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.

Keywords: CD63; CD81; HSV-1; biogenesis of extracellular vesicles; exocytosis; extracellular vesicles; tetraspanins.

FIG 1

Elimination of intracellular CD63 during HSV-1 infection. (A) HEL cells were exposed to HSV-1(F) (1 PFU/cell). The cells were harvested at 3, 9, 24, and 48 h after infection, and equal amounts of proteins were analyzed by immunoblot analysis using an anti-CD63 antibody. β-Actin served as a loading control. Lighter and longer exposures of the ECL Western blotting detection reagent-treated membrane after incubation with the anti-CD63 antibody are depicted. (B) The experiment whose results are presented in panel A was repeated in HEp-2 cells. (C) The experiment whose results are presented in panel A was repeated in HEK-293 cells. p.i., postinfection. The numbers to the left of the gels in panels A to C are molecular masses (in kilodaltons). (D) HEL cells were uninfected or exposed to HSV-1(F) (10 PFU/cell). The cells were harvested at 30 min and 1, 3, 5, and 9 h postinfection, and quantification of the CD63 transcripts was done by real-time PCR analysis. The 18S rRNA was used for normalization. (E) HEL cells were uninfected or exposed to HSV-1(F) (1 PFU/cell). The cells were harvested at 0, 2, 5, 9, 15, and 24 h postinfection, and quantification of the CD63 transcripts was done as described above. *, P ≤ 0.05; **, P ≤ 0.01.

FIG 2

HSV-1-infected cells release higher numbers of EVs enriched in CD63 protein. (A) Quantity of EVs derived from infected or uninfected HEL cells using NTA, as detailed in Materials and Methods. (B) Concentration and size distribution of EVs from the experiment whose results are presented in panel A, as determined by NTA. (C) HEL cells were exposed to HSV-1(F) (1 PFU/cell). The cells and the supernatant were harvested at 3, 24, and 48 h after infection and analyzed for CD63 protein. The supernatant was concentrated through a 10-kDa-molecular-mass-cutoff filter, as described in Materials and Methods, and analyzed for the presence of CD63 protein by immunoblot analysis. The cells were lysed, and equal amounts of proteins were analyzed by immunoblot analysis using an anti-CD63 antibody. β-Actin served as a loading control for the cell lysates. (D) EVs were isolated from uninfected and infected HEL cells (0.1 PFU/cell, 48 h), and equal number of EVs, determined by NTA, were analyzed by immunoblot analysis using antibodies against CD63, Flotillin-2, Alix, and CD81. (E to G) HEL cells were exposed to HSV-1(F) (1 PFU/cell). The cells were harvested at 3, 24, and 48 h after infection, and equal amounts of proteins were analyzed by immunoblot analysis using the CD81, Flotillin-2, TSG101, and Alix antibodies. The ICP0 protein was used as a control to monitor the infection. β-Actin served as a loading control. (H) HEL cells were infected with HSV-1(F) (10 PFU/cell). CHX (100 μg/ml) was added to the cultures at 12 h postinfection. The cells were harvested at 1, 7, 12, 18, and 24 h postinfection and 6 or 12 h after the addition of CHX. Equal amounts of proteins from total cell lysates were analyzed for the presence of the CD63 and CD81 proteins. ICP0 served as a control of the infection, and β-actin served as a loading control. The numbers to the left of the gels in panels C to G are molecular masses (in kilodaltons).

FIG 3

Viral replication is required for exocytosis of CD63 during HSV-1 infection. (A) HEL cells were either not infected or exposed to HSV-1(F) or various HSV-1 mutants (the ΔICP27, ΔICP0, RF, D199A, ΔUL46, Δγ₁34.5, and ΔVHS mutants) (1 PFU/cell). The cells were harvested at 24 h after infection, and the amount of intracellular CD63 was assessed in equal amounts of cell lysates by immunoblot analysis using an anti-CD63 antibody. The expression of ICP0 is also depicted. β-Actin served as a loading control. (B) HEL cells were either not infected or exposed to HSV-1(F) (1 PFU/cell). PAA was added to the cultures at the moment of infection (500 μg/ml). The cells were harvested at 3, 24, and 48 h postinfection, and the amount of intracellular CD63 was assessed as described above. Expression of gD is also depicted. β-Actin served as a loading control. (C) HEL cells were exposed to HSV-1(F) or a ΔICP8 virus (1 PFU/cell). The cells were harvested at 3, 24, and 48 h after infection, and equal amounts of proteins were analyzed by immunoblot analysis using antibodies against CD63, Alix, UL42, and ICP0. β-Actin served as a loading control. The numbers to the left of the gels in panels A to C are molecular masses (in kilodaltons). (D) EVs were derived from the supernatant of HEL cells exposed to HSV-1(F) or ΔICP8 virus (0.1 PFU/cell, 48 h), as detailed in Materials and Methods. Equal amount of EVs, determined by NTA, were analyzed using an anti-CD63 antibody. (E and F) EVs derived from uninfected or HSV-1(F)- or ΔICP8-infected cells, as described in the legend to panel C, were quantified using a NanoSight LM10 instrument, and their concentrations and quantities are depicted. The results represent the average for three independent isolations of EVs. P was equal to 8,0425 × 10⁻¹⁵ for mock-infected versus HSV-1(F)-infected cells, and P was equal to 2,020 × 10⁻¹⁷ for mock-infected versus ΔICP8-infected cells. P values were determined by a two-tailed, unpaired t test.

FIG 4

Exogenous expression of CD63 negatively impacts HSV-1 virus yields. (A) HEL cells and their derivatives expressing Flag-CD63 were exposed to HSV-1(F) (0.01 PFU/cell). The cells were harvested at 3, 24, and 48 h after infection, and titration of progeny viruses was done in Vero cells. (B) HEp-2 cells and their derivatives expressing either EYFP-CD63 or Flag-CD63 were infected as described in the legend to panel A, and progeny virus production was quantified as described in the legend to panel A. (C, D) Expression of Flag-CD63 and EYFP-CD63 in the established cell lines. The numbers to the left of the gels in panels C and D are molecular masses (in kilodaltons). (E) HEp-2 cells and their derivatives expressing Flag-CD63 were exposed to HSV-1(F) (0.1 PFU/cell). The cells were harvested at 1, 5, 15, and 24 h after infection, and quantification of the viral genome was done by quantitative PCR analysis using primer pairs against the gI region of the viral genome or the β-actin gene, which served as a normalization control. P was equal to 0.033 for HEp-2 versus HEp-2-expressing Flag-CD63 at 24 h postinfection (as determined by a one-tailed, unpaired t test). *, P ≤ 0.05.