Structural Basis of Low-pH-Dependent Lysosomal Cholesterol Egress by NPC1 and NPC2.

Lysosomal cholesterol egress requires two proteins, NPC1 and NPC2, whose defects are responsible for Niemann-Pick disease type C (NPC). Here, we present systematic structural characterizations that reveal the molecular basis for low-pH-dependent cholesterol delivery from NPC2 to the transmembrane (TM) domain of NPC1. At pH 8.0, similar structures of NPC1 were obtained in nanodiscs and in detergent at resolutions of 3.6 Å and 3.0 Å, respectively. A tunnel connecting the N-terminal domain (NTD) and the transmembrane sterol-sensing domain (SSD) was unveiled. At pH 5.5, the NTD exhibits two conformations, suggesting the motion for cholesterol delivery to the tunnel. A putative cholesterol molecule is found at the membrane boundary of the tunnel, and TM2 moves toward formation of a surface pocket on the SSD. Finally, the structure of the NPC1-NPC2 complex at 4.0 Å resolution was obtained at pH 5.5, elucidating the molecular basis for cholesterol handoff from NPC2 to NPC1(NTD).

ß-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

Lymphocyte Circadian Clocks Control Lymph Node Trafficking and Adaptive Immune Responses.

Lymphocytes circulate through lymph nodes (LN) in search for antigen in what is believed to be a continuous process. Here, we show that lymphocyte migration through lymph nodes and lymph occurred in a non-continuous, circadian manner. Lymphocyte homing to lymph nodes peaked at night onset, with cells leaving the tissue during the day. This resulted in strong oscillations in lymphocyte cellularity in lymph nodes and efferent lymphatic fluid. Using lineage-specific genetic ablation of circadian clock function, we demonstrated this to be dependent on rhythmic expression of promigratory factors on lymphocytes. Dendritic cell numbers peaked in phase with lymphocytes, with diurnal oscillations being present in disease severity after immunization to induce experimental autoimmune encephalomyelitis (EAE). These rhythms were abolished by genetic disruption of T cell clocks, demonstrating a circadian regulation of lymphocyte migration through lymph nodes with time-of-day of immunization being critical for adaptive immune responses weeks later.

Regulation of calcium entry by cyclic GMP signaling in Toxoplasma gondii.

Ca2+ signaling impacts almost every aspect of cellular life. Ca2+ signals are generated through the opening of ion channels that permit the flow of Ca2+ down an electrochemical gradient. Cytosolic Ca2+ fluctuations can be generated through Ca2+ entry from the extracellular milieu or release from intracellular stores. In Toxoplasma gondii, Ca2+ ions play critical roles in several essential functions for the parasite, like invasion of host cells, motility, and egress. Plasma membrane Ca2+ entry in T. gondii was previously shown to be activated by cytosolic calcium and inhibited by the voltage-operated Ca2+ channel blocker nifedipine. However, Ca2+ entry in T. gondii did not show the classical characteristics of store regulation. In this work, we characterized the mechanism by which cytosolic Ca2+ regulates plasma membrane Ca2+ entry in extracellular T. gondii tachyzoites loaded with the Ca2+ indicator Fura-2. We compared the inhibition by nifedipine with the effect of the broad spectrum TRP channel inhibitor, anthranilic acid or ACA, and we find that both inhibitors act on different Ca2+ entry activities. We demonstrate, using pharmacological and genetic tools, that an intracellular signaling pathway engaging cyclic GMP, protein kinase G, Ca2+, and the phosphatidyl inositol phospholipase C affects Ca2+ entry and we present a model for crosstalk between cyclic GMP and cytosolic Ca2+ for the activation of T. gondii's lytic cycle traits.

Disruption of a Plasmodium falciparum patatin-like phospholipase delays male gametocyte exflagellation.

An essential process in transmission of the malaria parasite to the Anopheles vector is the conversion of mature gametocytes into gametes within the mosquito gut, where they egress from the red blood cell (RBC). During egress, male gametocytes undergo exflagellation, leading to the formation of eight haploid motile microgametes, while female gametes retain their spherical shape. Gametocyte egress depends on sequential disruption of the parasitophorous vacuole membrane and the host cell membrane. In other life cycle stages of the malaria parasite, phospholipases have been implicated in membrane disruption processes during egress, however their importance for gametocyte egress is relatively unknown. Here, we performed comprehensive functional analyses of six putative phospholipases for their role during development and egress of Plasmodium falciparum gametocytes. We localize two of them, the prodrug activation and resistance esterase (PF3D7_0709700) and the lysophospholipase 1 (PF3D7_1476700), to the parasite plasma membrane. Subsequently, we show that disruption of most of the studied phospholipase genes does neither affect gametocyte development nor egress. The exception is the putative patatin-like phospholipase 3 (PF3D7_0924000), whose gene deletion leads to a delay in male gametocyte exflagellation, indicating an important, albeit not essential, role of this enzyme in male gametogenesis.

Comparative proteomics of vesicles essential for the egress of Plasmodium falciparum gametocytes from red blood cells.

Transmission of malaria parasites to the mosquito is mediated by sexual precursor cells, the gametocytes. Upon entering the mosquito midgut, the gametocytes egress from the enveloping erythrocyte while passing through gametogenesis. Egress follows an inside-out mode during which the membrane of the parasitophorous vacuole (PV) ruptures prior to the erythrocyte membrane. Membrane rupture requires exocytosis of specialized egress vesicles of the parasites; that is, osmiophilic bodies (OBs) involved in rupturing the PV membrane, and vesicles that harbor the perforin-like protein PPLP2 (here termed P-EVs) required for erythrocyte lysis. While some OB proteins have been identified, like G377 and MDV1/Peg3, the majority of egress vesicle-resident proteins is yet unknown. Here, we used high-resolution imaging and BioID methods to study the two egress vesicle types in Plasmodium falciparum gametocytes. We show that OB exocytosis precedes discharge of the P-EVs and that exocytosis of the P-EVs, but not of the OBs, is calcium sensitive. Both vesicle types exhibit distinct proteomes with the majority of proteins located in the OBs. In addition to known egress-related proteins, we identified novel components of OBs and P-EVs, including vesicle-trafficking proteins. Our data provide insight into the immense molecular machinery required for the inside-out egress of P. falciparum gametocytes.

Moving on: How malaria parasites exit the liver.

An essential step in the life cycle of malaria parasites is their egress from hepatocytes, which enables the transition from the asymptomatic liver stage to the pathogenic blood stage of infection. To exit the liver, Plasmodium parasites first disrupt the parasitophorous vacuole membrane that surrounds them during their intracellular replication. Subsequently, parasite-filled structures called merosomes emerge from the infected cell. Shrouded by host plasma membrane, like in a Trojan horse, parasites enter the vasculature undetected by the host immune system and travel to the lung where merosomes rupture, parasites are released, and the blood infection stage begins. This complex, multi-step process must be carefully orchestrated by the parasite and requires extensive manipulation of the infected host cell. This review aims to outline the known signaling pathways that trigger exit, highlight Plasmodium proteins that contribute to the release of liver-stage merozoites, and summarize the accompanying changes to the hepatic host cell.

Cell death induction facilitates egress of Coxiella burnetii from infected host cells at late stages of infection.

Intracellular bacteria have evolved mechanisms to invade host cells, establish an intracellular niche that allows survival and replication, produce progeny, and exit the host cell after completion of the replication cycle to infect new target cells. Bacteria exit their host cell by (i) initiation of apoptosis, (ii) lytic cell death, and (iii) exocytosis. While bacterial egress is essential for bacterial spreading and, thus, pathogenesis, we currently lack information about egress mechanisms for the obligate intracellular pathogen C. burnetii, the causative agent of the zoonosis Q fever. Here, we demonstrate that C. burnetii inhibits host cell apoptosis early during infection, but induces and/or increases apoptosis at later stages of infection. Only at later stages of infection did we observe C. burnetii egress, which depends on previously established large bacteria-filled vacuoles and a functional intrinsic apoptotic cascade. The released bacteria are not enclosed by a host cell membrane and can infect and replicate in new target cells. In summary, our data argue that C. burnetii egress in a non-synchronous way at late stages of infection. Apoptosis-induction is important for C. burnetii egress, but other pathways most likely contribute.

Tools and mechanisms of vacuolar escape leading to host egress in Legionella pneumophila infection: Emphasis on bacterial phospholipases.

The phenomenon of host cell escape exhibited by intracellular pathogens is a remarkably versatile occurrence, capable of unfolding through lytic or non-lytic pathways. Among these pathogens, the bacterium Legionella pneumophila stands out, having adopted a diverse spectrum of strategies to disengage from their host cells. A pivotal juncture that predates most of these host cell escape modalities is the initial escape from the intracellular compartment. This critical step is increasingly supported by evidence suggesting the involvement of several secreted pathogen effectors, including lytic proteins. In this intricate landscape, L. pneumophila emerges as a focal point for research, particularly concerning secreted phospholipases. While nestled within its replicative vacuole, the bacterium deftly employs both its type II (Lsp) and type IVB (Dot/Icm) secretion systems to convey phospholipases into either the phagosomal lumen or the host cell cytoplasm. Its repertoire encompasses numerous phospholipases A (PLA), including three enzymes-PlaA, PlaC, and PlaD-bearing the GDSL motif. Additionally, there are 11 patatin-like phospholipases A as well as PlaB. Furthermore, the bacterium harbors three extracellular phospholipases C (PLCs) and one phospholipase D. Within this comprehensive review, we undertake an exploration of the pivotal role played by phospholipases in the broader context of phagosomal and host cell egress. Moreover, we embark on a detailed journey to unravel the established and potential functions of the secreted phospholipases of L. pneumophila in orchestrating this indispensable process.

Where is the EXIT? Phenotypic screens for new egress factors in apicomplexan parasites.

Apicomplexans, such as Plasmodium and Toxoplasma are obligate intracellular parasites that invade, replicate and finally EXIT their host cell. During replication within a parasitophorous vacuole (PV), the parasites establish an extensive F-actin-containing network that connects individual parasites and is required for material exchange, recycling and the final steps of daughter cell assembly. After multiple rounds of replication, the parasites exit the host cell involving multiple signalling cascades, disassembly of the network, secretion of microneme proteins and activation of the acto-myosin motor. Blocking the host cell EXIT process leads to the formation of large PVs, making the screening for genes involved in exiting the cell relatively straightforward. Given that apicomplexans are highly diverse from other eukaryotes, approximately 30% of all genes are annotated as hypothetical, some apicomplexan-specific factors are likely to be critical during EXIT. This motivated several labs to design and perform forward genetic and phenotypic screens using various approaches, such as random insertion mutagenesis, temperature-sensitive mutants and, more recently, CRISPR/Cas9-mediated targeted editing and conditional mutagenesis. Here we will provide an overview of the technological developments over recent years and the most successful stories that led to the identification of new critical factors in Toxoplasma gondii.

Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor.

Malaria parasite egress from host erythrocytes (RBCs) is regulated by discharge of a parasite serine protease called SUB1 into the parasitophorous vacuole (PV). There, SUB1 activates a PV-resident cysteine protease called SERA6, enabling host RBC rupture through SERA6-mediated degradation of the RBC cytoskeleton protein ß-spectrin. Here, we show that the activation of Plasmodium falciparum SERA6 involves a second, autocatalytic step that is triggered by SUB1 cleavage. Unexpectedly, autoproteolytic maturation of SERA6 requires interaction in multimolecular complexes with a distinct PV-located protein cofactor, MSA180, that is itself a SUB1 substrate. Genetic ablation of MSA180 mimics SERA6 disruption, producing a fatal block in ß-spectrin cleavage and RBC rupture. Drug-like inhibitors of SERA6 autoprocessing similarly prevent ß-spectrin cleavage and egress in both P. falciparum and the emerging zoonotic pathogen P. knowlesi. Our results elucidate the egress pathway and identify SERA6 as a target for a new class of antimalarial drugs designed to prevent disease progression.

Role of the membrane-associated accessory protein (MAAP) in adeno-associated virus (AAV) infection.

Adeno-associated viruses (AAVs) package a single-stranded (ss) DNA genome of 4.7 kb in their capsid of ~20 nm in diameter. AAV replication requires co-infection of a helper virus, such as adenovirus. During the optimization of recombinant AAV production, a small viral nonstructural protein, membrane-associated accessory protein (MAAP), was identified. However, the function of the MAAP in the context of AAV infection remains unknown. Here, we investigated the expression strategy and function of the MAAP during infection of both AAV2 and AAV5 in human embryonic kidney (HEK)293 cells. We found that AAV2 MAAP2 and AAV5 MAAP5 are expressed from the capsid gene (cap)-transcribing mRNA spliced from the donor to the second splice site that encodes VP2 and VP3. Thus, this AAV cap gene transcribes a multicistronic mRNA that can be translated to four viral proteins, MAAP, VP2, AAP, and VP3 in order. In AAV2 infection, MAAP2 predominantly localized in the cytoplasm, alongside the capsid, near the nuclear and plasma membranes, but a fraction of MAAP2 exhibited nuclear localization. In AAV5 infection, MAAP5 revealed a distinct pattern, predominantly localizing within the nucleus. In the cells infected with an MAAP knockout mutant of AAV2 or AAV5, both viral DNA replication and virus replication increased, whereas virus egress decreased, and the decrease in virus egress can be restored by providing MAAP in trans. In summary, MAAP, a novel AAV nonstructural protein translated from a multicistronic viral cap mRNA, not only facilitates cellular egress of AAV but also likely negatively affects viral DNA replication during infection. IMPORTANCE: Recombinant adeno-associated virus (rAAV) has been used as a gene delivery vector in clinical gene therapy. In current gene therapies employing rAAV, a high dose of the vector is required. Consequently, there is a high demand for efficient and high-purity vector production systems. In this study, we demonstrated that membrane-associated accessory protein (MAAP), a small viral nonstructural protein, is translated from the same viral mRNA transcript encoding VP2 and VP3. In AAV-infected cells, apart from its prevalent expression in the cytoplasm with localization near the plasma and nuclear membranes, the MAAP also exhibits notable localization within the nucleus. During AAV infection, MAAP expression increases the cellular egress of progeny virions and decreases viral DNA replication and progeny virion production. Thus, the choice of MAAP expression has pros and cons during AAV infection, which could provide a guide to rAAV production.

Multifaceted interactions between host ESCRT-III and budded virus-related proteins involved in entry and egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus.

The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.

Antibody inhibition of influenza A virus assembly and release.

Antibodies are frontline defenders against influenza virus infection, providing protection through multiple complementary mechanisms. Although a subset of monoclonal antibodies (mAbs) has been shown to restrict replication at the level of virus assembly and release, it remains unclear how potent and pervasive this mechanism of protection is, due in part to the challenge of separating this effect from other aspects of antibody function. To address this question, we developed imaging-based assays to determine how effectively a broad range of mAbs against the IAV surface proteins can specifically restrict viral egress. We find that classically neutralizing antibodies against hemagglutinin are broadly multifunctional, inhibiting virus assembly and release at concentrations 1-20-fold higher than the concentrations at which they inhibit viral entry. These antibodies are also capable of altering the morphological features of shed virions, reducing the proportion of filamentous particles. We find that antibodies against neuraminidase and M2 also restrict viral egress and that inhibition by anti-neuraminidase mAbs is only partly attributable to a loss in enzymatic activity. In all cases, antigen crosslinking-either on the surface of the infected cell, between the viral and cell membrane, or both-plays a critical role in inhibition, and we are able to distinguish between these modes experimentally and through a structure-based computational model. Together, these results provide a framework for dissecting antibody multifunctionality that could help guide the development of improved therapeutic antibodies or vaccines and that can be extended to other viral families and antibody isotypes.IMPORTANCEAntibodies against influenza A virus provide multifaceted protection against infection. Although sensitive and quantitative assays are widely used to measure inhibition of viral attachment and entry, the ability of diverse antibodies to inhibit viral egress is less clear. We address this challenge by developing an imaging-based approach to measure antibody inhibition of virus release across a panel of monoclonal antibodies targeting the influenza A virus surface proteins. Using this approach, we find that inhibition of viral egress is common and can have similar potency to the ability of an antibody to inhibit viral entry. Insights into this understudied aspect of antibody function may help guide the development of improved countermeasures.

Individual herpes simplex virus 1 (HSV-1) particles exit by exocytosis and accumulate at preferential egress sites.

The human pathogen herpes simplex virus 1 (HSV-1) produces a lifelong infection in the majority of the world's population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect are cell type dependent, not virus dependent. This preferential egress and clustering appear to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor.IMPORTANCEAlpha herpesviruses produce lifelong infections in their human and animal hosts. The majority of people in the world are infected with herpes simplex virus 1 (HSV-1), which typically causes recurrent oral or genital lesions. However, HSV-1 can also spread to the central nervous system, causing severe encephalitis, and might also contribute to the development of neurodegenerative diseases. Many of the steps of how these viruses infect and replicate inside host cells are known in depth, but the final step, exiting from the infected cell, is not fully understood. In this study, we engineered a novel variant of HSV-1 that allows us to visualize how individual virus particles exit from infected cells. With this imaging assay, we investigated preferential egress site formation in certain cell types and their contribution to the cell-cell spread of HSV-1.

Computational modeling of protracted HCMV replication using genome substrates and protein temporal profiles.

Human cytomegalovirus (HCMV) is a major cause of illness in immunocompromised individuals. The HCMV lytic cycle contributes to the clinical manifestations of infection. The lytic cycle occurs over ∼96 h in diverse cell types and consists of viral DNA (vDNA) genome replication and temporally distinct expression of hundreds of viral proteins. Given its complexity, understanding this elaborate system can be facilitated by the introduction of mechanistic computational modeling of temporal relationships. Therefore, we developed a multiplicity of infection (MOI)-dependent mechanistic computational model that simulates vDNA kinetics and late lytic replication based on in-house experimental data. The predictive capabilities were established by comparison to post hoc experimental data. Computational analysis of combinatorial regulatory mechanisms suggests increasing rates of protein degradation in association with increasing vDNA levels. The model framework also allows expansion to account for additional mechanisms regulating the processes. Simulating vDNA kinetics and the late lytic cycle for a wide range of MOIs yielded several unique observations. These include the presence of saturation behavior at high MOIs, inefficient replication at low MOIs, and a precise range of MOIs in which virus is maximized within a cell type, being 0.382 IU to 0.688 IU per fibroblast. The predicted saturation kinetics at high MOIs are likely related to the physical limitations of cellular machinery, while inefficient replication at low MOIs may indicate a minimum input material required to facilitate infection. In summary, we have developed and demonstrated the utility of a data-driven and expandable computational model simulating lytic HCMV infection.

Human herpesvirus 6A nuclear matrix protein U37 interacts with heat shock transcription factor 1 and activates the heat shock response.

Nascent nucleocapsids of herpesviruses acquire a primary envelope during their nuclear export by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This process is mediated by a conserved viral heterodimeric complex designated the nuclear egress complex, which consists of the nuclear matrix protein and the nuclear membrane protein. In addition to its essential roles during nuclear egress, the nuclear matrix protein has been shown to interact with intracellular signaling pathway molecules including NF-κB and IFN-ß to affect viral or cellular gene expression. The human herpesvirus 6A (HHV-6A) U37 gene encodes a nuclear matrix protein, the role of which has not been analyzed. Here, we show that HHV-6A U37 activates the heat shock element promoter and induces the accumulation of the molecular chaperone Hsp90. Mechanistically, HHV-6A U37 interacts with heat shock transcription factor 1 (HSF1) and induces its phosphorylation at Ser-326. We report that pharmacological inhibition of HSF1, Hsp70, or Hsp90 decreases viral protein accumulation and viral replication. Taken together, our results lead us to propose a model in which HHV-6A U37 activates the heat shock response to support viral gene expression and replication. IMPORTANCE Human herpesvirus 6A (HHV-6A) is a dsDNA virus belonging to the Roseolovirus genus within the Betaherpesvirinae subfamily. It is frequently found in patients with neuroinflammatory disease, although its pathogenetic role, if any, awaits elucidation. The heat shock response is important for cell survival under stressful conditions that disrupt homeostasis. Our results indicate that HHV-6A U37 activates the heat shock element promoter and leads to the accumulation of heat shock proteins. Next, we show that the heat shock response is important for viral replication. Overall, our findings provide new insights into the function of HHV-6A U37 in host cell signaling and identify potential cellular targets involved in HHV-6A pathogenesis and replication.

Extragenic suppression of an HSV-1 UL34 nuclear egress mutant reveals role for pUS9 as an inhibitor of epithelial cell-to-cell spread.

IMPORTANCE: Herpesviruses are able to disseminate in infected hosts despite development of a strong immune response. Their ability to do this relies on a specialized process called cell-to-cell spread in which newly assembled virus particles are trafficked to plasma membrane surfaces that abut adjacent uninfected cells. The mechanism of cell-to-cell spread is obscure, and little is known about whether or how it is regulated in different cells. We show here that a viral protein with a well-characterized role in promoting spread from neurons has an opposite, inhibitory role in other cells.

The role of lysosomes as intermediates in betacoronavirus PHEV egress from nerve cells.

IMPORTANCE: Betacoronaviruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. However, whether all betacoronaviruses members use the same pathway to exit cells remains unknown. Here, we demonstrated that porcine hemagglutinating encephalomyelitis virus (PHEV) egress occurs by Arl8b-dependent lysosomal exocytosis, a cellular egress mechanism shared by SARS-CoV-2 and MHV. Notably, PHEV acidifies lysosomes and activates lysosomal degradative enzymes, while SARS-CoV-2 and MHV deacidify lysosomes and limit the activation of lysosomal degradative enzymes. In addition, PHEV release depends on V-ATPase-mediated lysosomal pH. Furthermore, this is the first study to evaluate ßCoV using lysosome for spreading through the body, and we have found that lysosome played a critical role in PHEV neural transmission and brain damage caused by virus infection in the central nervous system. Taken together, different betacoronaviruses could disrupt lysosomal function differently to exit cells.

SARS-CoV-2 egress from Vero cells: a morphological approach.

Despite being extensively studied because of the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interactions with mammalian cells are still poorly understood. Furthermore, little is known about this coronavirus cycle within the host cells, particularly the steps that lead to viral egress. This study aimed to shed light on the morphological features of SARS-CoV-2 egress by utilizing transmission and high-resolution scanning electron microscopy, along with serial electron tomography, to describe the route of nascent virions towards the extracellular medium. Electron microscopy revealed that the clusters of viruses in the paracellular space did not seem to result from collective virus release. Instead, virus accumulation was observed on incurved areas of the cell surface, with egress primarily occurring through individual vesicles. Additionally, our findings showed that the emission of long membrane projections, which could facilitate virus surfing in Vero cells infected with SARS-CoV-2, was also observed in non-infected cultures, suggesting that these are constitutive events in this cell lineage.