The pUL51 Tegument Protein Is Essential for Marek's Disease Virus Growth In Vitro and Bears a Function That Is Critical for Pathogenesis In Vivo.

pUL51 is a minor tegument protein important for viral assembly and cell-to-cell spread (CCS) but dispensable for replication in cell culture of all Herpesviruses for which its role has been investigated. Here, we show that pUL51 is essential for the growth of Marek's disease virus, an oncogenic alphaherpesvirus of chickens that is strictly cell-associated in cell culture. MDV pUL51 localized to the Golgi apparatus of infected primary skin fibroblasts, as described for other Herpesviruses. However, the protein was also observed at the surface of lipid droplets in infected chicken keratinocytes, hinting at a possible role of this compartment for viral assembly in the unique cell type involved in MDV shedding in vivo. Deletion of the C-terminal half of pUL51 or fusion of GFP to either the N- or C-terminus were sufficient to disable the protein's essential function(s). However, a virus with a TAP domain fused at the C-terminus of pUL51 was capable of replication in cell culture, albeit with viral spread reduced by 35% and no localization to lipid droplets. In vivo, we observed that although the replication of this virus was moderately impacted, its pathogenesis was strongly impaired. This study describes for the first time the essential role of pUL51 in the biology of a herpesvirus, its association to lipid droplets in a relevant cell type, and its unsuspected role in the pathogenesis of a herpesvirus in its natural host. IMPORTANCE Viruses usually spread from cell to cell through two mechanisms: cell-released virus and/or cell-to-cell spread (CCS). The molecular determinants of CCS and their importance in the biology of viruses during infection of their natural host are unclear. Marek's disease virus (MDV) is a deadly and highly contagious herpesvirus of chickens that produces no cell-free particles in vitro, and therefore, spreads only through CCS in cell culture. Here, we show that viral protein pUL51, an important factor for CCS of Herpesviruses, is essential for MDV growth in vitro. We demonstrate that the fusion of a large tag at the C-terminus of the protein is sufficient to moderately impair viral replication in vivo and almost completely abolish pathogenesis while only slightly reducing viral growth in vitro. This study thus uncovers a role for pUL51 associated with virulence, linked to its C-terminal half, and possibly independent of its essential functions in CCS.

In vivo imaging reveals novel replication sites of a highly oncogenic avian herpesvirus in chickens.

In vivo bioluminescence imaging facilitates the non-invasive visualization of biological processes in living animals. This system has been used to track virus infections mostly in mice and ferrets; however, until now this approach has not been applied to pathogens in avian species. To visualize the infection of an important avian pathogen, we generated Marek's disease virus (MDV) recombinants expressing firefly luciferase during lytic replication. Upon characterization of the recombinant viruses in vitro, chickens were infected and the infection visualized in live animals over the course of 14 days. The luminescence signal was consistent with the known spatiotemporal kinetics of infection and the life cycle of MDV, and correlated well with the viral load measured by qPCR. Intriguingly, this in vivo bioimaging approach revealed two novel sites of MDV replication, the beak and the skin of the feet covered in scales. Feet skin infection was confirmed using a complementary fluorescence bioimaging approach with MDV recombinants expressing mRFP or GFP. Infection was detected in the intermediate epidermal layers of the feet skin that was also shown to produce infectious virus, regardless of the animals' age at and the route of infection. Taken together, this study highlights the value of in vivo whole body bioimaging in avian species by identifying previously overlooked sites of replication and shedding of MDV in the chicken host.

The Tegument Protein pUL47 of Marek's Disease Virus Is Necessary for Horizontal Transmission and Is Important for Expression of Glycoprotein gC.

Viral tropism and transmission of herpesviruses are best studied in their natural host for maximal biological relevance. In the case of alphaherpesviruses, few reports have focused on those aspects, primarily because of the few animal models available as natural hosts that are compatible with such studies. Here, using Marek's disease virus (MDV), a highly contagious and deadly alphaherpesvirus of chickens, we analyze the role of tegument proteins pUL47 and pUL48 in the whole life cycle of the virus. We report that a virus lacking the UL48 gene (vΔUL48) is impaired in growth in cell culture and has diminished virulence in vivo In contrast, a virus lacking UL47 (vΔUL47) is unaffected in its growth in vitro and is as virulent in vivo as the wild-type (WT) virus. Surprisingly, we observed that vΔUL47 was unable to be horizontally transmitted to naive chickens, in contrast to the WT virus. In addition, we show that pUL47 is important for the splicing of UL44 transcripts encoding glycoprotein gC, a protein known as being essential for horizontal transmission of MDV. Importantly, we observed that the levels of gC are lower in the absence of pUL47. Notably, this phenotype is similar to that of another transmission-incompetent mutant ΔUL54, which also affects the splicing of UL44 transcripts. This is the first study describing the role of pUL47 in both viral transmission and the splicing and expression of gC.IMPORTANCE Host-to-host transmission of viruses is ideally studied in vivo in the natural host. Veterinary viruses such as Marek's disease virus (MDV) are, therefore, models of choice to explore these aspects. The natural host of MDV, the chicken, is small, inexpensive, and economically important. MDV is a deadly and contagious herpesvirus that can kill infected animals in less than 4 weeks. The virus naturally infects epithelial cells of the feather follicle epithelium from where it is shed into the environment. In this study, we demonstrate that the viral protein pUL47 is an essential factor for bird-to-bird transmission of the virus. We provide some molecular basis to this function by showing that pUL47 enhances the splicing and the expression of another viral gene, UL44, which is essential for viral transmission. pUL47 may have a similar function in human herpesviruses such as varicella-zoster virus or herpes simplex viruses.

Dynamic organization of Herpesvirus glycoproteins on the viral envelope revealed by super-resolution microscopy.

The processes of cell attachment and membrane fusion of Herpes Simplex Virus 1 involve many different envelope glycoproteins. Viral proteins gC and gD bind to cellular receptors. Upon binding, gD activates the gH/gL complex which in turn activates gB to trigger membrane fusion. Thus, these proteins must be located at the point of contact between cellular and viral envelopes to interact and allow fusion. Using super-resolution microscopy, we show that gB, gH/gL and most of gC are distributed evenly round purified virions. In contrast, gD localizes essentially as clusters which are distinct from gB and gH/gL. Upon cell binding, we observe that all glycoproteins, including gD, have a similar ring-like pattern, but the diameter of these rings was significantly smaller than those observed on cell-free viruses. We also observe that contrary to cell-free particles, gD mostly colocalizes with other glycoproteins on cell-bound particles. The differing patterns of localization of gD between cell-free and cell-bound viruses indicates that gD can be reorganized on the viral envelope following either a possible maturation of the viral particle or its adsorption to the cell. This redistribution of glycoproteins upon cell attachment could contribute to initiate the cascade of activations leading to membrane fusion.

Insights into herpesvirus tegument organization from structural analyses of the 970 central residues of HSV-1 UL36 protein.

The tegument of all herpesviruses contains a capsid-bound large protein that is essential for multiple viral processes, including capsid transport, decapsidation at the nuclear pore complex, particle assembly, and secondary envelopment, through mechanisms that are still incompletely understood. We report here a structural characterization of the central 970 residues of this protein for herpes simplex virus type 1 (HSV-1 UL36, 3164 residues). This large fragment is essentially a 34-nm-long monomeric fiber. The crystal structure of its C terminus shows an elongated domain-swapped dimer. Modeling and molecular dynamics simulations give a likely molecular organization for the monomeric form and extend our findings to alphaherpesvirinae. Hence, we propose that an essential feature of UL36 is the existence in its central region of a stalk capable of connecting capsid and membrane across the tegument and that the ability to switch between monomeric and dimeric forms may help UL36 fulfill its multiple functions.

Le transport intracellulaire des herpèsvirus.

Intracellular trafficking of viruses is a vital part of the viral life cycle because of the density of the cytosol and of important distances between organelles necessary for viral replication. Viral transport is active, regulated and requires the host machinery. Transport issues are particularly important for neurotropic alpha-herpesviruses whose large capsids travel through large distances in highly specialized cells such as neurons or epithelial cells. In this review, I will summarize the knowledge accumulated for the last 15 years on the intracellular trafficking of these viruses. In this fairly recent field of research, they are among the best described because they are easily genetically modified and also because they recruit the cellular transport machinery very efficiently.

Differing effects of herpes simplex virus 1 and pseudorabies virus infections on centrosomal function.

Efficient intracellular transport of the capsid of alphaherpesviruses, such as herpes simplex virus 1 (HSV-1), is known to be dependent upon the microtubule (MT) network. Typically, the MT network radiates from an MT-organizing center (MTOC), which is, in most cases, the centrosome. During herpesvirus egress, it has been assumed that capsids travel first from the nucleus to the centrosome and then from the centrosome to the site of envelopment. Here we report that the centrosome is no longer a primary MTOC in HSV-1-infected cells, but it retains this function in cells infected by another alphaherpesvirus, pseudorabies virus (PrV). As a result, MTs formed at late times after infection with PrV grow from a major, centralized MTOC, while those formed after HSV-1 infection arise from dispersed locations in the cytoplasm, indicating the presence of alternative and minor MTOCs. Thus, loss of the principal MT nucleating center in cells following HSV-1 infection raises questions about the mechanism of HSV-1 capsid egress. It is possible that, rather than passing via the centrosome, capsids may travel directly to the site of envelopment after exiting the nucleus. We suggest that, in HSV-1-infected cells, the disruption of centrosomal functions triggers reorganization of the MT network to favor noncentrosomal MTs and promote efficient viral spread.

Herpesvirus tegument protein pUL37 interacts with dystonin/BPAG1 to promote capsid transport on microtubules during egress.

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that travels long distances through cells using the microtubule network. Its 125-nm-diameter capsid is a large cargo which efficiently recruits molecular motors for movement. Upon entry, capsids reach the centrosome by minus-end-directed transport. From there, they are believed to reach the nucleus by plus-end-directed transport. Plus-end-directed transport is also important during egress, when capsids leave the nucleus to reach the site of envelopment in the cytoplasm. Although capsid interactions with dynein and kinesins have been described in vitro, the actual composition of the cellular machinery recruited by herpesviruses for capsid transport in infected cells remains unknown. Here, we identify the spectraplakin protein, dystonin/BPAG1, an important cytoskeleton cross-linker involved in microtubule-based transport, as a binding partner of the HSV-1 protein pUL37, which has been implicated in capsid transport. Viral replication is delayed in dystonin-depleted cells, and, using video microscopy of living infected cells, we show that dystonin depletion strongly inhibits capsid movement in the cytoplasm during egress. This study provides new insights into the cellular requirements for HSV-1 capsid transport and identifies dystonin as a nonmotor protein part of the transport machinery.

Dystonin/BPAG1 promotes plus-end-directed transport of herpes simplex virus 1 capsids on microtubules during entry.

During infection by herpes simplex virus 1 (HSV-1), the viral capsid is transported around the cytoplasm along the microtubule (MT) network. Although molecular motors have been implicated in this process, the composition of the molecular machinery required for efficient directional transport is unknown. We previously showed that dystonin (BPAG1) is recruited to HSV-1 capsids by the capsid-bound tegument protein pUL37 to promote efficient cytoplasmic transport of capsids during egress. Dystonin is a cytoskeleton cross-linker which localizes at MT plus ends and has roles in retrograde and anterograde transport in neurons. In this study, we investigated the role of dystonin during the entry stages of HSV-1 infection. Because of the way in which the MT network is organized, capsids are required to change their direction of motion along the MTs as they travel from the point of entry to the nucleus, where replication takes place. Thus, capsids first travel to the centrosome (the principal microtubule organizing center) by minus-end-directed transport and then switch polarity and travel to the nucleus by plus-end-directed transport. We observed that transport of capsids toward the centrosome was slowed, but not blocked, by dystonin depletion. However, transport of capsids away from the centrosome was significantly impaired, causing them to accumulate in the vicinity of the centrosome and reducing the numbers reaching the nucleus. We conclude that, during entry of HSV-1, dystonin has a specific role in plus-ended transport of capsids from the centrosome to the nucleus.

Automatic measurements of left ventricular volumes and ejection fraction by artificial intelligence: clinical validation in real time and large databases.

AIMS: Echocardiography is a cornerstone in cardiac imaging, and left ventricular (LV) ejection fraction (EF) is a key parameter for patient management. Recent advances in artificial intelligence (AI) have enabled fully automatic measurements of LV volumes and EF both during scanning and in stored recordings. The aim of this study was to evaluate the impact of implementing AI measurements on acquisition and processing time and test-retest reproducibility compared with standard clinical workflow, as well as to study the agreement with reference in large internal and external databases. METHODS AND RESULTS: Fully automatic measurements of LV volumes and EF by a novel AI software were compared with manual measurements in the following clinical scenarios: (i) in real time use during scanning of 50 consecutive patients, (ii) in 40 subjects with repeated echocardiographic examinations and manual measurements by 4 readers, and (iii) in large internal and external research databases of 1881 and 849 subjects, respectively. Real-time AI measurements significantly reduced the total acquisition and processing time by 77% (median 5.3 min, P < 0.001) compared with standard clinical workflow. Test-retest reproducibility of AI measurements was superior in inter-observer scenarios and non-inferior in intra-observer scenarios. AI measurements showed good agreement with reference measurements both in real time and in large research databases. CONCLUSION: The software reduced the time taken to perform and volumetrically analyse routine echocardiograms without a decrease in accuracy compared with experts.

Real-time guiding by deep learning during echocardiography to reduce left ventricular foreshortening and measurement variability.

Aims: Apical foreshortening leads to an underestimation of left ventricular (LV) volumes and an overestimation of LV ejection fraction and global longitudinal strain. Real-time guiding using deep learning (DL) during echocardiography to reduce foreshortening could improve standardization and reduce variability. We aimed to study the effect of real-time DL guiding during echocardiography on measures of LV foreshortening and inter-observer variability. Methods and results: Patients (n = 88) in sinus rhythm referred for echocardiography without indication for contrast were included. All participants underwent three echocardiograms. The first two examinations were performed by sonographers, and the third by cardiologists. In Period 1, the sonographers were instructed to provide high-quality echocardiograms. In Period 2, the DL guiding was used by the second sonographer. One blinded expert measured LV length in all recordings. Tri-plane recordings by cardiologists were used as reference. Apical foreshortening was calculated at the end-diastole. Both sonographer groups significantly foreshortened the LV in Period 1 (mean foreshortening: Sonographer 1: 4 mm; Sonographer 2: 3 mm, both P < 0.001 vs. reference) and reduced foreshortening in Period 2 (2 and 0 mm, respectively. Period 1 vs. Period 2, P < 0.05). Sonographers using DL guiding did not foreshorten more than cardiologists (P ≥ 0.409). Real-time guiding did not improve intra-class correlation (ICC) [LV end-diastolic volume ICC, (95% confidence interval): DL guiding 0.87 (0.77-0.93) vs. no guiding 0.92 (0.88-0.95)]. Conclusion: Real-time guiding reduced foreshortening among experienced operators and has the potential to improve image standardization. Even though the effect on inter-operator variability was minimal among experienced users, real-time guiding may improve test-retest variability among less experienced users. Clinical trial registration: ClinicalTrials.gov, Identifier: NCT04580095.

Real-Time Echocardiography Guidance for Optimized Apical Standard Views.

Measurements of cardiac function such as left ventricular ejection fraction and myocardial strain are typically based on 2-D ultrasound imaging. The reliability of these measurements depends on the correct pose of the transducer such that the 2-D imaging plane properly aligns with the heart for standard measurement views and is thus dependent on the operator's skills. We propose a deep learning tool that suggests transducer movements to help users navigate toward the required standard views while scanning. The tool can simplify echocardiography for less experienced users and improve image standardization for more experienced users. Training data were generated by slicing 3-D ultrasound volumes, which permits simulation of the movements of a 2-D transducer. Neural networks were further trained to calculate the transducer position in a regression fashion. The method was validated and tested on 2-D images from several data sets representative of a prospective clinical setting. The method proposed the adequate transducer movement 75% of the time when averaging over all degrees of freedom and 95% of the time when considering transducer rotation solely. Real-time application examples illustrate the direct relation between the transducer movements, the ultrasound image and the provided feedback.

Real-time guidance by deep learning of experienced operators to improve the standardization of echocardiographic acquisitions.

Aims: Impaired standardization of echocardiograms may increase inter-operator variability. This study aimed to determine whether the real-time guidance of experienced sonographers by deep learning (DL) could improve the standardization of apical recordings. Methods and results: Patients (n = 88) in sinus rhythm referred for echocardiography were included. All participants underwent three examinations, whereof two were performed by sonographers and the third by cardiologists. In the first study period (Period 1), the sonographers were instructed to provide echocardiograms for the analyses of the left ventricular function. Subsequently, after brief training, the DL guidance was used in Period 2 by the sonographer performing the second examination. View standardization was quantified retrospectively by a human expert as the primary endpoint and the DL algorithm as the secondary endpoint. All recordings were scored in rotation and tilt both separately and combined and were categorized as standardized or non-standardized. Sonographers using DL guidance had more standardized acquisitions for the combination of rotation and tilt than sonographers without guidance in both periods (all P ≤ 0.05) when evaluated by the human expert and DL [except for the apical two-chamber (A2C) view by DL evaluation]. When rotation and tilt were analysed individually, A2C and apical long-axis rotation and A2C tilt were significantly improved, and the others were numerically improved when evaluated by the echocardiography expert. Furthermore, all, except for A2C rotation, were significantly improved when evaluated by DL (P < 0.01). Conclusion: Real-time guidance by DL improved the standardization of echocardiographic acquisitions by experienced sonographers. Future studies should evaluate the impact with respect to variability of measurements and when used by less-experienced operators. ClinicalTrialsgov Identifier: NCT04580095.

Genetic editing of herpes simplex virus 1 and Epstein-Barr herpesvirus genomes by human APOBEC3 cytidine deaminases in culture and in vivo.

Human APOBEC3 cytidine deaminases target and edit single-stranded DNA, which can be of viral, mitochondrial, or nuclear origin. Retrovirus genomes, such as human immunodeficiency virus (HIV) genomes deficient in the vif gene and the hepatitis B virus genome, are particularly vulnerable. The genomes of some DNA viruses, such as human papillomaviruses, can be edited in vivo and in transfection experiments. Accordingly, herpesviruses should be no exception. This is indeed the case for herpes simplex virus 1 (HSV-1) in tissue culture, where APOBEC3C (A3C) overexpression can reduce virus titers and the particle/PFU ratio ∼10-fold. Nonetheless, A3A, A3G, and AICDA can edit what is presumably a small fraction of HSV genomes in an experimental setting without seriously impacting the viral titer. Hyperediting was found in HSV genomes recovered from 4/8 uncultured buccal lesions. The phenomenon is not restricted to HSV, since hyperedited Epstein-Barr virus (EBV) genomes were readily recovered from 4/5 established cell lines, indicating that episomes are vulnerable to editing. These findings suggest that the widely expressed A3C cytidine deaminase can function as a restriction factor for some human herpesviruses. That the A3C gene is not induced by type I interferons begs the question whether some herpesviruses encode A3C antagonists.

Inner tegument protein pUL37 of herpes simplex virus type 1 is involved in directing capsids to the trans-Golgi network for envelopment.

Secondary envelopment of herpes simplex virus type 1 has been demonstrated as taking place at the trans-Golgi network (TGN). The inner tegument proteins pUL36 and pUL37 and the envelope glycoproteins gD and gE are known to be important for secondary envelopment. We compared the cellular localizations of capsids from a virus mutant lacking the UL37 gene with those of a virus mutant lacking the genes encoding gD and gE. Although wild-type capsids accumulated at the TGN, capsids of the pUL37(-) mutant were distributed throughout the cytoplasm and showed no association with TGN-derived vesicles. This was in contrast to capsids from a gD(-)gE(-) mutant, which accumulated in the vicinity of TGN vesicles, but did not colocalize with them, suggesting that they were transported to the TGN but were unable to undergo envelopment. We conclude that the inner tegument protein pUL37 is required for directing capsids to the TGN, where secondary envelopment occurs.

Herpesvirus capsid association with the nuclear pore complex and viral DNA release involve the nucleoporin CAN/Nup214 and the capsid protein pUL25.

After penetrating the host cell, the herpesvirus capsid is transported to the nucleus along the microtubule network and docks to the nuclear pore complex before releasing the viral DNA into the nucleus. The viral and cellular interactions involved in the docking process are poorly characterized. However, the minor capsid protein pUL25 has recently been reported to be involved in viral DNA uncoating. Here we show that herpes simplex virus type 1 (HSV-1) capsids interact with the nucleoporin CAN/Nup214 in infected cells and that RNA silencing of CAN/Nup214 delays the onset of viral DNA replication in the nucleus. We also show that pUL25 interacts with CAN/Nup214 and another nucleoporin, hCG1, and binds to the pUL36 and pUL6 proteins, two other components of the herpesvirus particle that are known to be important for the initiation of infection and viral DNA release. These results identify CAN/Nup214 as being a nuclear receptor for the herpesvirus capsid and pUL25 as being an interface between incoming capsids and the nuclear pore complex and as being a triggering element for viral DNA release into the nucleus.

Differing roles of inner tegument proteins pUL36 and pUL37 during entry of herpes simplex virus type 1.

Studies with herpes simplex virus type 1 (HSV-1) have shown that secondary envelopment and virus release are blocked in mutants deleted for the tegument protein gene UL36 or UL37, leading to the accumulation of DNA-containing capsids in the cytoplasm of infected cells. The failure to assemble infectious virions has meant that the roles of these genes in the initial stages of infection could not be investigated. To circumvent this, cells infected at a low multiplicity were fused to form syncytia, thereby allowing capsids released from infected nuclei access to uninfected nuclei without having to cross a plasma membrane. Visualization of virus DNA replication showed that a UL37-minus mutant was capable of transmitting infection to all the nuclei within a syncytium as efficiently as the wild-type HSV-1 strain 17(+) did, whereas infection by UL36-minus mutants failed to spread. Thus, these inner tegument proteins have differing functions, with pUL36 being essential during both the assembly and uptake stages of infection, while pUL37 is needed for the formation of virions but is not required during the initial stages of infection. Analysis of noninfectious enveloped particles (L-particles) further showed that pUL36 and pUL37 are dependent on each other for incorporation into tegument.

Nucleocytoplasmic shuttling of the rabies virus P protein requires a nuclear localization signal and a CRM1-dependent nuclear export signal.

Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal part of P (172-297). This domain contains a short lysine-rich stretch ((211)KKYK(214)) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to beta-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products.