it is the only single-domain common APOBEC3 enzyme that restricts HIV-1. APOBEC3H exists in humans as seven haplotypes (I-VII) with different cellular stabilities. Amongst three stable APOBEC3H haplotypes, haplotype II and V occur most frequently in the population. I characterized the single-stranded DNA scanning mechanisms that haplotype II and V use to search their single-stranded substrate for cytosine-containing deamination motif. APOBEC3H haplotype II was able to processively deaminate its substrate using Brownian motion-driven movements termed sliding, jumping and intersegmental transfer, whereas haplotype V showed compromised sliding and intersegmental transfer abilities but was able to jump along single-stranded DNA. Since an Asp or Glu at amino acid 178 differentiates these two haplotypes, these data suggest this amino acid on predicted helix 6 contributes to processivity. I found the optimal processivity on ssDNA also required dimerization of APOBEC3H through the β2 strands. The diminished processivity of APOBEC3H haplotype V did not result in a reduced efficiency to restrict HIV-1 replication in single-cycle infectivity assay. This suggests a redundancy in the contribution of jumping and intersegmental transfer to mutagenic efficiency. VifHXB2, but not VifIIIB, can cause degradation of APOBEC3H even though APOBEC3H interacts with both Vif variants. APOBEC3G is degraded after interaction with both of these Vif variants. To define the parameters for efficient Vif-induced degradation of an APOBEC3 enzyme, I used an in vitro quantitative method to determine the binding strength of APOBEC3G and APOBEC3H with Vif variant heterotetramers (Vif/CBFβ/EloB/EloC), the most stable form of Vif. Our biochemical analysis, along with cellular experiments to determine Vif-induced degradation efficiency and APOBEC3-Vif interactions in cells, support a model in which the degradation efficiency of Vifs correlates with both the APOBEC3-Vif binding strength and APOBEC3-Vif interface. I also investigated how APOBEC3 enzymes restrict the replication of retrotransposon LINE-1. Retrotransposons are DNA sequences that replicate using a “copy-and-paste” mechanism through an RNA intermediate. The degradation of deaminated L1 cDNA rendered it difficult to detect any APOBEC3-induced G-to-A mutations while the addition of uracil DNA glycosylase inhibitor allowed for the recovery of the APOBEC3-mediated deamination events. I found that two stable A3H haplotypes (haplotype II and haplotype V) use a deamination-independent mechanism to restrict L1 mobilization and compared the ability of APOBEC3H’s to inhibit LINE1 with that of APOBEC3A and APOBEC3G, two APOBEC3s whose LINE1 restriction ability have been previously characterized. Taken together, these studies of the molecular mechanisms that APOBEC3G and APOBEC3H use to inhibit HIV-1 and LINE1 have allowed us to better understand their biological properties as cytidine deaminases and the determinants in APOBEC3s that made them efficient host innate immune restriction factors

Epi-genomic determinants of HIV-1 integration in primary CD4+ T cells and macrophages

The infection with HIV-1 nowadays does not represent a condition with a deadly outcome. Due to current therapeutic approaches, the infection with HIV-1 represents a chronic condition in which viral load is kept at undetectable levels, but patients depend on a lifelong therapy without a chance of cure. The eradication of integrated viral DNA still remains the biggest challenge in curing HIV-1. The aim of this work was to contribute to a better understanding and definition of genomic regions and epi-genomic features that HIV-1 targets for integration, and give a detailed description on the importance of chromatin accessibility, as well as the importance of certain genomic features in the process of HIV-1 integration. The first part of this project deals with the importance of HMT G9a activity and H3K9me2 histone mark distribution and deposition in the context of HIV-1 integration in primary CD4+ T cells, which was studied by the application of G9a inhibitor BIX0129, also known as a very potent latency reversing agent. The significance of G9a activity and facultative heterochromatin mark H3K9me2 deposition has previously been shown to affect T cell development and impact shaping of the nuclear architecture. In this work it was demonstrated that the chemical inhibition of G9a and depletion of H3K9me2 by BIX01294 has an increasing effect on HIV-1 integration. The increase in integration was also followed by increased viral transcriptional activity, as well as spatial repositioning of the provirus from the preferred nuclear periphery towards the nuclear center. Similar spatial repositioning has been demonstrated for genes highly and recurrently targeted by HIV-1 for integration (RIGs). However, genic nuclear repositioning upon BIX01294 treatment did not affect transcriptional profiles of HIV-1 RIGs, as demonstrated by RNA microarray analysis, but other groups of genes mainly involved in iron metabolism and inflammatory response were upregulated upon BIX01294 treatment. In addition, HIV-1 integration patterns were shown not to be affected by H3K9me2 depletion, and the virus was still targeting similar genic regions for integration. The analysis of chromatin mark distribution and chromatin binding elements upon BIX01294 treatment on RIGs revealed increased binding profiles of open chromatin mark H3K36me3 which is followed by increased LEDGF/p75 binding upon H3K9me2 depletion. The observed phenomenon might provide an explanation for the observed increased viral integration upon BIX01294 treatment, considering that LEDGF/p75 is a prominent host cell factor involved in the viral integration process. Overall, the first part of this study clearly demonstrated that chromatin accessibility significantly affects HIV-1 integration levels which are directly proportional to viral expression levels and viral activity. The second part of this study deals with the relevance of R-loops, as specific genomic structures, as sites selected for HIV-1 integration in primary CD4+ T cells and macrophages. It was demonstrated that the GFP tagged IN enzyme of HIV-1, in a high occurrence, colocalizes with R-loops in cells, and that for the occurrence of this process a functionally active IN is required. This finding implicated that the observed colocalization is not randomly taking place and that HIV-1 is actively docked to R-loop forming genomic sites. In addition, biochemical as well as computational meta data analysis revealed that HIV-1 RIGs are enriched in R-loops and that R-loop forming sites can accommodate integrated viral DNA. Further on, it was demonstrated that HIV-1 IN has R-loop binding capacity and is also capable of performing the strand transfer reaction on R-loop containing DNA templates. It was also demonstrated that R-loop depletion by RNase H1 overexpression in several cell lines, as well as in primary cells, significantly impairs HIV-1 integration, indicating that R-loop presence is crucial for efficient HIV-1 integration. In line with this result was the finding that RIGs expression was not affected by R-loop removal, indicating that only the presence of R-loops, as structural genomic elements, is more affecting HIV-1 integration compared to gene expression levels. The final finding is also in line with previous work from our lab. In summary, the second part of this study provides strong evidence that R-loops represent structural genomic elements targeted by HIV-1 for integration and also gives new insight into HIV-1 IN functional III features which have not been addressed before

Innate Immune Responses in HIV-1 Infected Macrophages

In this study M-CSF differentiated human monocyte derived macrophages were used to investigate HIV-1 interactions with macrophage innate immune responses. Macrophages may be an important host cell for HIV-1. HIV-1 can infect and replicate within these cells without causing host cell cytopathicity unlike in T cells. Macrophages also aid in the spread of the virus and are likely to act as a viral reservoir protected from antivirals and immune responses due to the unique localisation of the virus within these cells. Surprisingly HIV-1 infection has little effect on the steady state transcriptome of MDM and despite the role of macrophages to detect incoming pathogens; no innate immune response to HIV-1 could be detected, in contrast with other viruses tested. The lack of immune response was not due to active viral suppression and addition of exogenous IFN or activation of the innate immune response at the time of HIV-1 infection can restrict viral infection in these cells, despite HIV-1 having a full complement of accessory proteins known to counteract IFN inducible restriction factors. This HIV-1 restriction induced by IFN was long lasting, likely for the lifetime of the MDM. IFN treatment of MDM with established HIV-1 infection however only transiently suppressed viral replication. Comparisons of MDM with other cell types which do show an innate immune IFN response to HIV-1 showed that MDM have relatively low levels of TLR7 gene expression, suggesting that MDM may lack one of the PRRs for detecting HIV-1. HIV-1 infection of MDM was found to attenuate NFκB activation in response to TLR stimulation and this attenuation could be reversed by priming the MDM with IFNγ. However this attenuation of the NFκB signal did not translate into decreased protein expression for a selection of proinflammatory cytokines examined

Physiology, syntrophy and viral interplay in the marine sponge holobiont

Holobionts result from intimate associations of eukaryotic hosts and microbes and are now widely accepted as ubiquitous and important elements of nature. Marine sponge holobionts combine simple morphology and complex microbiology whilst diverging early in the animal kingdom. As filter feeders, sponges feed on planktonic bacteria, but also harbour stable species-specific microbial consortia. This interaction with bacteria renders sponges to exciting systems to study basal determinants of animal-microbe symbioses. While inventories of symbiont taxa and gene functions continue to grow, we still know little about the symbiont physiology, cellular interactions and metabolic currencies within sponges. This limits our mechanistic understanding of holobiont stability and function. Therefore, this PhD thesis set out to study the questions of what individual symbionts actually do and how they interact. The first part of this thesis focuses on the cell physiology of cosmopolitan sponge symbionts. For the first time, I characterised the ultrastructure of dominant sponge symbiont clades within sponge tissue by establishing fluorescence in situ hybridization-correlative light and electron microscopy (FISH-CLEM). In combination with genome-centred metatranscriptomics, this approach revealed structural adaptations of symbionts to process complex holobiont-derived nutrients (i.e., bacterial microcompartments and bipolar storage polymers). Next, we unravelled complementary symbiont physiologies and cell co-localisation indicating vivid symbiont-symbiont metabolic interactions within the holobiont. This suggests strategies of nutritional resource partitioning and syntrophy to dominate over spatial segregation to avoid competitive exclusion- a mechanistic framework to sustain high microbial diversity. By combining stable isotope pulse-chase experiments with metabolic imaging, we demonstrated that symbionts can account for up to 60 % of the heterotrophic carbon and nitrogen assimilation in sponges. Thus, sponge symbiont action determines sponge-driven biochemical cycles in marine ecosystems. Finally, I explored the role of phages in the sponge holobiont focussing on tripartie phage-microbe-host interplay. Sponges appeared as rich reservoirs of novel viral diversity with 491 previously unidentified genus-level viral clades. Further, sponges harboured highly individual, yet species-specific viral communities. Importantly, I discovered that phages, termed “Ankyphages”, abundantly encode ankyrin proteins. Such “Ankyphages” I found to be widespread in host-associated environments, including humans. Using macrophage infection assays I showed that phage ankyrins aid bacteria in eukaryote immune evasion by downregulating eukaryotic antibacterial immunity. Thus, I identified a potentially widespread mechanism of tripartite phage-prokaryote-host interplay where phages foster animal-microbe symbioses. Altogether, I draw three main conclusions: The sponge holobiont is a metabolically intertwined ecosystem, with symbiont action impacting the environment, and tripartite phage-prokaryote-eukaryote interplay fostering symbiosis

Spumaretroviruses

Foamy viruses, currently referred to as spumaretroviruses, are the most ancient retroviruses as evidenced by traces of viral sequences dispersed in all vertebrate classes from fish to mammals. Additionally, infectious foamy viruses circulate in a variety of mammalian species including simian, bovine, equine, caprine, and feline. Foamy viruses have many unique features which led to the division of the retrovirus family into two subfamilies, the Orthoretrovirinae and Spumaretrovirinae. In vitro, foamy viruses have a broad host range and in vivo, human infections have been described due to cross-species transmission from infected nonhuman primates. Thus far, there are no reports of virus-induced disease in humans or in the natural host species. These unique properties of foamy viruses have led researchers to develop foamy viruses as gene therapy vectors to study virus–virus and virus–host interactions for identifying factors involved in virus replication, transmission, and immune regulation that could influence potential clinical outcomes in humans as well as for using endogenous foamy virus sequences in the analysis of host species evolution