Diverse Populations of Extracellular Vesicles with Opposite Functions during Herpes Simplex Virus 1 Infection.

Extracellular vesicles (EVs) are released by all types of cells as a means of intercellular communication. Their significance lies in the fact that they can alter recipient cell functions, despite their limited capacity for cargo. We have previously demonstrated that herpes simplex virus 1 (HSV-1) infection influences the cargo and functions of EVs released by infected cells and that these EVs negatively impact a subsequent HSV-1 infection. In the present study, we have implemented cutting-edge technologies to further characterize EVs released during HSV-1 infection. We identified distinct EV populations that were separable through a gradient approach. One population was positive for the tetraspanin CD63 and was distinct from EVs carrying components of the endosomal sorting complexes required for transport (ESCRT). Nanoparticle tracking analysis (NTA) combined with protein analysis indicated that the production of CD63+ EVs was selectively induced upon HSV-1 infection. The ExoView platform supported these data and suggested that the amount of CD63 per vesicle is larger upon infection. This platform also identified EV populations positive for other tetraspanins, including CD81 and CD9, whose abundance decreased upon HSV-1 infection. The stimulator of interferon genes (STING) was found in CD63+ EVs released during HSV-1 infection, while viral components were found in ESCRT+ EVs. Functional characterization of these EVs demonstrated that they have opposite effects on the infection, but the dominant effect was negative. Overall, we have identified the dominant population of EVs, and other EV populations produced during HSV-1 infection, and we have provided information about potential roles.IMPORTANCE Extracellular vesicles mediate cell-to-cell communication and convey messages important for cell homeostasis. Pathways of EV biogenesis are often hijacked by pathogens to facilitate their dissemination and to establish a favorable microenvironment for the infection. We have previously shown that HSV-1 infection alters the cargo and functions of the released EVs, which negatively impact the infection. We have built upon our previous findings by developing procedures to separate EV populations from HSV-1-infected cells. We identified the major population of EVs released during infection, which carries the DNA sensor STING and has an antiviral effect. We also identified an EV population that carries selected viral proteins and has a proviral role. This is the first study to characterize EV populations during infection. These data indicate that the complex interactions between the virus and the host are extended to the extracellular environment and could impact HSV-1 dissemination and persistence in the host.

Biogenesis of Extracellular Vesicles during Herpes Simplex Virus 1 Infection: Role of the CD63 Tetraspanin.

Herpes simplex virus 1 (HSV-1) infections afflict more than 80% of the population worldwide. The virus primarily infects mucoepithelial cells and establishes latent reservoirs in neurons in sensory ganglia. Frequent reactivation has been linked to severe diseases, especially in immunocompromised individuals. Earlier, we reported that viral and host factors are packaged in extracellular vesicles (EVs) and delivered to uninfected cells, where they activate antiviral responses and restrict virus infection. Here, we interrogated the effect of HSV-1 infection on EV biogenesis. We found that HSV-1 infection causes a decrease in the amount of intracellular CD63 protein with a concomitant increase in extracellular CD63. This observation correlates with our previous finding that infected cells release more CD63-positive EVs than uninfected cells. The stimulation of CD63 exocytosis requires virus replication. CD63 is a member of the tetraspanin family of proteins that traffics between the plasma membrane and endosomal compartments and has a role in sorting cargo into the EVs. Previously, we reported that in cells depleted of CD63, HSV-1 virus yields increased, and here we provide data showing that in cells overexpressing CD63, HSV-1 virus yields decreased. Taken together, our data indicate that CD63 negatively impacts HSV-1 infection and that the CD63-positive EVs could control the dissemination of the virus in the host. Perhaps EV release by HSV-1-infected cells is a mechanism that controls virus dissemination.IMPORTANCE Intercellular communication, especially in neurons, largely relies on EVs, and modulation of EVs is known to impact physiological processes. Here, we present evidence that HSV-1 infection causes major alterations in the biogenesis of EVs, including an increase in their number and an increase in the CD63-positive population of EVs. These alterations result in an enrichment of the milieu of infection with EVs carrying signatures from infected cells. In addition to changes in the origin and type, EVs released by infected cells have differences in cargo, as they carry viral and host factors determined by the virus. The tetraspanin CD63 negatively impacts the infection, as demonstrated by CD63-knockdown and overexpression assays. A proposed mechanism involves the activation of antiviral responses in cells receiving CD63-positive EVs released by infected cells. Overall, HSV-1 causes major alterations in EVs that could contribute to HSV-1 persistence and pathogenesis.

Discovery of Small-Molecule Inhibitors Targeting the E3 Ubiquitin Ligase Activity of the Herpes Simplex Virus 1 ICP0 Protein Using an In Vitro High-Throughput Screening Assay.

Herpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ΔICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-ß)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors.IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infection in vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.

Cbl E3 Ligase Mediates the Removal of Nectin-1 from the Surface of Herpes Simplex Virus 1-Infected Cells.

The Cbl E3 ligase has been linked to the down-modulation of surface signaling responses by inducing internalization of surface receptors. The adaptor protein CIN85 is a partner of Cbl that augments many of these interactions. Previously, an interaction was demonstrated between ICP0 and CIN85, which results in the removal of epidermal growth factor receptor (EGFR) from the surface of the infected cells with a concomitant attenuation of EGFR signaling. Here, we examined whether Cbl mediates the removal of the herpes simplex virus 1 (HSV-1) entry receptor Nectin-1 from the surface of infected cells. We found the following: (i) that Cbl, Nectin-1, and the viral glycoprotein D (gD) form a complex in infected cells; (ii) that during infection Nectin-1 is removed from the surface of the infected cells but is retained on the surface of cells that have been depleted of Cbl; and (iii) that in cells infected with a ΔICP0 mutant virus, Nectin-1 remained on the cell surface. Thus, Cbl is necessary but not sufficient for the removal of Nectin-1 from the cell surface. In addition, we observed that in Cbl-depleted cells there was enhanced entry after infection. These cells were susceptible to secondary infections by HSV-1. Viral entry in CIN85-depleted cells was only moderately enhanced compared to that in the Cbl-depleted cells, suggesting that the Cbl-Nectin-1 interaction is likely the key to the downregulation of surface Nectin-1. The removal of the HSV-1 entry receptor Nectin-1 from the surface of the infected cells may be part of the strategy of the virus to efficiently spread to uninfected cells.IMPORTANCE The Cbl E3 ligase suppresses surface signaling responses by inducing internalization of surface components. The targets of Cbl include such components as immune system receptors, growth factor receptors, adhesion, and cell-to-cell contact molecules. The immediate early protein ICP0 of herpes simplex virus 1 (HSV-1) interacts with CIN85, an adaptor protein that augments Cbl functions. The consequence of this interaction is the removal of the epidermal growth factor receptor (EGFR) from the surface of the infected cells with concomitant suppression of the EGF ligand signaling. The viral entry receptor Nectin-1 is also internalized during HSV-1 infection in a Cbl-dependent mechanism, and that increases the opportunity of the virus to spread to uninfected cells. The diversion of the Cbl/CIN85 endocytic machinery may be a strategy utilized by the virus to alter the cell surface pattern to prevent detrimental host responses.

Tracing the STING exocytosis pathway during herpes viruses infection.

The STimulator of INterferon Genes (STING) constitutes a major DNA-sensing pathway that restricts HSV-1 infection in different models by activating type I interferon and pro-inflammatory responses. To counteract STING, HSV-1 has evolved numerous strategies including mechanisms to interfere with its oligomerization, post-translational modifications, and downstream signaling. Previously, we demonstrated that STING is packaged in extracellular vesicles (EVs) produced from HSV-1-infected cells. These EVs activated antiviral responses in uninfected recipient cells and suppressed a subsequent HSV-1 infection in a STING-dependent manner. Here, we provide information on the packaging of STING in EVs and its exocytosis. We found that STING exocytosis did not occur in CD63 knockdown cells supporting that STING follows the CD63 exocytosis pathway. Consistently, we found that STING co-localized with CD63 in cytoplasmic globular structures and exosomal STING and CD63 co-fractionated. Both golgicide A and brefeldin A prevented STING exocytosis during HSV-1 infection suggesting that STING trafficking through the Golgi is required. A STING ligand was insufficient for STING exocytosis, and downstream signaling through TBK1 was not required. However, STING palmitoylation and tethering to the ER by STIM1 were required for STING exocytosis. Finally, we found that HSV-1 replication/late gene expression triggered CD63 exocytosis that was required for STING exocytosis. Surprisingly, HSV-2 strain G did not trigger CD63 or STING exocytosis as opposed to VZV and HCMV. Also, EVs from HSV-1(F)- and HSV-2(G)-infected cells displayed differences in their ability to restrict these viruses. Overall, STING exocytosis is induced by certain viruses and shapes the microenvironment of infection.IMPORTANCEExtracellular vesicles (EVs) are released by all types of cells as they constitute a major mechanism of intercellular communication. The packaging of specific cargo in EVs and the pathway of exocytosis are not fully understood. STING is a sensor of a broad spectrum of pathogens and a key component of innate immunity. STING exocytosis during HSV-1 infection has been an intriguing observation, raising questions of whether this is a virus-induced process, the purpose it serves, and whether it is observed after infection with other viruses. Here, we have provided insights into the pathway of STING exocytosis and determined factors involved. STING exocytosis is a virus-induced process and not a response of the host to the infection. Besides HSV-1, other herpes viruses triggered STING exocytosis, but HSV-2(G) did not. HSV-1 EVs displayed different restriction capabilities compared with HSV-2(G) EVs. Overall, STING exocytosis is triggered by viruses to shape the microenvironment of infection.

"Non-Essential" Proteins of HSV-1 with Essential Roles In Vivo: A Comprehensive Review.

Viruses encode for structural proteins that participate in virion formation and include capsid and envelope proteins. In addition, viruses encode for an array of non-structural accessory proteins important for replication, spread, and immune evasion in the host and are often linked to virus pathogenesis. Most virus accessory proteins are non-essential for growth in cell culture because of the simplicity of the infection barriers or because they have roles only during a state of the infection that does not exist in cell cultures (i.e., tissue-specific functions), or finally because host factors in cell culture can complement their absence. For these reasons, the study of most nonessential viral factors is more complex and requires development of suitable cell culture systems and in vivo models. Approximately half of the proteins encoded by the herpes simplex virus 1 (HSV-1) genome have been classified as non-essential. These proteins have essential roles in vivo in counteracting antiviral responses, facilitating the spread of the virus from the sites of initial infection to the peripheral nervous system, where it establishes lifelong reservoirs, virus pathogenesis, and other regulatory roles during infection. Understanding the functions of the non-essential proteins of herpesviruses is important to understand mechanisms of viral pathogenesis but also to harness properties of these viruses for therapeutic purposes. Here, we have provided a comprehensive summary of the functions of HSV-1 non-essential proteins.

Cloaked Viruses and Viral Factors in Cutting Edge Exosome-Based Therapies.

Extracellular vesicles (EVs) constitute a heterogeneous group of vesicles released by all types of cells that play a major role in intercellular communication. The field of EVs started gaining attention since it was realized that these vesicles are not waste bags, but they carry specific cargo and they communicate specific messages to recipient cells. EVs can deliver different types of RNAs, proteins, and lipids from donor to recipient cells and they can influence recipient cell functions, despite their limited capacity for cargo. EVs have been compared to viruses because of their size, cell entry pathways, and biogenesis and to viral vectors because they can be loaded with desired cargo, modified, and re-targeted. These properties along with the fact that EVs are stable in body fluids, they can be produced and purified in large quantities, they can cross the blood-brain barrier, and autologous EVs do not appear to cause major adverse effects, have rendered them attractive for therapeutic use. Here, we discuss the potential for therapeutic use of EVs derived from virus infected cells or EVs carrying viral factors. We have focused on six major concepts: (i) the role of EVs in virus-based oncolytic therapy or virus-based gene delivery approaches; (ii) the potential use of EVs for developing viral vaccines or optimizing already existing vaccines; (iii) the role of EVs in delivering RNAs and proteins in the context of viral infections and modulating the microenvironment of infection; (iv) how to take advantage of viral features to design effective means of EV targeting, uptake, and cargo packaging; (v) the potential of EVs in antiviral drug delivery; and (vi) identification of novel antiviral targets based on EV biogenesis factors hijacked by viruses for assembly and egress. It has been less than a decade since more attention was given to EV research and some interesting concepts have already been developed. In the coming years, additional information on EV biogenesis, how they are hijacked and utilized by pathogens, and their impact on the microenvironment of infection is expected to indicate avenues to optimize existing therapeutic tools and develop novel approaches.