Interplay between Autophagy and Herpes Simplex Virus Type 1: ICP34.5, One of the Main Actors.

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that occasionally may spread to the central nervous system (CNS), being the most common cause of sporadic encephalitis. One of the main neurovirulence factors of HSV-1 is the protein ICP34.5, which although it initially seems to be relevant only in neuronal infections, it can also promote viral replication in non-neuronal cells. New ICP34.5 functions have been discovered during recent years, and some of them have been questioned. This review describes the mechanisms of ICP34.5 to control cellular antiviral responses and debates its most controversial functions. One of the most discussed roles of ICP34.5 is autophagy inhibition. Although autophagy is considered a defense mechanism against viral infections, current evidence suggests that this antiviral function is only one side of the coin. Different types of autophagic pathways interact with HSV-1 impairing or enhancing the infection, and both the virus and the host cell modulate these pathways to tip the scales in its favor. In this review, we summarize the recent progress on the interplay between autophagy and HSV-1, focusing on the intricate role of ICP34.5 in the modulation of this pathway to fight the battle against cellular defenses.

Herpes Simplex Virus 1 Spread in Oligodendrocytic Cells Is Highly Dependent on MAL Proteolipid.

Myelin and lymphocyte protein (MAL) is a tetraspan integral membrane protein that resides in detergent-insoluble membrane fractions enriched in condensed membranes. MAL is expressed in oligodendrocytes, in Schwann cells, where it is essential for the stability of myelin, and at the apical membrane of epithelial cells, where it has a critical role in transport. In T lymphocytes, MAL is found at the immunological synapse and plays a crucial role in exosome secretion. However, no involvement of MAL in viral infections has been reported so far. Here, we show that herpes simplex virus 1 (HSV-1) virions travel in association with MAL-positive structures to reach the end of cellular processes, which contact uninfected oligodendrocytes. Importantly, the depletion of MAL led to a significant decrease in infection, with a drastic reduction in the number of lytic plaques in MAL-silenced cells. These results suggest a significant role for MAL in viral spread at cell contacts. The participation of MAL in the cell-to-cell spread of HSV-1 may shed light on the involvement of proteolipids in this process.IMPORTANCE Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establish latent infections in neurons. HSV-1 may spread from infected to uninfected cells by two main routes: by cell-free virus or by cell-to-cell spread. In the first case, virions exit into the extracellular space and then infect another cell from the outside. In the second case, viral transmission occurs through cell-to-cell contacts via a mechanism that is still poorly understood. A third mode of spread, using extracellular vesicles, also exists. In this study, we demonstrate the important role for a myelin protein, myelin and lymphocyte protein (MAL), in the process of cell-to-cell viral spread in oligodendrocytes. We show that MAL is involved in trafficking of virions along cell processes and that MAL depletion produces a significant alteration in the viral cycle, which reduces cell-to cell spread of HSV-1.

HSV-1 and Endogenous Retroviruses as Risk Factors in Demyelination.

Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that can infect the peripheral and central nervous systems, and it has been implicated in demyelinating and neurodegenerative processes. Transposable elements (TEs) are DNA sequences that can move from one genomic location to another. TEs have been linked to several diseases affecting the central nervous system (CNS), including multiple sclerosis (MS), a demyelinating disease of unknown etiology influenced by genetic and environmental factors. Exogenous viral transactivators may activate certain retrotransposons or class I TEs. In this context, several herpesviruses have been linked to MS, and one of them, HSV-1, might act as a risk factor by mediating processes such as molecular mimicry, remyelination, and activity of endogenous retroviruses (ERVs). Several herpesviruses have been involved in the regulation of human ERVs (HERVs), and HSV-1 in particular can modulate HERVs in cells involved in MS pathogenesis. This review exposes current knowledge about the relationship between HSV-1 and human ERVs, focusing on their contribution as a risk factor for MS.

The Role of Herpes Simplex Virus Type 1 Infection in Demyelination of the Central Nervous System.

Herpes simplex type 1 (HSV-1) is a neurotropic virus that infects the peripheral and central nervous systems. After primary infection in epithelial cells, HSV-1 spreads retrogradely to the peripheral nervous system (PNS), where it establishes a latent infection in the trigeminal ganglia (TG). The virus can reactivate from the latent state, traveling anterogradely along the axon and replicating in the local surrounding tissue. Occasionally, HSV-1 may spread trans-synaptically from the TG to the brainstem, from where it may disseminate to higher areas of the central nervous system (CNS). It is not completely understood how HSV-1 reaches the CNS, although the most accepted idea is retrograde transport through the trigeminal or olfactory tracts. Once in the CNS, HSV-1 may induce demyelination, either as a direct trigger or as a risk factor, modulating processes such as remyelination, regulation of endogenous retroviruses, or molecular mimicry. In this review, we describe the current knowledge about the involvement of HSV-1 in demyelination, describing the pathways used by this herpesvirus to spread throughout the CNS and discussing the data that suggest its implication in demyelinating processes.

The Role of Extracellular Vesicles in Demyelination of the Central Nervous System.

It is being increasingly demonstrated that extracellular vesicles (EVs) are deeply involved in the physiology of the central nervous system (CNS). Processes such as synaptic activity, neuron-glia communication, myelination and immune response are modulated by EVs. Likewise, these vesicles may participate in many pathological processes, both as triggers of disease or, on the contrary, as mechanisms of repair. EVs play relevant roles in neurodegenerative disorders such as Alzheimer's or Parkinson's diseases, in viral infections of the CNS and in demyelinating pathologies such as multiple sclerosis (MS). This review describes the involvement of these membrane vesicles in major demyelinating diseases, including MS, neuromyelitis optica, progressive multifocal leukoencephalopathy and demyelination associated to herpesviruses.

Role of Microvesicles in the Spread of Herpes Simplex Virus 1 in Oligodendrocytic Cells.

Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in the neurons of sensory ganglia. In some cases, the virus spreads into the central nervous system, causing encephalitis or meningitis. Cells infected with several different types of viruses may secrete microvesicles (MVs) containing viral proteins and RNAs. In some instances, extracellular microvesicles harboring infectious virus have been found. Here we describe the features of shedding microvesicles released by the human oligodendroglial HOG cell line infected with HSV-1 and their participation in the viral cycle. Using transmission electron microscopy, we detected for the first time microvesicles containing HSV-1 virions. Interestingly, the Chinese hamster ovary (CHO) cell line, which is resistant to infection by free HSV-1 virions, was susceptible to HSV-1 infection after being exposed to virus-containing microvesicles. Therefore, our results indicate for the first time that MVs released by infected cells contain virions, are endocytosed by naive cells, and lead to a productive infection. Furthermore, infection of CHO cells was not completely neutralized when virus-containing microvesicles were preincubated with neutralizing anti-HSV-1 antibodies. The lack of complete neutralization and the ability of MVs to infect nectin-1/HVEM-negative CHO-K1 cells suggest a novel way for HSV-1 to spread to and enter target cells. Taken together, our results suggest that HSV-1 could spread through microvesicles to expand its tropism and that microvesicles could shield the virus from neutralizing antibodies as a possible mechanism to escape the host immune response.IMPORTANCE Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in neurons. Extracellular vesicles are a heterogeneous group of membrane vesicles secreted by most cell types. Microvesicles, which are extracellular vesicles which derive from the shedding of the plasma membrane, isolated from the supernatant of HSV-1-infected HOG cells were analyzed to find out whether they were involved in the viral cycle. The importance of our investigation lies in the detection, for the first time, of microvesicles containing HSV-1 virions. In addition, virus-containing microvesicles were endocytosed into CHO-K1 cells and were able to actively infect these otherwise nonpermissive cells. Finally, the infection of CHO cells with these virus-containing microvesicles was not completely neutralized by anti-HSV-1 antibodies, suggesting that these extracellular vesicles might shield the virus from neutralizing antibodies as a possible mechanism of immune evasion.

Pseudorabies virus uses clathrin mediated endocytosis to enter PK15 swine cell line.

Pseudorabies virus (PRV), a herpesvirus responsible for Aujeszky's disease, causes high mortality in swine populations. To develop effective and novel antiviral strategies, it is essential to understand the mechanism of entry used by PRV to infect its host. Viruses have different ways of entering host cells. Among others, they can use endocytosis, a fundamental cellular process by which substances from the external environment are internalized into the cell. This process is classified into clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE), depending on the role of clathrin. Although the involvement of cholesterol-rich lipid rafts in the entry of PRV has already been described, the importance of other endocytic pathways involving clathrin remains unexplored to date. Here, we characterize the role of CME in PRV entry into the PK15 swine cell line. By using CME inhibitory drugs, we report a decrease in PRV infection when the CME pathway is blocked. We also perform the shRNA knockdown of the µ-subunit of the adaptor protein AP-2 (AP2M1), which plays an important role in the maturation of clathrin-coated vesicles, and the infection is greatly reduced when this subunit is knocked down. Furthermore, transmission electron microscopy images report PRV virions inside clathrin-coated vesicles. Overall, this study suggests for the first time that CME is a mechanism used by PRV to enter PK15 cells and provides valuable insights into its possible routes of entry.

Herpes Simplex Virus type 1 inhibits autophagy in glial cells but requires ATG5 for the success of viral replication.

Herpes Simplex Virus type 1 (HSV-1) 1 is a neurotropic virus that has been associated with neurodegenerative disorders. The dysregulation of autophagy by HSV-1 has been proposed as a potential cause of neurodegeneration. While studies have extensively tackled the interaction between autophagy and HSV-1 in neurons, research in glial cells is currently limited. Our studies demonstrate that HSV-1 inhibits, but not completely blocks, the formation of autophagosomes in human oligodendroglioma- and astrocytoma- derived cell lines. These findings have been confirmed in murine oligodendrocyte precursor cells (OPCs). Finally, this study investigates the impact of autophagy on HSV-1 infection in glial cells. While the lack of basal autophagy in LC3B knockout glial cells does not have a significant effect on viral infection, cells without the autophagy-related protein ATG5 exhibit reduced viral production. The absence of ATG5 leads to a decrease in the transcription and replication of viral genes, as well as a delay in the initial stages of the formation of HSV-1 replication compartments. These findings indicate that while autophagy may not play a significant role in antiviral defense in glial cells, HSV-1 may be inhibiting autophagy to exploit non-canonical functions of certain components of the autophagic machinery, such as ATG5, to benefit its lifecycle.

Liposomal Lactoferrin Exerts Antiviral Activity against HCoV-229E and SARS-CoV-2 Pseudoviruses In Vitro.

A limited number of effective therapies are currently available to treat human coronavirus SARS-CoV-2 and other human coronaviruses, which are responsible for nearly a third of global cases of the common cold. The possibility of new emerging coronaviruses demands powerful new antiviral strategies. Lactoferrin is a well-known protein that possesses anti-inflammatory and immunomodulatory activities, and it has previously shown antiviral activity against several viruses, including SARS-CoV-2. To increase this antiviral activity, here we present bovine liposomal lactoferrin. Liposomal encapsulation of the compound was proven to increase permeability, bioavailability, and time release. In the present work, we compare the antiviral activity of free and liposomal bovine lactoferrin against HCoV229E and SARS-CoV-2 in vitro and in human primary bronchial epithelial cells, and we demonstrated that the liposomal form exerts a more potent antiviral activity than its free form at non-cytotoxic doses.

Dextran sulfate from Leuconostoc mesenteroides B512F exerts potent antiviral activity against SARS-CoV-2 in vitro and in vivo.

The emergent human coronavirus SARS-CoV-2 and its resistance to current drugs makes the need for new potent treatments for COVID-19 patients strongly necessary. Dextran sulfate (DS) polysaccharides have long demonstrated antiviral activity against different enveloped viruses in vitro. However, their poor bioavailability has led to their abandonment as antiviral candidates. Here, we report for the first time the broad-spectrum antiviral activity of a DS-based extrapolymeric substance produced by the lactic acid bacterium Leuconostoc mesenteroides B512F. Time of addition assays with SARS-CoV-2 pseudoviruses in in vitro models confirm the inhibitory activity of DSs in the early stages of viral infection (viral entry). In addition, this exopolysaccharide substance also reports broad-spectrum antiviral activity against several enveloped viruses such as SARS-CoV-2, HCoV229E, HSV-1, in in vitro models and in human lung tissue. The toxicity and antiviral capacity of DS from L. mesenteroides was tested in vivo in mouse models which are susceptible to SARS-CoV-2 infection. The described DS, administered by inhalation, a new route of administration for these types of polymers, shows strong inhibition of SARS-CoV-2 infection in vivo, significantly reducing animal mortality and morbidity at non-toxic doses. Therefore, we suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2.

Role of the small GTPase Rab27a during herpes simplex virus infection of oligodendrocytic cells.

BACKGROUND: The morphogenesis of herpes simplex virus type 1 (HSV-1) comprises several events, of which some are not completely understood. It has been shown that HSV-1 glycoproteins accumulate in the trans-Golgi network (TGN) and in TGN-derived vesicles. It is also accepted that HSV-1 acquires its final morphology through a secondary envelopment by budding into TGN-derived vesicles coated with viral glycoproteins and tegument proteins. Nevertheless, several aspects of this process remain elusive. The small GTPase Rab27a has been implicated in regulated exocytosis, and it seems to play a key role in certain membrane trafficking events. Rab27a also seems to be required for human cytomegalovirus assembly. However, despite the involvement of various Rab GTPases in HSV-1 envelopment, there is, to date, no data reported on the role of Rab27a in HSV-1 infection. RESULTS: Herein, we show that Rab27a colocalized with GHSV-UL46, a tegument-tagged green fluorescent protein-HSV-1, in the TGN. In fact, this small GTPase colocalized with viral glycoproteins gH and gD in that compartment. Functional analysis through Rab27a depletion showed a significant decrease in the number of infected cells and viral production in Rab27a-silenced cells. CONCLUSIONS: Altogether, our results indicate that Rab27a plays an important role in HSV-1 infection of oligodendrocytic cells.

Extracellular Polymeric Substances: Still Promising Antivirals.

Sulfated polysaccharides and other polyanions have been promising candidates in antiviral research for decades. These substances gained attention as antivirals when they demonstrated a high inhibitory effect in vitro against human immunodeficiency virus (HIV) and other enveloped viruses. However, that initial interest was followed by wide skepticism when in vivo assays refuted the initial results. In this paper we review the use of sulfated polysaccharides, and other polyanions, in antiviral therapy, focusing on extracellular polymeric substances (EPSs). We maintain that, in spite of those early difficulties, the use of polyanions and, specifically, the use of EPSs, in antiviral therapy should be reconsidered. We base our claim in several points. First, early studies showed that the main disadvantage of sulfated polysaccharides and polyanions is their low bioavailability, but this difficulty can be overcome by the use of adequate administration strategies, such as nebulization of aerosols to gain access to respiratory airways. Second, several sulfated polysaccharides and EPSs have demonstrated to be non-toxic in animals. Finally, these macromolecules are non-specific and therefore they might be used against different variants or even different viruses.

Potent Virucidal Activity In Vitro of Photodynamic Therapy with Hypericum Extract as Photosensitizer and White Light against Human Coronavirus HCoV-229E.

The emergent human coronavirus SARS-CoV-2 and its high infectivity rate has highlighted the strong need for new virucidal treatments. In this sense, the use of photodynamic therapy (PDT) with white light, to take advantage of the sunlight, is a potent strategy for decreasing the virulence and pathogenicity of the virus. Here, we report the virucidal effect of PDT based on Hypericum extract (HE) in combination with white light, which exhibits an inhibitory activity of the human coronavirus HCoV-229E on hepatocarcinoma Huh-7 cells. Moreover, despite continuous exposure to white light, HE has long durability, being able to maintain the prevention of viral infection. Given its potent in vitro virucidal capacity, we propose HE in combination with white light as a promising candidate to fight against SARS-CoV-2 as a virucidal compound.

Nebulized CLODOS Technology Shows Clear Virucidal Properties against the Human Coronavirus HCoV-229E at Non-Cytotoxic Doses.

The emergent human coronavirus SARS-CoV-2 and its high infectivity rate has highlighted the strong need for new disinfection systems. Evidence has proven that airborne transmission is an important route of spreading for this virus. Therefore, this short communication introduces CLODOS Technology®, a novel strategy to disinfect contaminated surfaces. It is a product based on stable and 99% pure chlorine dioxide, already certified as a bactericide, fungicide and virucide against different pathogens. In this study, CLODOS Technology®, by direct contact or thermonebulization, showed virucidal activity against the human coronavirus HCoV-229E at non-cytotoxic doses. Different conditions such as nebulization, exposure time and product concentration have been tested to standardize and optimize this new feasible method for disinfection.

Membrane Rafts: Portals for Viral Entry.

Membrane rafts are dynamic, small (10-200 nm) domains enriched with cholesterol and sphingolipids that compartmentalize cellular processes. Rafts participate in roles essential to the lifecycle of different viral families including virus entry, assembly and/or budding events. Rafts seem to participate in virus attachment and recruitment to the cell surface, as well as the endocytic and non-endocytic mechanisms some viruses use to enter host cells. In this review, we will introduce the specific role of rafts in viral entry and define cellular factors implied in the choice of one entry pathway over the others. Finally, we will summarize the most relevant information about raft participation in the entry process of enveloped and non-enveloped viruses.

The Valproic Acid Derivative Valpromide Inhibits Pseudorabies Virus Infection in Swine Epithelial and Mouse Neuroblastoma Cell Lines.

Pseudorabies virus (PRV) infection of swine can produce Aujeszky's disease, which causes neurological, respiratory, and reproductive symptoms, leading to significant economic losses in the swine industry. Although humans are not the natural hosts of PRV, cases of human encephalitis and endophthalmitis caused by PRV infection have been reported between animals and workers. Currently, a lack of specific treatments and the emergence of new PRV strains against which existing vaccines do not protect makes the search for effective antiviral drugs essential. As an alternative to traditional nucleoside analogues such as acyclovir (ACV), we studied the antiviral effect of valpromide (VPD), a compound derived from valproic acid, against PRV infection in the PK15 swine cell line and the neuroblastoma cell line Neuro-2a. First, the cytotoxicity of ACV and VPD in cells was compared, demonstrating that neither compound was cytotoxic at a specific concentration range after 24 h exposure. Furthermore, the lack of direct virucidal effect of VPD outside of an infected cell environment was demonstrated. Finally, VPD was shown to have an antiviral effect on the viral production of two strains of pseudorabies virus (wild type NIA-3 and recombinant PRV-XGF) at the concentrations ranging from 0.5 to 1.5 mM, suggesting that VPD could be a suitable alternative to nucleoside analogues as an antiherpetic drug against Aujeszky's disease.

Characterization of the MAL2-positive compartment in oligodendrocytes.

Oligodendrocytes (OLs), the myelin-producing cells of the central nervous system, segregate different surface subdomains at the plasma membrane as do other differentiated cells such as polarized epithelia and neurons. To generate the complex membrane system that characterizes myelinating OLs, large amounts of membrane proteins and lipids need to be synthesized and correctly targeted. In polarized epithelia, a considerable fraction of apical proteins are transported by an indirect pathway involving a detour to the basolateral membrane before being internalized and transported across the cell to the apical membrane by a process known as transcytosis. The apical recycling endosome (ARE) or its equivalent, the subapical compartment (SAC), of hepatocytes is an intracellular trafficking station involved in the transcytotic pathway. MAL2, an essential component of the machinery for basolateral-to-apical transcytosis, is an ARE/SAC resident protein. Here, we show that, after differentiation, murine oligodendrocyte precursor and human oligodendroglioma derived cell lines, Oli-neu and HOG, respectively, up-regulate the expression of MAL2 and accumulate it in an intracellular compartment, exhibiting a peri-centrosomal localization. In these oligodendrocytic cell lines, this compartment shares some of the main features of the ARE/SAC, such as colocalization with Rab11a, sensitivity to disruption of the microtubule cytoskeleton with nocodazole, and lack of internalized transferrin. Therefore, we suggest that the MAL2-positive compartment in oligodendrocytic cells could be a structure analogous to the ARE/SAC and might have an important role in the sorting of proteins and lipids for myelin assembly during oligodendrocyte differentiation.

Isolation/Analysis of Extracellular Microvesicles from HSV-1-Infected Cells.

Extracellular vesicles (EVs) are secreted membrane vesicles, derived from endosomes or from the plasma membrane, which have been isolated from most cell types and biological fluids. Although EVs are highly heterogeneous and their classification is complex, two major categories can be distinguished: microvesicles (MVs), which derive from the shedding of the plasma membrane, and exosomes, which correspond to intraluminal vesicles released to the extracellular milieu upon fusion of multivesicular bodies (MVBs) with the plasma membrane. Cells infected with viruses may secrete MVs containing viral proteins, RNAs and, in some instances, infectious virions. A recent study carried out by our laboratory has shown that MVs released by cells infected with HSV-1 contained virions and were endocytosed by naïve cells leading to a productive infection. This suggests that HSV-1 may use MVs for spreading, expanding its tropism and evading the host immune response. Here we describe in detail the methods used to isolate and analyse the MVs released from HSV-1-infected cells.

Extracellular Vesicles in Viral Spread and Antiviral Response.

Viral spread by both enveloped and non-enveloped viruses may be mediated by extracellular vesicles (EVs), including microvesicles (MVs) and exosomes. These secreted vesicles have been demonstrated to be an efficient mechanism that viruses can use to enter host cells, enhance spread or evade the host immune response. However, the complex interplay between viruses and EVs gives rise to antagonistic biological tasks-to benefit the viruses, enhancing infection and interfering with the immune system or to benefit the host, by mediating anti-viral responses. Exosomes from cells infected with herpes simplex type 1 (HSV-1) may transport viral and host transcripts, proteins and innate immune components. This virus may also use MVs to expand its tropism and evade the host immune response. This review aims to describe the current knowledge about EVs and their participation in viral infection, with a specific focus on the role of exosomes and MVs in herpesvirus infections, particularly that of HSV-1.

Hsv-1 Endocytic Entry into a Human Oligodendrocytic Cell Line is Mediated by Clathrin and Dynamin but Not Caveolin.

Endocytosis is a pathway used by viruses to enter cells that can be classified based on the proteins involved, such as dynamin, clathrin or caveolin. Although the entry of herpes simplex type 1 (HSV-1) by endocytosis has been documented in different cell types, its dependence on clathrin has not been described whereas its dependence on dynamin has been shown according to the cell line used. The present work shows how clathrin-mediated endocytosis (CME) is one way that HSV-1 infects the human oligodendroglial (HOG) cell line. Partial dynamin inhibition using dynasore revealed a relationship between decrease of infection and dynamin inhibition, measured by viral titration and immunoblot. Co-localization between dynamin and HSV-1 was verified by immunofluorescence at the moment of viral entry into the cell. Inhibition by chlorpromazine revealed that viral progeny also decreased when clathrin was partially inhibited in our cell line. RT-qPCR of immediately early viral genes, specific entry assays and electron microscopy all confirmed clathrin's participation in HSV-1 entry into HOG cells. In contrast, caveolin entry assays showed no effect on the entry of this virus. Therefore, our results suggest the participation of dynamin and clathrin during endocytosis of HSV-1 in HOG cells.