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

Abstract

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.

The ICP0 Protein of Herpes Simplex Virus 1 (HSV-1) Downregulates Major Autophagy Adaptor Proteins Sequestosome 1 and Optineurin during the Early Stages of HSV-1 Infection

“…Immunoblot analysis. The procedures for immunoblotting were described elsewhere (50,86). Briefly, cells were solubilized in triple-detergent buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 100 g of phenylmethylsulfonyl fluoride ml Ϫ1 ) supplemented with phosphatase inhibitors (10 mM NaF, 10 mM ␤-glycerophosphate, 0.1 mM sodium vanadate) and protease inhibitor cocktail (Sigma) and briefly sonicated.…”

Participation of Extracellular Vesicles from Zika-Virus-Infected Mosquito Cells in the Modification of Naïve Cells’ Behavior by Mediating Cell-to-Cell Transmission of Viral Elements

“…First, the presence of CD63-like tetraspanin at the membrane surface of C6/36 cells was evaluated as well. The point time of the CD63 membrane decreased (CD63 internalization) to select the best condition for the identification of the sEVs marker (inside cells) and, likewise, the time for the optimal isolation of sEVs CD63-like+ [45]. The tetraspanin detection (FACS) was evaluated at 24, 48, 72, 96, and 120 h PI ( Figure 3A).…”

Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules

“…Interestingly, alphaherpesvirus gB contains a conserved and still unexplored YP(L/I/V)X4L motif in the C-terminal domain, which resembles cryptic (non-canonical) L-domain motifs YPx(n)L/I from Ebola and Marburg virus-encoded VP40 proteins, known for their EVs localization [68]. What is also interesting is that CD63 can regulate EVs and HSV-1 secretion during virus infection [4,69].…”