- Volume 1, Issue 1A, 2019

Herpes simplex virus 1 (HSV-1) establishes latency in sensory neurons, allowing it to persist for the lifetime of the host. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 2.0 kb major LAT intron and several microRNAs. These non-coding RNAs (ncRNAs) have been reported to influence latency, possibly through limiting apoptosis of infected cells. As these studies have used animal models or non-neuronal cell culture, we have developed a differentiated human neuroblastoma (SH-SY5Y cells) model to examine their effect in human neuronal cells. We have infected these neuronal cultures with replication-defective HSV-1, which establishes a quiescent infection and strongly expresses the latency ncRNAs. We show that quiescent HSV-1 infection reproducibly protects differentiated SH-SY5Y from etoposide-induced apoptosis. We are also further defining the contribution of different LAT ncRNAs using recombinant lentiviruses to drive expression of the LAT intron or microRNAs. Furthermore, we are also currently exploring the mechanisms of this anti-apoptosis effect, and broader virus-neuron interactions by characterising whether the human neuronal transcriptome is altered by LAT RNA expression. Improving our understanding of the molecular interactions underpinning HSV-1 latency in neurons could help develop novel therapies to target HSV-1 latency.

High-risk human papillomavirus (HR-HPV) infects epithelial cells and is the major cause of anogenital and oropharyngeal cancers. HR-HPV oncogenic activity is through E6 control of p53, but E6 binds and degrades PDZ proteins such as the tumour suppress protein hDlg (human homologue of DrosophilaDiscs Large). The E6/hDlg complex also contains Connexin 43 (Cx43) (MacDonald, Sun et al. 2012), the major building block of gap junctions that allow intercellular molecular communication. In HPV16-positive non-tumour cervical keratinocytes (W12NT: low E6 levels) Cx43/hDlg is on the plasma membrane but in the cytoplasm in W12T tumour epithelial cells (W12T: high E6 levels) correlating with loss of gap junction cell-cell communication. E6 siRNA depletion in W12T cells restored Cx43 to the cell membrane, while overexpressing E6 in HPV-negative cervical cancer cells C33a resulted in Cx43 moving to the cytoplasm. E6 could control Cx43 trafficking through controlling hDlg or by altering cell signalling. In the absence of E6 in HEK293, HaCaT and normal immortalised keratinocytes (NIKS), Cx43 and hDlg could be co-immunoprecipitated and they co-localised on the plasma membrane. Thus, the Cx43/hDlg interaction is not carcinoma cell-specific, is not dependent on HR-HPV E6, and may have a functional role in non-cancer cells. siRNA depletion of hDlg, led to reduction in Cx43 protein levels and some relocation to the cytoplasm. This indicates that HPV E6 controls Cx43 through interaction with and degradation of hDlg. However, we cannot discount that E6 itself may have additional effects on Cx43 levels and trafficking besides via hDlg.

Events controlling herpesvirus nuclear genome uncoating, nuclear transport, and the onset of transcription remain poorly understood. We have now developed procedures to examine these processes within individual cells and at the single molecule level for both the genome and the transcripts produced from it. We have combined two novel techniques of, firstly, bioorthogonal chemistry to visualise genomes which incorporate an alkyne-nucleoside analogue (ethynyl deoxycytidine, EdC) and secondly, single molecule RNA in-situ hybridisation (smFISH) which allows detection of individual mRNA transcripts. Using these techniques simultaneously, we can now qualitatively and quantitatively analyse individual transcript abundances and their intracellular localisation, in relation to the genome itself at single molecule resolution during the progression of infection. Moreover, we are able to examine these parameters when a single genome infects a cell. We have examined the transcripts of the immediate-early mRNA for ICP0, and features revealed from this work include; transcriptional ‘bursting’ with clustered transcripts around individual genomes; mean mRNA transcript number, variance, and intracellular localisation produced from a single genome; the progressive abundant ICP0 transcription occurring selectively from replicated genomes; an increasing bottleneck in cytoplasmic transport of transcripts emanating from replicated genomes; and increased transcription bursts from virtually every uncoated genome when protein synthesis is suppressed. Further, by multiplexing probes, we can simultaneously analyse distinct transcription outputs from different genes of the same or classes, and genomes, in the same individual cell. Our results reveal completely new perspectives on the very early events of genome presentation and transcription from those genomes.

Epstein-Barr virus (EBV), is an oncogenic gamma-herpesvirus, which is associated with malignant diseases of B cells, T cells, and epithelial cells. EB viruses have large DNA genomes of more than 170 kb that are difficult to clone and manipulate. Here we describe 2 different approaches for cloning whole EBV genomes of diverse strains for reverse genetics studies. The first approach used CRISPR/Cas9-mediated cloning of the entire EBV genome into a bacterial artificial chromosome (BAC) vector using homologous recombination in B cells. This method allowed the cloning of the type 2 EBV strain Jijoye for the first time, but the BAC-clones are unstable. This strategy is being modified by recoding the homology regions to make the clones more stable. The second approach involves transformation-associated recombination (TAR) cloning of EBV fragments and their assembly in yeast, which will allow for mixing and matching DNA regions from different EBV strains for functional studies. This approach is based on TAR cloning of the EBV genome as 10 overlapping fragments, which average 17 kilobases long, using the natural homologous recombination processes of the yeast. Subsequent assembly of all the overlapping fragments is undertaken in yeast or by Gibson assembly to reconstitute the infectious EBV clone. Two fragments from EBV strains B95-8 and AG876 were captured and isolated successfully, but at low efficiency. We are currently improving the TAR cloning efficiency by increasing the size of the capture homology regions to approximately 500 bp coupled with CRISPR/Cas-9-mediated fragmentation of the EBV genome.

Hepatitis B virus causes chronic liver infection in 257 million people worldwide. Current treatments against HBV can control, but not cure HBV infection. Therefore, new treatments for chronic HBV infection need to be developed. The APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3) proteins are cellular restriction factors, which have been shown to restrict viral replication for a number of viruses (e.g. HIV) and for retrotransposons. The aim of this study is to elucidate the role of APOBEC3 proteins in inhibiting Hepatitis B virus replication. Quantitative polymerase chain reaction (qPCR) was used to evaluate the impact of APOBEC3 family members on the Hepatitis B virus replication in HepG2.2.15 cells. The highest inhibition of intracellular capsid associated HBV DNA, extracellular virion associated HBV DNA was induced by APOBEC3DE, APOBEC3F, and APOBEC3G as compared to the other APOBEC3 proteins. However, APOBEC3DE showed no inhibition of HBV total RNA, whereas the highest inhibition of HBV total RNA was induced by APOBEC3F and APOBEC3G. The sub-cellular localisation of APOBEC3 proteins was determined by immunofluorescence using confocal microscopy. It was found that APOBEC3DE, APOBEC3F, and APOBEC3G localise to the cytoplasm, suggesting a crucial role of these proteins in HBV replication in the cytoplasm. Nevertheless, APOBEC3A and APOBEC3B localise to the nucleus. The expression and co-localisation of APOBEC3 proteins and viral and host proteins (Uracil DNA Glycosylase UNG and ATP-dependent RNA helicase DDX3) in HepG2.2.15 and HEK293T cells are being investigated in order to determine the interaction between these proteins.

To evade innate immunity, many viruses express interferon-antagonists that target STATs, critical mediators of immune signalling. Virus-STAT interfaces may provide new therapeutic targets but progress is hindered by a lack of direct structural data, owing to poor tractability of antagonists/full-length STATs for structural/biophysical approaches. By applying cross-saturation transfer NMR, we report the first direct structural analysis of binding of full-length STAT1 to an interferon-antagonist of a human pathogenic virus, the first such study of the virus-host interface. Analysis using mutation of the interface, biophysical characterization, immune signalling/protein-protein interaction assays including PCA, reverse genetics and animal infection demonstrated the significance of this interface in immune signaling suppression, and in disease caused by a pathogenic field-strain lyssavirus. Importantly, NMR/mutagenesis also revealed that the interface comprises multiple surfaces/domains in both the viral and cellular partners, indicating that antagonism involves extensive interactions consistent with a multifaceted inhibitory mechanism, distinct from ‘simple’ mechanisms such as tethering. Furthermore, by elucidating the spatial relationship of interactions critical to immune evasion and replication, the data provide insight into how ostensibly simple viruses can regulate these central functions via a single multifunctional protein. These data provide novel insights into fundamental viral biology, and potential exploitation of these mechanisms as new targets for antivirals and vaccine development. The study also demonstrates the power of biophysical/NMR approaches to elucidate the atomic interface of full-length STATs with regulatory proteins, providing a framework for studies to reveal immune evasion mechanisms of other pathogens in their full complexity.

The accessory protein Vpr of Human Immunodeficiency Virus type 1 (HIV-1) enhances replication of the virus in macrophages. Virus particle packaged Vpr is released in target cells shortly after entry, suggesting it is required early in infection. Why it is required for infection of macrophages and not cycling T-cells and why it induces G2/M arrest in cycling cells are unknown. Here we observe, by co-immunoprecipitation assay, an interaction between Vpr and endogenous REAF (RNA-associated Early-stage Antiviral Factor, RPRD2), a protein shown previously to potently restrict HIV infection. After HIV-1 infects macrophages, within 30 min of viral entry, Vpr induces the degradation of REAF. Subsequently, as replication continues, REAF expression is upregulated – a response which is curtailed by Vpr. REAF is more highly expressed in differentiated macrophages than in cycling T-cells. Expression in cycling cells is cell-cycle dependent and knockdown induces cell-cycle perturbation. Therefore, our results support the long held hypothesis that Vpr induces the degradation of a factor involved in the cell cycle that impedes HIV infection in macrophages.

Tegument protein pUL83 is the most abundant component of human cytomegalovirus (hCMV) particles. The viral protein is predicted to be composed of three domains: a pyrin association domain (PAD), a carboxy-terminal domain (CTD), and an intrinsically disordered linker domain (amino acids 388–479) located between the PAD and CTD. Although pUL83 has been shown to antagonize interferon (IFN) responses, it has not been fully elucidated how the viral protein may contribute to hCMV replication. In this study we demonstrate that pUL83 associates broadly with viral and host chromatin including condensed chromosomes during mitosis. We further show that the linker domain in pUL83 is both required and sufficient for host chromatin targeting, and that this interaction depends on two evolutionary conserved arginine residues (R453 and R455) in the viral protein. Our data indicate that the pUL83 linker domain specifically associates with human core histones (but not linker histones). Furthermore, pUL83 inhibits IFN-beta and IFN-lambda gene induction, but not expression of other cytokine genes, via a mechanism that largely depends on the linker domain including R453/455. Although earlier studies suggested that pUL83 is dispensable for productive hCMV infection in fibroblasts, we find that the viral protein is necessary for efficient plaque formation in these cells, specifically in the presence of IFN. Finally, the pUL83 linker domain including R453/455 contributes significantly to the plaque size in hCMV-infected fibroblasts. Overall, we propose that pUL83 promotes spread of hCMV by selectively inhibiting induction of IFN gene expression via a novel chromatin-based molecular mechanism involving core histones.

Type I interferons (IFN) are potent inducers of an anti-viral state in response to infection and have been demonstrated to inhibit cytomegalovirus (CMV) replication both in vitro and in vivo. CMV, like all herpes viruses, has the capacity to establish lifelong infections of host through the establishment of latency. As the very early stages of viral entry can trigger IFN responses we investigated the impact of IFN on the establishment of latent human CMV (HCMV) in myeloid progenitor cells. Here we show that priming of myeloid THP1 cells with type I IFN prior to infection skews infection towards a more efficient establishment of latency. This is evidenced by detection of reduced lytic gene expression, increased latent gene expression, and increased levels of reactivation following differentiation. Blockade of IFN signalling with neutralising antibodies antagonised the latent phenotype suggesting that endogenous IFN production upon infection contributed to the effect observed. Intriguingly, whilst both IFNα2 and IFNβ can drive latent infection individually, their effects were dose-dependent and demonstrated a biphasic impact on the establishment of latency, with the highest doses of IFN preventing both lytic and latent infection. These data demonstrate that the HCMV derives an unexpected benefit from IFN production. They support a hypothesis that, although anti-viral in nature, concentration-specific effects of IFN may be evident in the cells which can modulate different outcomes post infection in persistent viruses such as HCMV. Future work is identifying the IFN concentration-specific effects responsible for a cellular environment that favours the establishment of latency.

DNA sensors including cGAS, STING and IFI16 are key components of the innate immune response to infection. However, the precise mechanisms of action, in particular the relative importance of direct suppression of replication versus paracrine signalling of an antiviral state to susceptible cells remains unclear. We examined the kinetics of herpes simplex virus infection and spread in a relevant cell type, human keratinocytes, lacking one or other of these DNA sensors using time-lapse microscopy. We also examine transcriptional induction of interferon from the native locus, at single cell and single molecule level using highly sensitive RNA FISH. Our results reveal distinct aspects of the roles of these factors and reveal outcomes not appreciated by other methods. Cells lacking either of these factors showed increased susceptibility to initial infection (prior to any downstream paracrine signalling) but with quite different outcomes. Lack of cGAS resulted in increased cellular migration and cell density at the infection focus. On the other hand, cells lacking STING showed lower cell density and significantly increased cytopathic effect likely curtailing virus yield. Initial results demonstrate that we can analyse interferon transcription at single cell level with exquisite sensitivity down to a few transcripts per cell and reveal profound spatial heterogeneity in responses to induction by PAMP ligands. Altogether, our results reveal new insight into the spatial landscape of the initiation and spread of HSV and key cellular responses which likely integrate pathways including innate immunity, apoptosis and cell migration.

African horse sickness is a major infectious disease of equids and is caused by African horse sickness virus (AHSV), a dsRNA virus with 10 genome segments encoding for 7 structural and 4/5 non-structural proteins. Here, we focused on the characterisation of the AHSV NS4, the latest protein found to be expressed by this virus. In silico analysis of available sequences confirmed the existence of two phylogenetically distinct AHSV clades: NS4-I and NS4-II. NS4-II is further divided into three subtypes (a, b and g). Confocal microscopy demonstrated that all AHSV NS4 types localised in the cytoplasm of infected cells, unlike the BTV NS4 which, has a strong nucleolar localisation. The replication kinetics of reverse genetics derived AHSV NS4 deletion mutants (AHSVDNS4) were similar to their wild type counterparts in insect (Kc) or interferon incompetent (BSR) cells. However, replication of AHSVDNS4 mutants in primary horse endothelial cells was restricted, in comparison to wild-type viruses. Importantly, primary cells restriction to AHSV replication was dependent on the JAK/STAT pathway. Furthermore, AHSVDNS4 mutants were not able to efficiently suppress the secretion of anti-viral cytokines from primary cells, while the wild-type viruses suppressed this response to varying degrees. Importantly, AHSVDNS4 mutants were less virulent than their wild type counterparts in a murine model of AHSV infection. These results indicate that AHSV NS4 has a role in interferon IFN antagonism and a determinant of viral virulence. We are currently carrying out mass spectrometry analyses to identify the cellular proteins interacting with the AHSV NS4.

Paramyxoviruses (PVs) are negative-sense RNA viruses that are important in human and animal health, and can cause severe zoonotic diseases. We screened a wide range of animal and human PV matrix proteins for host interactions, and found that the long (L) isoform of the host protein ZAP (zinc-finger antiviral protein) interacted with all the tested viral matrixes. ZAP-L is constitutively expressed, has a prenlyation motif for membrane-localization, and primarily mediates antiviral activity by binding and degrading viral RNAs through the exosome complex. However, ZAP restriction of PVs has not been demonstrated in the literature. We found that knockdown of ZAP-L results in increased replication of a panel of five human and animal PVs. Overexpression of ZAP-L (but not ZAP-short) restricts replication of PVs in a reciprocal pattern – with the notable exception of Sendai virus (SeV) – and as with other viruses, mutating the prenylation motif of ZAP-L abolishes restriction. RT-qPCR of PV RNAs and pulled-down RNAs does not indicate specific targeting of a viral transcript by ZAP-L, although overall genome abundance is reduced. Finally, immunoprecipitation of ZAP shows an additional RNA-independent interaction between ZAP-L and SeV-nucleocapsid not found with HPIV3-nucleocapsid, a closely-related PV. Thus, we have observed that ZAP-L interacts with the matrix of, and restricts replication of, a wide range of paramyxoviruses. A PV that is not restricted (SeV), has an additional interaction with ZAP via its nucleocapsid protein that may ameliorate ZAP restriction. Investigating ZAP-related restriction differences between closely-related PVs may shed light on anti-viral mechanisms of ZAP.

Louping ill virus (LIV; Flavivirus, Flaviviridae) is an important—but poorly characterized—animal pathogen of significant economic concern within the UK. Transmitted by ticks, LIV predominantly causes disease in ruminants and grouse, resulting in heavy losses. LIV is closely related to another flavivirus—tick-borne encephalitis virus (TBEV) which, unlike LIV, is a significant human pathogen. The molecular mechanisms that underpin host restriction in these viruses are poorly understood however, previously it has been shown that the TBEV non-structural (NS) proteins do not act as type-I interferon (IFN) antagonists, unlike many other Flavivirus NS proteins. Therefore, to facilitate comparison with LIV we investigated the possible antagonistic actions of the LIV NS proteins using a luciferase-based IFN reporter assay. Utilising this assay we identified six LIV NS proteins that function as antagonists throughout the IFN induction cascade. We also identified and modelled a subgenomic flavivirus RNA (sfRNA) that is produced during LIV infection and is similar in structure to TBEV sfRNA. We found that the LIV and TBEV sfRNAs antagonise RIG-I, indicating that the IFN antagonistic ability of sfRNA is not limited to mosquito-borne flaviviruses. Finally, we established the first LIV reverse genetics system using circular polymerase extension reaction (CPER). This powerful tool can be used to produce chimeric viruses which will allow further investigation into the factors governing host restriction and virulence in tick-borne flaviviruses. Investigating the mechanisms that underlie LIV infection aids our understanding of interferon antagonism in flaviviruses and the molecular determinants of host restriction.

The Birnaviridae family is comprised of non-enveloped viruses with a double-stranded RNA genome that is divided into two segments, A and B. Birnaviruses are responsible for major economic losses to the poultry and aquaculture industries, and reassortment complicates their epidemiology and control. However, little is known about the nature of theirreplication in cells, or the molecular mechanism underpinning reassortment. In order to address this, we rescued two recombinant infectious bursal disease (IBD) viruses, with either a GFP11 or Tetracysteine (TC) tag at the 3’ end of segment B (IBDV-GFP11 and IBDV-TC, respectively). DF-1 cells were either transfected with GFP1-10 prior to IBDV-GFP11 infection, or stained with ReAsH following IBDV-TC infection, which led to the apprearance of green or red foci in the cytoplasm, respectively. Foci co-localised with VP3 and dsRNA, suggesting these were virus factories (VFs). The average number of VFs significantly decreased from 60 to 5 per cell between 10 and 24 h post infection (P<0.01), while the average area significantly increased from 1.24 µm2 to 45.01 µm2 (P<0.01), suggesting VFs coalesce in the cytoplasm over time. Red, green and yellow foci were observed in the cytoplasm of co-infected cells, suggesting that VFs are initially derived from distinct input viruses prior to coalescence. Live cell imaging revealed that larger VFs were more static while smaller VFs were more mobile, and fusion events were observed. We speculate that VF coalescence is required for birnavirus reassortment, and current work is aimed at determining the cellular factors that drive coalescence.

The pneumoviruses human and bovine respiratory syncytial virus (RSV) are significant global respiratory viral pathogens responsible for causing lower respiratory tract infections in humans and cattle, respectively. The formation of progeny virions at the cell surface requires the coordinated assembly of glycoproteins trafficked to the apical surface of cells via the host secretary pathway, other viral protein complexes assembled in cytoplasm inclusion bodies and lastly viral ribonucleoproteins. Although some host proteins and pathways have been implicated in pneumovirus budding and assembly (e.g. HSP90, Rab-11 and apical recycling endosomes), the full spectrum of virus-host interactions has not been fully elucidated. Using a siRNA library targeting membrane trafficking and the Incucyte® Live Cell Imaging System, we have developed and optimised a high-throughput siRNA protocol for characterising RSV egress. The siRNA library targets human proteins which are known, or predicted, to be involved in membrane trafficking or remodelling, while the Incucyte® System allows near real-time imaging over an extended time-course, generating high quality data that allows the monitoring and quantification of multiple parameters such as cell viability, viral replication and syncytia formation. Using a recombinant human RSV expressing a GFP reporter we have identified a number of proteins involved in pneumovirus trafficking in infected cells. Our techniques provide a robust and sensitive mechanism for genetic screening and the identification of pneumovirus-protein interactions.

Bluetongue virus (BTV), in family Reoviridae, is an insect-borne, double capsid virus causing haemorrhagic disease in livestock around the world. Here, we elucidate how outer capsid proteins VP2 and VP5 coordinate cell entry of BTV. The recently solved high-resolution structures reveal unique features of BTV VP2 and VP5. To identify key functional residues, we used atomic-level structural data to guide mutagenesis of VP2 and VP5 and a series of biological and biochemical approaches, including site-directed mutagenesis, reverse genetics-based virus recovery, expression and characterization of individual recombinant mutant proteins, and various in vitro and in vivo assays. We demonstrate the dynamic nature of the conformational change process, revealing that a unique zinc finger (CCCH) in VP2 acts as the major low pH sensor, coordinating VP2 detachment, subsequently allowing VP5 to sense low pH via specific histidine residues at key positions. We show that single substitution of only certain histidine residues has a lethal effect, indicating that the location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show that the VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveal a novel, multiconformational process that overcomes entry barriers faced by this multicapsid nonenveloped virus.

BK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that can establish a lifelong, silently persistent infection in the kidney and is estimated to infect 70–90 % of the world’s population. In immunocompromised individuals, particularly bone marrow and kidney transplant patients, increases in BKPyV replication can result in significant pathological conditions. In the case of kidney transplant patients, this can result in nephropathy, which in severe cases can result in the deterioration of allograft function and loss of the transplanted organ. There are currently no antiviral treatments with strong evidence of clinical efficacy against BKPyV. Though little is known about BKPyV egress from infected cells, we have evidence showing that BKPyV can be released in a non-lytic manner by an unconventional cellular secretory pathway that bypasses the Golgi apparatus. Here, we investigate the mechanisms behind BKPyV non-lytic egress through studying the effects of knocking out candidate host proteins involved in unconventional secretory pathways on BKPyV release, examining the effects of BKPyV infection on host cell protein secretion, and ascertaining which host proteins are essential for the BKPyV life cycle via a whole-genome CRISPR screen. These experiments are uncovering novel virus-host interactions that, when targeted, could lead to antiviral effects.