Extracellular Vesicles Released by Herpes Simplex Virus 1-Infected Cells Block Virus Replication in Recipient Cells in a STING-Dependent Manner.

Herpes simplex virus 1 (HSV-1)-infected cells release extracellular vesicles (EVs) that deliver to uninfected cells viral factors and host components, such as the stimulator of interferon genes (STING), which activates type I interferon upon foreign DNA sensing. The functions of EVs released by HSV-1-infected cells have remained unknown. Here, we describe a procedure to separate the EVs from HSV-1 virions that is based on an iodixanol/sucrose gradient. STING, along with the EV markers CD63 and CD9, was found in light-density fractions, while HSV components accumulated in heavy-density fractions. HSV-1 infection stimulated the release of EVs from the cells. The EVs derived from infected cells, but not from uninfected cells, activated innate immunity in recipient cells and suppressed viral gene expression and virus replication. Moreover, only the EVs derived from infected cells stimulated the expression of a subset of M1-type markers in recipient macrophages. Conversely, EVs derived from STING-knockdown cells failed to stimulate the expression of these M1-type markers, they activated innate immune responses to a lesser extent in recipient cells, and they did not sustain the inhibition of virus replication. These data suggest that STING from the EV donor cells contributes to the antiviral responses in cells receiving EVs from HSV-1-infected cells. Perturbations in the biogenesis of EVs by silencing CD63 or blocking the activity of the neutral spingomyelinase-2 (nSMase-2) increased the HSV-1 yields. Overall, our data suggest that the EVs released from HSV-1-infected cells negatively impact the infection and could control the dissemination of the virus.IMPORTANCE Extracellular vesicles (EVs) are released by all types of cells as they constitute major mechanism of intercellular communication and have the capacity to alter the functions of recipient cells despite their limited capacity for cargo. How the EVs released by HSV-infected cells could alter the surrounding microenvironment and influence the infection currently remains unknown. The cargo of EVs reflects the physiological state of the cells in which they were produced, so the content of EVs originating from infected cells is expected to be substantially different from that of healthy cells. Our studies indicate that the EVs released by HSV-1-infected cells carry innate immune components such as STING and other host and viral factors; they can activate innate immune responses in recipient cells and inhibit HSV-1 replication. The implication of these data is that the EVs released by HSV-1-infected cells could control HSV-1 dissemination promoting its persistence in the host.

Deschamps T ，Kalamvoki M 《-》

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.

Dogrammatzis C ，Saleh S ，Deighan C ，Kalamvoki M ... -  《-》

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.

Dogrammatzis C ，Deschamps T ，Kalamvoki M 《-》

Tracing the STING exocytosis pathway during herpes viruses infection.

Dogrammatzis C ，Saud R ，Waisner H ，Lasnier S ，Suma SM ，Grieshaber B ，Kalamvoki M ... -  《mBio》

Extracellular vesicles during Herpes Simplex Virus type 1 infection: an inquire.

Kalamvoki M ，Deschamps T 《-》