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Organisations' increasing adoption of lightweight automation, such as robotic process automation (RPA), raises concerns about the associated systems' robustness and security, with data-security concerns becoming further accentuated when tools of this sort are deployed for handling of potentially sensitive data. However, literature on designing these tools in a manner mitigating risks related to organisational data security has remained scarce. This paper addresses this gap by presenting a study in which RPA was successfully designed for a process wherein the software robot handles sensitive personal data. Informed by work on the mindlessness of automation, sociotechnical envelopment, and security by design, this empirical study, employing action design research at Wärtsilä Corporation, pointed to three design principles, related to envelopment, access rights, and audit trails. By adhering to these, Wärtsilä created envelopes around the robot that afford the automation's safe operation and processing of the sensitive data. This research advances the theory of sociotechnical envelopment's design and deployment by introducing a novel approach in security by envelopment to elaborate on the security-oriented envelopment of mindless automation agents. The paper also discusses the practical utility of the artefact designed, in terms of both design and evaluation. [ABSTRACT FROM AUTHOR]

Critical engagement with spaces of exposure is an important research agenda in the contemporary social sciences and humanities. Developing and extending this agenda, this paper offers an account of how scenes of exposure at border zones are mediated materially, aesthetically, and politically by forms of envelopment. Specifically, it discusses the geographies of exposure and envelopment that unfold through the use and re‐use of the emergency blanket at these zones. Fabricated from metallised polymer films, emergency blankets are used commonly to provide thermal protection for bodies at risk of exposure in a range of situations. More than functional, however, these objects also have a distinctive aesthetic allure. By attending to the material and aesthetic qualities of the emergency blanket, this paper explores its visibility and significance in scenes of exposure at border zones. Highlighting how the blanket is deployed as a device of minimal comfort, the paper then considers artistic works that repurpose this object as part of a creative critique of conditions at these zones. Drawing on the work of Stacy Alaimo and Ronak Kapadia, among others, the paper develops the concept of insurgent envelopment to understand how artists use the emergency blanket in works that simultaneously foreground, disrupt, and reimagine relations between exposure and envelopment. [ABSTRACT FROM AUTHOR]

Herpesvirus assembly requires the cytoplasmic association of large macromolecular and membrane structures that derive from both the nucleus and cytoplasmic membrane systems. Results from the study of human cytomegalovirus (HCMV) in cells where it organizes a perinuclear cytoplasmic virus assembly compartment (cVAC) show a clear requirement for the minus-end-directed microtubule motor, dynein, for virus assembly. In contrast, the assembly of herpes simplex virus -1 (HSV-1) in epithelial cells where it forms multiple dispersed, peripheral assembly sites is only mildly inhibited by the microtubule-depolymerizing agent, nocodazole. Here, we make use of a neuronal cell line system in which HSV-1 forms a single cVAC and show that dynein and its co-factor dynactin localize to the cVAC, and dynactin is associated with membranes that contain the virion tegument protein pUL11. We also show that the virus membraneassociated structural proteins pUL51 and the viral envelope glycoprotein gE arrive at the cVAC by different routes. Specifically, gE arrives at the cVAC after retrieval from the plasma membrane, suggesting the need for an intact retrograde transport system. Finally, we demonstrate that inhibition of dynactin function profoundly inhibits cVAC formation and virus production during the cytoplasmic assembly phase of infection. IMPORTANCE Many viruses reorganize cytoplasmic membrane systems and macromolecular transport systems to promote the production of progeny virions. Clarifying the mechanisms by which they accomplish this may reveal novel therapeutic strategies and illustrate mechanisms that are critical for normal cellular organization. Here, we explore the mechanism by which HSV-1 moves macromolecular and membrane cargo to generate a virus assembly compartment in the infected cell. We find that the virus makes use of a well-characterized, microtubule-based transport system that is stabilized against drugs that disrupt microtubules. [ABSTRACT FROM AUTHOR]

Abstract Duck plague virus (DPV) is one of the major infectious and fatal diseases of geese, ducks, and other wild waterfowl. The DPV UL49 gene product VP22 is one of the most abundant tegument proteins. However, the role of the DPV VP22 is enigmatic to be clarified. In this study, we found deletion of the UL49 gene resulted in reduced viral growth curve and smaller plaque size in duck embryo fibroblast (DEF) cells, confirming that DPV VP22 is required for efficient viral growth in vitro. In addition, deletion of the UL49 gene inhibited the secondary envelopment of the virus, the release of viral particles, and the spread of viruses between cells. Our study signified the importance of VP22 for DPV secondary envelopment, release, cell-to-cell spread, and accumulation of viral RNA. These findings provide a basis for further study of the function of VP22 in DPV or other herpesviruses.

Duck plague virus (DPV) is one of the major infectious and fatal diseases of geese, ducks, and other wild waterfowl. The DPV UL49 gene product VP22 is one of the most abundant tegument proteins. However, the role of the DPV VP22 is enigmatic to be clarified. In this study, we found deletion of the UL49 gene resulted in reduced viral growth curve and smaller plaque size in duck embryo fibroblast (DEF) cells, confirming that DPV VP22 is required for efficient viral growth in vitro. In addition, deletion of the UL49 gene inhibited the secondary envelopment of the virus, the release of viral particles, and the spread of viruses between cells. Our study signified the importance of VP22 for DPV secondary envelopment, release, cell-to-cell spread, and accumulation of viral RNA. These findings provide a basis for further study of the function of VP22 in DPV or other herpesviruses. [ABSTRACT FROM AUTHOR]

The tegument is the structure between the envelope and nucleocapsid of herpesvirus particles. Viral (and cellular) proteins accumulate to create the layers of the tegument. Some Epstein-Barr virus (EBV) tegument proteins are conserved widely in Herpesviridae, but others are shared only by members of the gamma-herpesvirus subfamily. As the interface to envelope and nucleocapsid, the tegument functions in virion morphogenesis and budding of the nucleocapsid during progeny production. When a virus particle enters a cell, enzymes such as kinase and deubiquitinase, and transcriptional activators are released from the virion to promote virus infection. Moreover, some EBV tegument proteins are involved in oncogenesis. Here, we summarize the roles of EBV tegument proteins, in comparison to those of other herpesviruses.

Literature from industrialized countries suggests that investments in innovative technologies such as Information Technology (IT) can boost performance and productivity growth. This has pushed severalinstitutions like banksworldwide to invest their resources and even their future in IT in order to gain advantage over their competitors within the same business environment. In solvingthis problem, the current study proposesthe usage of a model that combines a two-stage Data Envelopment Analysis (DEA) model, Multinomial Regression Analysis (MRA) and Decision Tree (DT) using C5.0 Algorithm, achieving favorable prediction rate of 88% and a good computational time compared to similar studies. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus and the leading cause of congenital disabilities as well as a significant cause of disease in immunocompromised patients. The envelopment and egress of HCMV particles is an essential step of the viral life cycle as it determines viral spread and potentially tropism. Here we review the current literature on HCMV envelopment and egress with a particular focus on the role of virus‐containing multivesicular body‐like vesicles for virus egress and spread. We discuss the difficulties of determining the cellular provenance of these structures in light of viral redistribution of cellular marker proteins and provide potential paths to illuminate their genesis. Finally, we discuss how divergent egress pathways could result in virions of different tropisms. [ABSTRACT FROM AUTHOR]

The assembly and egress of alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), within neurons is poorly understood. A key unresolved question is the structure of the viral particle that moves by anterograde transport along the axon, and two alternative mechanisms have been described. In the "married" model, capsids acquire their envelopes in the cell body and then traffic along axons as enveloped virions within a bounding organelle. In the "separate" model, nonenveloped capsids travel from the cell body into and along the axon, eventually encountering their envelopment organelles at a distal site, such as the nerve cell terminal. Here, we describe an "envelopment trap" to test these models using the dominant negative terminal endosomal sorting complex required for transport (ESCRT) component VPS4-EQ. Green fluorescent protein (GFP)- tagged VPS4-EQ was used to arrest HSV-1 or PRV capsid envelopment, inhibit downstream trafficking, and GFP-label envelopment intermediates. We found that GFPVPS4- EQ inhibited trafficking of HSV-1 capsids into and along the neurites and axons of mouse CAD cells and rat embryonic primary cortical neurons, consistent with egress via the married pathway. In contrast, transport of HSV-1 capsids was unaffected in the neurites of human SK-N-SH neuroblastoma cells, consistent with the separate mechanism. Unexpectedly, PRV (generally thought to utilize the married pathway) also appeared to employ the separate mechanism in SK-N-SH cells. We propose that apparent differences in the methods of HSV-1 and PRV egress are more likely a reflection of the host neuron in which transport is studied rather than true biological differences between the viruses themselves. [ABSTRACT FROM AUTHOR]

Interactions between the N-terminal (assembly) domain (NTD), the linker region of the hepatitis B virus (HBV) capsid protein, and the large (L) envelope protein are required for virion formation, which occurs via budding of cytoplasmic mature nucleocapsids (NCs) containing the relaxed circular (RC) DNA genome into an intracellular membrane compartment containing viral envelope proteins. L-capsid interactions also negatively regulate covalently closed circular (CCC) DNA formation, which occurs after RC DNA release from mature NCs and nuclear import. We have now found that L could increase RC DNA in cytoplasmic mature NCs that are destabilized due to mutations in the NTD or the linker, even in those that apparently fail to support secretion of complete virions extracellularly. Other mutations in the capsid linker could block the effects of L on both cytoplasmic NC DNA and nuclear CCC DNA. Furthermore, the maturity of RC DNA in cytoplasmic NCs that was enhanced by L or found in secreted virions was modulated by the capsid linker sequence. The level and maturity of the cytoplasmic RC DNA were further influenced by the efficiency of extracellular virion secretion dependent on viral genotype-specific envelope proteins. These results suggest that interactions between the capsid and envelope proteins regulate one or more steps during virion secretion beyond initial capsid envelopment and highlight the critical role of the capsid linker in regulating capsid-envelope interaction, including the timing of envelopment during NC maturation. [ABSTRACT FROM AUTHOR]

The "Blue Whale" Auditorium in Buenos Aires opened in 2015. Designed to be the headquarters of the National Symphony Orchestra of Argentina, its goal was to become the city's main space for symphonic music. The architectural program posed several challenges from an acoustic point of view, as 2000 people had to be accommodated in a square space into which none of the usual architectural typologies fit properly. It was decided, therefore, to place in this space an "ad-hoc" hall. The design centered around three main premises: to achieve an enveloping acoustic field, to establish an adequate reverberation decay, and to combine reflective and diffusing surfaces to attain a similar acoustic field through the entire audience area. This work details the design process of the Auditorium, during which the final shape was deduced from the established acoustic premises, and some acoustical measurements made in the finished hall. [ABSTRACT FROM AUTHOR]

α-herpesviruses have been very successful, principally because they establish lifelong latency in sensory ganglia. An essential piece of the lifecycle of α-herpesviruses involves the capacity to travel from sensory neurons to epithelial tissues following virus reactivation from latency, a process known as anterograde transport. Virus particles formed in neuron cell bodies hitchhike on kinesin motors that run along microtubules, the length of axons. Herpes simplex virus (HSV) and pseudorabies virus (PRV) have been intensely studied to elucidate anterograde axonal transport. Both viruses use similar strategies for anterograde transport, although there are significant differences in the form of virus particles transported in axons, the identity of the kinesins that transport viruses, and how certain viral membrane proteins, gE/gI and US9, participate in this process. This review compares the older models for HSV and PRV anterograde transport with recent results, which are casting a new light on several aspects of this process. • α−herpesvirus anterograde axonal transport can involve unenveloped or enveloped capsids or both. • α−herpesviruses use at least two families of kinesin motors for anterograde transport. • Membrane proteins US9 and gE/gI promote virus assembly in the cytoplasm and sorting of viruses into axons. [ABSTRACT FROM AUTHOR]

This paper analyzes the entry of digital platforms such as Google into complementary hardware markets (e.g., internet service provision) using a game-theoretic model. Access to a platform's services often requires consumers to use a complementary hardware product or service, for example, internet service is needed to access the YouTube platform. Typically, such access products are provided by third-party firms. More recently, however, some major platforms such as Google have themselves ventured into providing these access products. For example, Google Fiber provides access to YouTube. In this paper, we examine the effect of a platform's entry into an access product market when the profits from the platform's advertising business depend upon the quality of the access products. We develop a theoretical model to study this context and find that such an entry by the platform (i) can lead the platform to provide a higher quality access product than the third-party firms at a lower price, (ii) may, in contrast to the entry by a third-party firm, lead to a higher quality access product by both the platform as well as the firms, (iii) improves the platform's profits because of increased advertising revenue and/or additional profits from the access product sales, and (iv) increases consumer surplus even though the platform becomes more dominant because of its entry into the access product market. All the results are driven by the positive association between the platform's advertising profits and the quality of access products in the market. [ABSTRACT FROM AUTHOR]

Glycerophospholipids are major components of cell membranes. Phosphatidylethanolamine (PE) is a glycerophospholipid that is involved in multiple cellular processes, such as membrane fusion, the cell cycle, autophagy, and apoptosis. In this study, we investigated the role of PE biosynthesis in herpes simplex virus 1 (HSV-1) infection by knocking out the host cell gene encoding phosphate cytidylyltransferase 2, ethanolamine (Pcyt2), which is a key rate-limiting enzyme in one of the two major pathways for PE biosynthesis. Pcyt2 knockout reduced HSV-1 replication and caused an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm of an HSV-1-infected cell culture. A similar phenotype was observed when infected cells were treated with meclizine, which is an inhibitor of Pcyt2. In addition, treatment of HSV-1- infected mice with meclizine significantly reduced HSV-1 replication in the mouse brains and improved their survival rates. These results indicated that PE biosynthesis mediated by Pcyt2 was required for efficient HSV-1 envelopment in the cytoplasm of infected cells and for viral replication and pathogenicity in vivo. The results also identified the PE biosynthetic pathway as a possible novel target for antiviral therapy of HSV-associated diseases and raised an interesting possibility for meclizine repositioning for treatment of these diseases, since it is an over-the-counter drug that has been used for decades against nausea and vertigo in motion sickness. [ABSTRACT FROM AUTHOR]

The herpes simplex virus (HSV) heterodimer gE/gI and another membrane protein, US9, which has neuron-specific effects, promote the anterograde transport of virus particles in neuronal axons. Deletion of both HSV gE and US9 blocks the assembly of enveloped particles in the neuronal cytoplasm, which explains why HSV virions do not enter axons. Cytoplasmic envelopment depends upon interactions between viral membrane proteins and tegument proteins that encrust capsids. We report that tegument protein UL16 is unstable, i.e., rapidly degraded, in neurons infected with a gE/US9 double mutant. Immunoprecipitation experiments with lysates of HSV-infected neurons showed that UL16 and three other tegument proteins, namely, VP22, UL11, and UL21, bound either to gE or gI. All four of these tegument proteins were also pulled down with US9. In neurons transfected with tegument proteins and gE/gI or US9, there was good evidence that VP22 and UL16 bound directly to US9 and gE/gI. However, there were lower quantities of these tegument proteins that coprecipitated with gE/gI and US9 from transfected cells than those of infected cells. This apparently relates to a matrix of several different tegument proteins formed in infected cells that bind to gE/gI and US9. In cells transfected with individual tegument proteins, this matrix is less prevalent. Similarly, coprecipitation of gE/gI and US9 was observed in HSV-infected cells but not in transfected cells, which argued against direct US9-gE/gI interactions. These studies suggest that gE/gI and US9 binding to these tegument proteins has neuronspecific effects on virus HSV assembly, a process required for axonal transport of enveloped particles. [ABSTRACT FROM AUTHOR]

The present study proposes a Fuzzy Data Envelopment Analysis (FDEA) approach for analyzing the performance of 8 selected paper mills of India. The proposed approach named FDEA considers the use of fuzzy weights in the objective function and makes use of alpha cut to decide the fuzzy interval. The efficiency of paper mills is evaluated based on 3 input parameters (raw material, energy consumption, and water consumption) and 4 output parameters (paper production, Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD) and Greenhouse Gas (GHG) emissions). Further, this paper also analyzes the effect of negative outputs, like BOD, COD, and GHG on the efficiency of paper mills. The study indicates that FDEA can be used efficiently for evaluating the performance of a particular sector under similar conditions. [ABSTRACT FROM AUTHOR]

(1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses.

Background : Capsids of herpes simplex virus 1 (HSV-1) are assembled in cell nuclei, released into the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope. Alternatively, capsids gain access to the cytoplasm via dilated nuclear pores. They are enveloped by Golgi membranes. Us3 is a non-essential viral kinase that is involved in nucleus-to-cytoplasm translocation, preventing apoptosis and regulation of phospholipid-biosynthesis. Us3-deletion mutants (HSV-1∆Us3) accumulate in the perinuclear space. Nuclear and Golgi membranes proliferate, and homogeneous, proteinaceous structures of unknown identity are deposited in nuclei and cytoplasm. Glycoprotein K (gK), a highly hydrophobic viral protein, is essential for production of infectious progeny virus but, according to the literature, exclusively vital for envelopment of capsids by Golgi membranes. In the absence of Us3, virions remain stuck in the perinuclear space but mature to infectivity without reaching Golgi membranes, suggesting further function of gK than assumed. Methods : We constructed a HSV-1∆Us3 mutant designated CK177∆Us3gK-HA, in which gK was hemagglutinin (HA) epitope-tagged in order to localize gK by immunolabeling using antibodies against HA for light and electron microscopy. Results : CK177∆Us3gK-HA-infected Vero cells showed similar alterations as those reported for other HSV-1∆Us3, including accumulation of virions in the perinuclear space, overproduction of nuclear and Golgi membranes containing electron dense material with staining property of proteins. Immunolabeling using antibodies against HA revealed that gK is overproduced and localized at nuclear membranes, perinuclear virions stuck in the perinuclear space, Golgi membranes and on protein deposits in cytoplasm and nuclei. Conclusions : Us3 is involved in proper assembly of membranes needed for envelopment and incorporation of gK. Without Us3, virions derived by budding at nuclear membranes remain stuck in the perinuclear space but incorporate gK into their envelope to gain infectivity.

Cytomegalovirus secondary envelopment occurs in a virus-induced cytoplasmic assembly compartment (vAC) generated via a drastic reorganization of the membranes of the secretory and endocytic systems. Dynamin is a eukaryotic GTPase that is implicated in membrane remodeling and endocytic membrane fission events; however, the role of dynamin in cellular trafficking of viruses beyond virus entry is only partially understood. Mouse embryonic fibroblasts (MEF) engineered to excise all three isoforms of dynamin were infected with mouse cytomegalovirus (MCMV-K181). Immediate-early (IE1; m123) viral protein was detected in these triple dynamin knockout (TKO) cells, as well as in mock-induced parental MEF, at early times postinfection, although levels were reduced in TKO cells, indicating that virus entry was affected but not eliminated. Levels of IE1 protein and another viral early protein (m04) were normalized by 48 h postinfection; however, late protein (m55; gB) expression was reduced in infected TKO cells compared to parental MEF. Ultrastructural analysis revealed intact stages of nuclear virus maturation in both cases with equivalent numbers of nucleocapsids containing packaged viral DNA (C-capsids), indicating successful viral DNA replication, capsid assembly, and genome packaging. Most importantly, severe defects in virus envelopment were visualized in TKO cells but not in parental cells. Dynamin inhibitor (dynasore)-treated MEF showed a phenotype similar to TKO cells upon mouse cytomegalovirus infection, confirming the role of dynamin in late maturation processes. In summary, dynamin-mediated endocytic pathways are critical for the completion of cytoplasmic stages of cytomegalovirus maturation. [ABSTRACT FROM AUTHOR]

Background: Herpesvirus capsids are assembled in the nucleus, translocated to the perinuclear space by budding, acquiring tegument and envelope, or released to the cytoplasm via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” is essential for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of alternative exit routes. Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1) or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1) by transmission and scanning electron microscopy, employing freezing technique protocols. Results: The Golgi complex is a compact entity in a juxtanuclear position covered by a membrane on the cis face. Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced. Conclusions: The data suggest that virions derived by budding at nuclear membranes are intraluminally transported from the perinuclear space via Golgi -endoplasmic reticulum transitions into Golgi cisternae for packaging. Virions derived by budding at nuclear membranes are infective like Us3 deletion mutants, which accumulate in the perinuclear space. Therefore, i) de-envelopment followed by re-envelopment is not essential for production of infective progeny virus, ii) the process taking place at the outer nuclear membrane is budding not fusion, and iii) naked capsids gain access to the cytoplasmic matrix via impaired nuclear envelope as reported earlier.

Herpes simplex virus type 1 (HSV-1) is a structurally complex enveloped dsDNA virus that has evolved to replicate in human neurons and epithelia. Viral gene expression, DNA replication, capsid assembly, and genome packaging take place in the infected cell nucleus, which mature nucleocapsids exit by envelopment at the inner nuclear membrane then de-envelopment into the cytoplasm. Once in the cytoplasm, capsids travel along microtubules to reach, dock, and envelope at cytoplasmic organelles. This generates mature infectious HSV-1 particles that must then be sorted to the termini of sensory neurons, or to epithelial cell junctions, for spread to uninfected cells. The focus of this review is upon our current understanding of the viral and cellular molecular machinery that enables HSV-1 to travel within infected cells during egress and to manipulate cellular organelles to construct its envelope.

Assembly of herpesvirus nucleocapsids shares significant similarities with the assembly of tailed dsDNA bacteriophages; however, important differences exist. A unique feature of herpesviruses is the presence of different mature capsid forms in the host cell nucleus during infection. These capsid forms, referred to as A-, B-, and C-capsids, represent empty capsids, scaffold containing capsids and viral DNA containing capsids, respectively. The C-capsids are the closest in form to those encapsidated into mature virions and are considered precursors to infectious virus. The evidence supporting A- and B-capsids as either abortive forms or assembly intermediates has been lacking. Interaction of specific capsid forms with viral tegument proteins has been proposed to be a mechanism for quality control at the point of nuclear egress of mature particles. Here, we will review the available literature on these capsid forms and present data to debate whether A- and B-capsids play an important or an extraneous role in the herpesvirus life cycle.

Herpes simplex virus 1 (HSV-1) capsids are assembled in the nucleus bud at the inner nuclear membrane into the perinuclear space, acquiring envelope and tegument. In theory, these virions are de-enveloped by fusion of the envelope with the outer nuclear membrane and re-enveloped by Golgi membranes to become infective. Us3 enables the nucleus to cytoplasm capsid translocation. Nevertheless, Us3 is not essential for the production of infective progeny viruses. Determination of phenotype distribution by quantitative electron microscopy, and calculation per mean nuclear or cell volume revealed the following: (i) The number of R7041(∆US3) capsids budding at the inner nuclear membrane was significantly higher than that of wild type HSV-1; (ii) The mean number of R7041(∆US3) virions per mean cell volume was 2726, that of HSV-1 virions 1460 by 24 h post inoculation; (iii) 98% of R7041(∆US3) virions were in the perinuclear space; (iv) The number of R7041(∆US3) capsids in the cytoplasm, including those budding at Golgi membranes, was significantly reduced. Cell associated R7041(∆US3) yields were 2.37 × 108 and HSV-1 yields 1.57 × 108 PFU/mL by 24 h post inoculation. We thus conclude that R7041(∆US3) virions, which acquire envelope and tegument by budding at the inner nuclear membrane into the perinuclear space, are infective.

Background: Herpesvirus capsids are assembled in the nucleus before they are translocated to the perinuclear space by budding, acquiring tegument and envelope, or releasing to the cytoplasm in a “naked” state via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” are essential steps for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of an alternative exit route. Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1) or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1) by transmission and scanning electron microscopy, employing freezing technique protocols that lead to improved spatial and temporal resolution. Results: Scanning electron microscopy showed the Golgi complex as a compact entity in a juxtanuclear position covered by a membrane on the cis face. Transmission electron microscopy revealed that Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced. Conclusions: The data strongly suggest that virions are intraluminally transported from the perinuclear space via Golgi complex-endoplasmic reticulum transitions into Golgi cisternae for packaging into transport vacuoles. Furthermore, virions derived by budding at nuclear membranes are infective as has been shown for HSV-1 Us3 deletion mutants, which almost entirely accumulate in the perinuclear space. Therefore, de-envelopment followed by re-envelopment is not essential for production of infective progeny virus.

Despite differences in the pathogenesis and host range of alphaherpesviruses, many stages of their morphogenesis are thought to be conserved. Here, an ultrastructural study of bovine herpesvirus 1 (BoHV-1) envelopment revealed profiles similar to those previously found for herpes simplex virus 1 (HSV-1), with BoHV-1 capsids associating with endocytic tubules. Consistent with the similarity of their genomes and envelopment strategies, the proteomic compositions of BoHV-1 and HSV-1 virions were also comparable. However, BoHV-1 morphogenesis exhibited a diversity in envelopment events. First, heterogeneous primary envelopment profiles were readily detectable at the inner nuclear membrane of BoHV-1-infected cells. Second, the BoHV-1 progeny comprised not just full virions but also an abundance of capsidless, noninfectious light particles (L-particles) that were released from the infected cells in numbers similar to those of virions and in the absence of DNA replication. Proteomic analysis of BoHV-1 L-particles and the much less abundant HSV-1 L-particles revealed that they contained the same complement of envelope proteins as virions but showed variations in tegument content. In the case of HSV-1, the UL46 tegument protein was reproducibly found to be >6-fold enriched in HSV-1 L-particles. More strikingly, the tegument proteins UL36, UL37, UL21, and UL16 were depleted in BoHV-1 but not HSV-1 L-particles. We propose that these combined differences reflect the presence of truly segregated "inner" and "outer" teguments in BoHV-1, making it a critical system for studying the structure and process of tegumentation and envelopment. IMPORTANCE The alphaherpesvirus family includes viruses that infect humans and animals. Hence, not only do they have a significant impact on human health, but they also have a substantial economic impact on the farming industry. While the pathogenic manifestations of the individual viruses differ from host to host, their relative genetic compositions suggest similarity at the molecular level. This study provides a side-byside comparison of the particle outputs from the major human pathogen HSV-1 and the veterinary pathogen BoHV-1. Ultrastructural and proteomic analyses have revealed that both viruses have broadly similar morphogenesis profiles and infectious virus compositions. However, the demonstration that BoHV-1 has the capacity to generate vast numbers of capsidless enveloped particles that differ from those produced by HSV-1 in composition implies a divergence in the cell biology of these viruses that impacts our general understanding of alphaherpesvirus morphogenesis. [ABSTRACT FROM AUTHOR]

Secondary envelopment of human cytomegalovirus (HCMV) occurs through a mechanism that is poorly understood. Many enveloped viruses utilize the endosomal sorting complexes required for transport (ESCRTs) for viral budding and envelopment. Although there are conflicting reports on the role of the ESCRT AAA ATPase protein VPS4 in HCMV infection, VPS4 may act in an envelopment role similar to its function during other viral infections. Because VPS4 is normally recruited by the ESCRT-III complex, we hypothesized that ESCRT-III subunits would also be required for HCMV infection. We investigated the role of ESCRT-III, the core ESCRT scission complex, during the late stages of infection. We show that inducible expression of dominant negative ESCRT-III subunits during infection blocks endogenous ESCRT function but does not inhibit virus production. We also show that HCMV forms enveloped intracellular and extracellular virions in the presence of dominant negative ESCRT-III subunits, suggesting that ESCRT-III is not involved in the envelopment of HCMV. We also found that as with ESCRT-III, inducible expression of a dominant negative form of VPS4A did not inhibit the envelopment of virions or reduce virus titers. Thus, HCMV does not require the ESCRTs for secondary envelopment. However, we found that ESCRT-III subunits are required for efficient virus spread. This suggests a role for ESCRT-III during the spread of HCMV that is independent of viral envelopment. IMPORTANCE Human cytomegalovirus (HCMV) is a prevalent opportunistic pathogen in the human population. For neonatal and immunocompromised patients, HCMV infection can cause severe and possibly life-threatening complications. It is important to define the mechanisms of the viral replication cycle in order to identify potential targets for new therapies. Secondary envelopment, or acquisition of the membrane envelope, of HCMV is a mechanism that needs further study. Using an inducible fibroblast system to carefully control for the toxicity associated with blocking ESCRT-III function, this study determines that the ESCRT proteins are not required for viral envelopment. However, the study does discover a nonenvelopment role for the ESCRTIII complex in the efficient spread of the virus. Thus, this study advances our understanding of an important process essential for the replication of HCMV. [ABSTRACT FROM AUTHOR]

In order to explore the mechanism by which Tween-80 enhances the production of curdlan produced by Agrobacterium sp., the effects of Tween-80 on the production and structure of curdlan and Agrobacterium sp. were evaluated. Maximum curdlan production (51.94 g/L) was achieved when 16 g/L Tween-80 was added at the beginning of the cell growth stage. The addition of Tween-80 at higher concentration inhibited cell growth. However, the addition of 16 g/L Tween-80 enhanced the production of curdlan with a looser ultrastructure, significantly weakened the envelopment of curdlan on Agrobacterium sp., altered the fine structure of cell membrane, and increased the cell membrane permeability. Moreover, the efficiency of oxygen and mass transport, respiration intensity, UTP regeneration, ATP regeneration, activity of curdlan synthetase, capacity of stress response and energy supply of Agrobacterium sp. were all greatly improved by the addition of Tween-80. These findings demonstrate the mechanisms by which Tween-80 enhances curdlan production and provide a cheap and feasible approach to weaken the envelopment of water-insoluble polysaccharides on bacteria. [ABSTRACT FROM AUTHOR]

Autophagy is an essential vacuolar process of the cell, leading to lysosomal degradation and recycling of proteins and organelles, which is extremely important in maintaining homeostasis. Multiple roles have been now associated with autophagy, in particular a pro-survival role in nutrient starvation or in stressful environments, a role in life span extension, in development, or in innate and adaptive immunity. This cellular process can also take over microorganisms or viral proteins inside autophagosomes and degrade them directly in autolysosomes and is then called xenophagy and virophagy, respectively. Several Herpesviruses have developed strategies to escape this degradation, by expression of specific anti-autophagic proteins. However, we are increasingly discovering that Herpesviruses hijack autophagy, rather than just fight it. This beneficial effect is obvious since inhibition of autophagy will lead to decreased viral titers for human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) or Varicella-Zoster virus (VZV), for example. Conversely, autophagy stimulation will improve viral multiplication. The autophagic machinery can be used in whole or in part, and can optimize viral propagation or persistence. Some viruses block maturation of autophagosomes to avoid the degradation step, then autophagosomal membranes are used to contribute to the envelopment and/or the egress of viral particles. On the other hand, VZV stimulates the whole process of autophagy to subvert it in order to use vesicles containing ATG (autophagy-related) proteins and resembling amphisomes for their transport in the cytoplasm. During latency, autophagy can also be activated by latent proteins encoded by different oncogenic Herpesviruses to promote cell survival and achieve long term viral persistence in vivo. Finally, reactivation of gammaherpesvirus Murid Herpesvirus 68 (MHV68) in mice appears to be positively modulated by autophagy, in order to control the level of inflammation. Therefore, Herpesviruses appear to behave more like thieves than fugitives. [ABSTRACT FROM AUTHOR]