TheraVision: Engineering platform technology for the development of oncolytic viruses based on herpes simplex virus type 1.

Viruses are able to efficiently penetrate cells, multiply, and eventually kill infected cells, release tumor antigens, and activate the immune system. Therefore, viruses are highly attractive novel agents for cancer therapy. Clinical trials with first generations of oncolytic viruses (OVs) are very promising but show significant need for optimization. The aim of TheraVision was to establish a broadly applicable engineering platform technology for combinatorial oncolytic virus and immunotherapy. Through genetic engineering, an attenuated herpes simplex virus type 1 (HSV1) was generated that showed increased safety compared to the wild-type strain. To demonstrate the modularity and the facilitated generation of new OVs, two transgenes encoding retargeting as well as immunomodulating single-chain variable fragments (scFvs) were integrated into the platform vector. The resulting virus selectively infected epidermal growth factor receptor (EGFR)-expressing cells and produced a functional immune checkpoint inhibitor against programmed cell death protein 1 (PD-1). Thus, both viral-mediated oncolysis and immune-cell-mediated therapy were combined into a single viral vector. Safety and functionality of the armed OVs have been shown in novel preclinical models ranging from patient-derived organoids and tissue-engineered human in vitro 3D tumor models to complex humanized mouse models. Consequently, a novel and proprietary engineering platform vector based on HSV1 is available for the facilitated preclinical development of oncolytic virotherapy.

The HSV1 Tail-Anchored Membrane Protein pUL34 Contains a Basic Motif That Supports Active Transport to the Inner Nuclear Membrane Prior to Formation of the Nuclear Egress Complex.

Herpes simplex virus type 1 nucleocapsids are released from the host nucleus by a budding process through the nuclear envelope called nuclear egress. Two viral proteins, the integral membrane proteins pUL34 and pUL31, form the nuclear egress complex at the inner nuclear membrane, which is critical for this process. The nuclear import of both proteins ensues separately from each other: pUL31 is actively imported through the central pore channel, while pUL34 is transported along the peripheral pore membrane. With this study, we identified a functional bipartite NLS between residues 178 and 194 of pUL34. pUL34 lacking its NLS is mislocalized to the TGN but retargeted to the ER upon insertion of the authentic NLS or a mimic NLS, independent of the insertion site. If co-expressed with pUL31, either of the pUL34-NLS variants is efficiently, although not completely, targeted to the nuclear rim where co-localization with pUL31 and membrane budding seem to occur, comparable to the wild-type. The viral mutant HSV1(17+)Lox-UL34-NLS mt is modestly attenuated but viable and associated with localization of pUL34-NLS mt to both the nuclear periphery and cytoplasm. We propose that targeting of pUL34 to the INM is facilitated by, but not dependent on, the presence of an NLS, thereby supporting NEC formation and viral replication.

Patterns of Autologous and Nonautologous Interactions Between Core Nuclear Egress Complex (NEC) Proteins of α-, ß- and γ-Herpesviruses.

Nuclear egress is a regulated process shared by α-, ß- and γ-herpesviruses. The core nuclear egress complex (NEC) is composed of the membrane-anchored protein homologs of human cytomegalovirus (HCMV) pUL50, murine cytomegalovirus (MCMV) pM50, Epstein-Barr virus (EBV) BFRF1 or varicella zoster virus (VZV) Orf24, which interact with the autologous NEC partners pUL53, pM53, BFLF2 or Orf27, respectively. Their recruitment of additional proteins leads to the assembly of a multicomponent NEC, coordinately regulating viral nucleocytoplasmic capsid egress. Here, the functionality of VZV, HCMV, MCMV and EBV core NECs was investigated by coimmunoprecipitation and confocal imaging analyses. Furthermore, a recombinant MCMV, harboring a replacement of ORF M50 by UL50, was analyzed both in vitro and in vivo. In essence, core NEC interactions were strictly limited to autologous NEC pairs and only included one measurable nonautologous interaction between the homologs of HCMV and MCMV. A comparative analysis of MCMV-WT versus MCMV-UL50-infected murine fibroblasts revealed almost identical phenotypes on the levels of protein and genomic replication kinetics. In infected BALB/c mice, virus spread to lung and other organs was found comparable between these viruses, thus stating functional complementarity. In conclusion, our study underlines that herpesviral core NEC proteins are functionally conserved regarding complementarity of core NEC interactions, which were found either virus-specific or restricted within subfamilies.

Cold atmospheric plasma as antiviral therapy - effect on human herpes simplex virus type 1.

In previous studies, cold atmospheric plasma (CAP) was explored as an antibacterial and antiviral agent for the treatment of chronic wounds. The aim of the present study was to investigate whether CAP may also be suitable as an antiviral therapy against herpes simplex virus type 1 (HSV-1). HSV-1 most frequently manifests as recurrent herpes labialis, but it can also cause encephalitis, conjunctivitis or herpes neonatorum as a perinatal infection. HSV-1 encoding the reporter gene GFP was propagated. The CAP dose for HSV-1 treatment was gradually increased, ranging from 0-150 s, and aciclovir was used as a positive control. After CAP treatment, the virus suspension was applied to a standard HSV research cell line (Vero cells) and the neuroblastoma cell line SH-SY5Y as a model for neuronal infection. The results showed that plasma treatment had a negligible antiviral effect on HSV-1 in both Vero- and SH-SY5Y cells at high dose. However, when we lowered the viral load 100-fold, we observed a significantly decreased number of internalized HSV-1 genomes 3 h post-infection for CAP-treated viruses. This difference was less pronounced with respect to GFP expression levels 24 h post-infection, which was in sharp contrast to the acyclovir-treated positive control, for which the viral load was reduced from 95 to 25%. In summary, we observed a low but measurable antiviral effect of CAP on HSV-1.

Comprehensive analysis of nuclear export of herpes simplex virus type 1 tegument proteins and their Epstein-Barr virus orthologs.

Morphogenesis of herpesviral virions is initiated in the nucleus but completed in the cytoplasm. Mature virions contain more than 25 tegument proteins many of which perform both nuclear and cytoplasmic functions suggesting they shuttle between these compartments. While nuclear import of herpesviral proteins was shown to be crucial for viral propagation, active nuclear export and its functional impact are still poorly understood. To systematically analyze nuclear export of tegument proteins present in virions of Herpes simplex virus type 1 (HSV1) and Epstein-Barr virus (EBV), the Nuclear EXport Trapped by RAPamycin (NEX-TRAP) was applied. Nine of the 22 investigated HSV1 tegument proteins including pUL4, pUL7, pUL11, pUL13, pUL21, pUL37d11, pUL47, pUL48 and pUS2 as well as 2 out of 6 EBV orthologs harbor nuclear export activity. A functional leucine-rich nuclear export sequence (NES) recognized by the export factor CRM1/Xpo1 was identified in six of them. The comparison between experimental and bioinformatic data indicates that experimental validation of predicted NESs is required. Mutational analysis of the pUL48/VP16 NES revealed its importance for herpesviral propagation. Together our data suggest that nuclear export is an important feature of the herpesviral life cycle required to co-ordinate nuclear and cytoplasmic processes.

Nuclear egress of TDP-43 and FUS occurs independently of Exportin-1/CRM1.

TDP-43 and FUS are nuclear proteins with multiple functions in mRNA processing. They play key roles in ALS (amyotrophic lateral sclerosis) and FTD (frontotemporal dementia), where they are partially lost from the nucleus and aggregate in the cytoplasm of neurons and glial cells. Defects in nucleocytoplasmic transport contribute to this pathology, hence nuclear import of both proteins has been studied in detail. However, their nuclear export routes remain poorly characterized and it is unclear whether aberrant nuclear export contributes to TDP-43 or FUS pathology. Here we show that predicted nuclear export signals in TDP-43 and FUS are non-functional and that both proteins are exported independently of the export receptor CRM1/Exportin-1. Silencing of Exportin-5 or the mRNA export factor Aly/REF, as well as mutations that abrogate RNA-binding do not impair export of TDP-43 and FUS. However, artificially enlarging TDP-43 or FUS impairs their nuclear egress, suggesting that they could leave the nucleus by passive diffusion. Finally, we found that inhibition of transcription causes accelerated nuclear egress of TDP-43, suggesting that newly synthesized RNA retains TDP-43 in the nucleus, limiting its egress into the cytoplasm. Our findings implicate reduced nuclear retention as a possible factor contributing to mislocalization of TDP-43 in ALS/FTD.

Venture from the Interior-Herpesvirus pUL31 Escorts Capsids from Nucleoplasmic Replication Compartments to Sites of Primary Envelopment at the Inner Nuclear Membrane.

Herpesviral capsid assembly is initiated in the nucleoplasm of the infected cell. Size constraints require that newly formed viral nucleocapsids leave the nucleus by an evolutionarily conserved vescular transport mechanism called nuclear egress. Mature capsids released from the nucleoplasm are engaged in a membrane-mediated budding process, composed of primary envelopment at the inner nuclear membrane and de-envelopment at the outer nuclear membrane. Once in the cytoplasm, the capsids receive their secondary envelope for maturation into infectious virions. Two viral proteins conserved throughout the herpesvirus family, the integral membrane protein pUL34 and the phosphoprotein pUL31, form the nuclear egress complex required for capsid transport from the infected nucleus to the cytoplasm. Formation of the nuclear egress complex results in budding of membrane vesicles revealing its function as minimal virus-encoded membrane budding and scission machinery. The recent structural analysis unraveled details of the heterodimeric nuclear egress complex and the hexagonal coat it forms at the inside of budding vesicles to drive primary envelopment. With this review, I would like to present the capsid-escort-model where pUL31 associates with capsids in nucleoplasmic replication compartments for escort to sites of primary envelopment thereby coupling capsid maturation and nuclear egress.

Begomoviral Movement Protein Effects in Human and Plant Cells: Towards New Potential Interaction Partners.

Geminiviral single-stranded circular DNA genomes replicate in nuclei so that the progeny DNA has to cross both the nuclear envelope and the plasmodesmata for systemic spread within plant tissues. For intra- and intercellular transport, two proteins are required: a nuclear shuttle protein (NSP) and a movement protein (MP). New characteristics of ectopically produced Abutilon mosaic virus (AbMV) MP (MPAbMV), either authentically expressed or fused to a yellow fluorescent protein or epitope tags, respectively, were determined by localization studies in mammalian cell lines in comparison to plant cells. Wild-type MPAbMV and the distinct MPAbMV: reporter protein fusions appeared as curled threads throughout mammalian cells. Co-staining with cytoskeleton markers for actin, intermediate filaments, or microtubules identified these threads as re-organized microtubules. These were, however, not stabilized by the viral MP, as demonstrated by nocodazole treatment. The MP of a related bipartite New World begomovirus, Cleome leaf crumple virus (ClLCrV), resulted in the same intensified microtubule bundling, whereas that of a nanovirus did not. The C-terminal section of MPAbMV, i.e., the protein's oligomerization domain, was dispensable for the effect. However, MP expression in plant cells did not affect the microtubules network. Since plant epidermal cells are quiescent whilst mammalian cells are proliferating, the replication-associated protein RepAbMV protein was then co-expressed with MPAbMV to induce cell progression into S-phase, thereby inducing distinct microtubule bundling without MP recruitment to the newly formed threads. Co-immunoprecipitation of MPAbMV in the presence of RepAbMV, followed by mass spectrometry identified potential novel MPAbMV-host interaction partners: the peptidyl-prolyl cis-trans isomerase NIMA-interacting 4 (Pin4) and stomatal cytokinesis defective 2 (SCD2) proteins. Possible roles of these putative interaction partners in the begomoviral life cycle and cytoskeletal association modes are discussed.

Herpesviral vectors and their application in oncolytic therapy, vaccination, and gene transfer.

Herpesviruses are enveloped DNA viruses that infect vertebrate cells. Their high potential cloning capacity and the lifelong persistence of their genomes in various host cells make them attractive platforms for vector-based therapy. In this review, we would like to highlight recent advances of three major areas of herpesvirus vector development and application: (i) oncolytic therapy, (ii) recombinant vaccines, and (iii) large capacity gene transfer vehicles.

Pathogens Inactivated by Low-Energy-Electron Irradiation Maintain Antigenic Properties and Induce Protective Immune Responses.

Inactivated vaccines are commonly produced by incubating pathogens with chemicals such as formaldehyde or ß-propiolactone. This is a time-consuming process, the inactivation efficiency displays high variability and extensive downstream procedures are often required. Moreover, application of chemicals alters the antigenic components of the viruses or bacteria, resulting in reduced antibody specificity and therefore stimulation of a less effective immune response. An alternative method for inactivation of pathogens is ionizing radiation. It acts very fast and predominantly damages nucleic acids, conserving most of the antigenic structures. However, currently used irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment from radioactivity or X-rays generated during the process. This excludes them from direct integration into biological production facilities. Here, low-energy electron irradiation (LEEI) is presented as an alternative inactivation method for pathogens in liquid solutions. LEEI can be used in normal laboratories, including good manufacturing practice (GMP)- or high biosafety level (BSL)-environments, as only minor shielding is necessary. We show that LEEI efficiently inactivates different viruses (influenza A (H3N8), porcine reproductive and respiratory syndrome virus (PRRSV), equine herpesvirus 1 (EHV-1)) and bacteria (Escherichia coli) and maintains their antigenicity. Moreover, LEEI-inactivated influenza A viruses elicit protective immune responses in animals, as analyzed by virus neutralization assays and viral load determination upon challenge. These results have implications for novel ways of developing and manufacturing inactivated vaccines with improved efficacy.

Asna1/TRC40 that mediates membrane insertion of tail-anchored proteins is required for efficient release of Herpes simplex virus 1 virions.

BACKGROUND: Herpes simplex virus type 1 (HSV1), a member of the alphaherpesvirinae, can cause recurrent facial lesions and encephalitis. Two membrane envelopment processes, one at the inner nuclear membrane and a second at cytoplasmic membranes are crucial for a productive viral infection. Depending on the subfamily, herpesviruses encode more than 11 different transmembrane proteins including members of the tail-anchored protein family. HSV1 encodes three tail-anchored proteins pUL34, pUL56 and pUS9 characterized by a single hydrophobic region positioned at their C-terminal end that needs to be released from the ribosome prior to posttranslational membrane insertion. Asna1/TRC40 is an ATPase that targets tail-anchored proteins to the endoplasmic reticulum in a receptor-dependent manner. Cell biological data point to a critical and general role of Asna1/TRC40 in tail-anchored protein biogenesis. With this study, we aimed to determine the importance of the tail-anchored insertion machinery for HSV1 infection. METHODS: To determine protein-protein interactions, the yeast-two hybrid system was applied. Asna1/TRC40 was depleted using RNA interference. Transient transfection and virus infection experiments followed by indirect immunofluorescence analysis were applied to analyse the localization of viral proteins as well as the impact of Asna1/TRC40 depletion on virus infection. RESULTS: All HSV1 tail-anchored proteins specifically bound to Asna1/TRC40 but independently localized to their target membranes. While non-essential for cell viability, Asna1/TRC40 is required for efficient HSV1 replication. We show that early events of the replication cycle like virion entry and overall viral gene expression were unaffected by depletion of Asna1/TRC40. Furthermore, equal amounts of infectious virions were formed and remained cell-associated. This indicated that both nuclear egress of capsids that requires the essential tail-anchored protein pUL34, and secondary envelopment to form infectious virions were successfully completed. Despite large part of the virus life cycle proceeding normally, viral propagation was more than 10 fold reduced. We show that depletion of Asna1/TRC40 specifically affected a step late in infection during release of infectious virions to the extracellular milieu. CONCLUSIONS: Asna1/TRC40 is required at a late step of herpesviral infection for efficient release of mature virions to the extracellular milieu. This study reveals novel tools to decipher exocytosis of newly formed virions as well as hitherto unknown cellular targets for antiviral therapy.

Subcellular Trafficking and Functional Relationship of the HSV-1 Glycoproteins N and M.

The herpes simplex virus type 1 (HSV-1) glycoprotein N (gN/UL49.5) is a type I transmembrane protein conserved throughout the herpesvirus family. gN is a resident of the endoplasmic reticulum that in the presence of gM is translocated to the trans Golgi network. gM and gN are covalently linked by a single disulphide bond formed between cysteine 46 of gN and cysteine 59 of gM. Exit of gN from the endoplasmic reticulum requires the N-terminal core of gM composed of eight transmembrane domains but is independent of the C-terminal extension of gM. Co-transport of gN and gM to the trans Golgi network also occurs upon replacement of conserved cysteines in gM and gN, suggesting that their physical interaction is mediated by covalent and non-covalent forces. Deletion of gN/UL49.5 using bacterial artificial chromosome (BAC) mutagenesis generated mutant viruses with wild-type growth behaviour, while full deletion of gM/UL10 resulted in an attenuated phenotype. Deletion of gN/UL49.5 in conjunction with various gM/UL10 mutants reduced average plaque sizes to the same extent as either single gM/UL10 mutant, indicating that gN is nonessential for the function performed by gM. We propose that gN functions in gM-dependent as well as gM-independent processes during which it is complemented by other viral factors.

Subcellular trafficking and functional importance of herpes simplex virus type 1 glycoprotein M domains.

Herpes simplex virus type 1 (HSV-1) glycoprotein M (gM/UL10) is a 473 aa type III transmembrane protein that resides in various membrane compartments. HSV-1 gM contains several putative trafficking motifs, but their functional relevance remains to be elucidated. We show here that transiently expressed gM 19­343 was sufficient for transport to the trans-Golgi network (TGN), whilst gM 133­473, where the first two transmembrane domains were deleted, and gM 1­342, which lacked the final residue of the last transmembrane domain, were retained in the endoplasmic reticulum (ER), indicating that all transmembrane domains are required for proper folding and ER exit. A series of bacterial artificial chromosome mutants revealed that in addition to the authentic start codon, translation of gM can be initiated at methionine 19 and 133/135. Whilst a protein lacking the first 18 residues supported WT-like growth, gM 133/135­473 resulted in reduced plaque diameters resembling a UL10 deletion mutant. An HSV-1 mutant encoding gM 1­342 showed similar growth characteristics and accumulated non-enveloped cytoplasmic particles, whilst gM 1­343 resulted in a gain of function, indicating that all transmembrane domains of the protein are important for viral growth. A C-terminal extension further supported viral propagation; however, the C-terminal trafficking motifs (residues 423­473) were completely dispensable. We propose a functional core within gM 19­343 comprised of all transmembrane domains that is sufficient to target the protein to the TGN, a favoured site for envelopment, and to support viral functions.

The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain.

Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+)Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment.

HCMV pUL135 remodels the actin cytoskeleton to impair immune recognition of infected cells.

Immune evasion genes help human cytomegalovirus (HCMV) establish lifelong persistence. Without immune pressure, laboratory-adapted HCMV strains have undergone genetic alterations. Among these, the deletion of the UL/b' domain is associated with loss of virulence. In a screen of UL/b', we identified pUL135 as a protein responsible for the characteristic cytopathic effect of clinical HCMV strains that also protected from natural killer (NK) and T cell attack. pUL135 interacted directly with abl interactor 1 (ABI1) and ABI2 to recruit the WAVE2 regulatory complex to the plasma membrane, remodel the actin cytoskeleton and dramatically reduce the efficiency of immune synapse (IS) formation. An intimate association between F-actin filaments in target cells and the IS was dispelled by pUL135 expression. Thus, F-actin in target cells plays a critical role in synaptogenesis, and this can be exploited by pathogens to protect against cytotoxic immune effector cells. An independent interaction between pUL135 and talin disrupted cell contacts with the extracellular matrix.

Single-stranded DNA catalyzes hybridization of PCR-products to microarray capture probes.

Since its development, microarray technology has evolved to a standard method in the biotechnological and medical field with a broad range of applications. Nevertheless, the underlying mechanism of the hybridization process of PCR-products to microarray capture probes is still not completely understood, and several observed phenomena cannot be explained with current models. We investigated the influence of several parameters on the hybridization reaction and identified ssDNA to play a major role in the process. An increase of the ssDNA content in a hybridization reaction strongly enhanced resulting signal intensities. A strong influence could also be observed when unlabeled ssDNA was added to the hybridization reaction. A reduction of the ssDNA content resulted in a massive decrease of the hybridization efficiency. According to these data, we developed a novel model for the hybridization mechanism. This model is based on the assumption that single stranded DNA is necessary as catalyst to induce the hybridization of dsDNA. The developed hybridization model is capable of giving explanations for several yet unresolved questions regarding the functionality of microarrays. Our findings not only deepen the understanding of the hybridization process, but also have immediate practical use in data interpretation and the development of new microarrays.

A high-throughput yeast two-hybrid protocol to determine virus-host protein interactions.

The yeast two-hybrid (Y2H) system is a powerful method to identify and analyze binary protein interactions. In the field of virology, the Y2H system has significantly increased our knowledge of structure and function of viral proteins by systematically assessing intraviral protein interactions. Several comprehensive approaches to determine virus-host interactions have provided insight into viral strategies to manipulate the host for efficient replication and to escape host-derived countermeasures. To expand our knowledge of intraviral and virus-host protein interactions, we here present a Y2H protocol that is well suited for high-throughput screening. Yeast mating followed by liquid handling in a 96-well format as well as fluorescent readout of the reporter system provides a highly standardized and fully automated screening situation. The protocol can either be applied to screen complex host cDNA libraries or protein pairs arrayed for cross-testing. The ease of use, the cost-effectiveness as well as the robotic handling allows for extensive and multiple rounds of screening providing high coverage of protein-protein interactions. Thus, this protocol represents an improved "deep" screening method for high-throughput Y2H assays.

Determination of HSV-1 infectivity by plaque assay and a luciferase reporter cell line.

Quantification of infectious virus is crucial to many experimental approaches in virological research. A broadly used and facile technique is the so-called "plaque assay" which provides precise information on the absolute quantity of infectivity in a given volume. Due to advances in the understanding of viral gene expression, transactivator-promoter pairs have been identified which can be used in transgenic cell lines as reporters of viral infection. Even though such "cellular reporter assay" systems are mostly restricted to relative quantification, they are attractive tools which can complement or replace the conventional plaque assay. Cellular reporter assays become especially interesting in state-of-the-art high-throughput screening approaches, as for instance RNAi and compound library screens, since they are often compatible with small-scale and automated experimentation. In this chapter, a regular plaque assay as well as a cellular reporter assay employing a luciferase reporter gene are described. As an example, HSV-1 infectivity is assessed with both methods yielding complementary information. Advantages and disadvantages of the two techniques and possible applications are discussed.

Comprehensive analysis of varicella-zoster virus proteins using a new monoclonal antibody collection.

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus's restricted host and cell type tropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251 monoclonal antibodies (MAbs) against 59 of the 71 (83%) currently known unique VZV proteins to characterize VZV protein expression in vitro and in situ. Using this new set of MAbs, 44 viral proteins were detected by Western blotting (WB) and indirect immunofluorescence (IF); 13 were detected by WB only, and 2 were detected by IF only. A large proportion of viral proteins was analyzed for the first time in the context of virus infection. Our study revealed the subcellular localization of 46 proteins, 14 of which were analyzed in detail by confocal microscopy. Seven viral proteins were analyzed in time course experiments and showed a cascade-like temporal gene expression pattern similar to those of other herpesviruses. Furthermore, selected MAbs tested positive on human skin lesions by using immunohistochemistry, demonstrating the wide applicability of the MAb collection. Finally, a significant portion of the VZV-specific antibodies reacted with orthologs of simian varicella virus (SVV), thus enabling the systematic analysis of varicella in a nonhuman primate model system. In summary, this study provides insight into the potential function of numerous VZV proteins and novel tools to systematically study VZV and SVV pathogenesis.

Mouse cytomegalovirus egress protein pM50 interacts with cellular endophilin-A2.

The herpesvirus replication cycle comprises maturation processes in the nucleus and cytoplasm of the infected cells. After their nuclear assembly viral capsids translocate via primary envelopment towards the cytoplasm. This event is mediated by the nuclear envelopment complex, which is composed by two conserved viral proteins belonging to the UL34 and UL31 protein families. Here, we generated recombinant viruses, which express affinity-tagged pM50 and/or pM53, the pUL34 and pUL31 homologues of the murine cytomegalovirus. We extracted pM50- and pM53-associated protein complexes from infected cells and analysed their composition after affinity purification by mass spectrometry. We observed reported interaction partners and identified new putative protein-protein interactions for both proteins. Endophilin-A2 was observed as the most prominent cellular partner of pM50. We found that endophilin-A2 binds to pM50 directly, and this interaction seems to be conserved in the pUL34 family.