The γ₁34.5 Neurovirulence Gene of Herpes Simplex Virus 1 Modifies the Exosome Secretion Profile in Epithelial Cells

LETTER

Recently, it has been demonstrated that herpes simplex virus 1 (HSV-1)-infected cells secrete exosomes that deliver to uninfected cells the innate immune sensor STING and viral RNAs (1, 2). Here, we report for the first time that the deletion of the viral γ₁34.5 neurovirulence gene affects HSV-induced exosome secretion.

HSV-1, lacking both copies of the γ₁34.5 neurovirulence gene, is a proficient gene therapy vector backbone for use in the nervous system. Although the γ₁34.5 mutants are nonneurovirulent, they can still spread in different cell types of the nervous system (3, 4). However, the possibility that exosomes copurify with HSV preparations raises concern about the vector purity and reproducibility in gene therapy use.

To elucidate the contents of our HSV stocks, ultracentrifuge gradients of two HSV wild-type (wt) strains, HSV-1(17+) and HSV-1(F), and four γ₁34.5 deletion mutants, H1052 (4), HSV-1(17+)Lox-Luc-Δγ₁34.5-Zeo (HSV-Zeo) (5), R3616 (6), and R3659 (7), were characterized and compared. H1052 and Zeo were derived from HSV-1(17+) (Fig. 1 [see next page]), and R3616 and R3659 were derived from HSV-1(F) (Fig. 2). Viruses were produced in human keratinocyte (HaCaT) cells, infected with 0.01 PFU/cell of the viruses. The cell-free culture medium was collected and passed through an 0.45-μm filter before ultracentrifugation and virus purification in a linear 10 to 40% Optiprep gradient. Fractions of 500 μl to 1 ml were collected, of which total protein concentrations and virus titers were determined. Five selected fractions were further analyzed in the Western blot assay (Fig. 1C and D and 2C and D) to examine whether exosome marker proteins, CD63 and Alix, are present in the virus fractions (positive for the capsid protein VP5). Strikingly, exosome marker proteins were abundant in the wt HSV-1(17+) and HSV-1(F) virus fractions but not in the corresponding γ₁34.5 deletion virus fractions (Fig. 1 and 2). To verify that the lack of exosome proteins resulted from the deletion of the γ₁34.5 neurovirulence gene and not from all mutations affecting the innate immunity evasion properties, an HSV-1 mutant lacking the Us3 gene (R7041) and its rescue virus (R7306) (8) were purified and characterized (Fig. 3). The viruses R7041 and R7306 were both copurified with exosome marker protein CD63 and, to a lesser extent, with Alix.

FIG 1.

CD63-positive exosomes copurify with wt HSV-1(17+) but not with its γ₁34.5 deletion mutants. wt HSV-1(17+) and its two γ₁34.5 deletion derivatives, H1052 (γ₁34.5 del) and HSV-1(17+)Lox-Luc-Δγ₁34.5-Zeo (HSV-Zeo), were produced in HaCaT cells and purified in an Optiprep 10 to 40% density gradient. Fractions were collected in 0.5- to 1-ml batches, and virus titer (A) and the total protein concentration (B) in each fraction were determined. Five selected fractions of each gradient from top to bottom were further analyzed by Western blotting (C and D) for the presence of viral proteins (gD, sc-21719; VP5, HA018-100) (red) and exosome proteins (CD63, sc-15363; Alix, sc-99010) (green) within the gradients. For secondary detection, IRDye antibodies 680 and 800 (926-68070 and 926-32211, respectively) from Li-Cor Biotechnology were used. Blots were visualized and quantified with Odyssey FC Imager and Image Studio Lite v4.0 program, and the fluorescence intensity of CD63 is shown in panel C. In all Western blot assays, 5 μg of protein was used per lane, and the exposure time for each channel was 2 min. gD, 61 kDa; VP5, 155 kDa; Alix, 95 kDa; CD63, 30 to 60 kDa. Chameleon Duo from Li-Cor was used as a molecular mass standard (std).

FIG 2.

CD63-positive exosomes copurify with wt HSV-1(F) but not with its γ₁34.5 deletion mutants. wt HSV-1(F) and its two γ₁34.5 deletion derivatives, R3616 (γ₁34.5 del) and R3659 (γ₁34.5 del), were produced in HaCaT cells and purified in an Optiprep 10 to 40% density gradient. Fractions were collected in 0.5- to 1-ml batches, and virus titer (A) and the total protein concentration (B) in each fraction were determined. Five selected fractions of each gradient from top to bottom were further analyzed by Western blotting (C and D) for the presence of viral proteins (gD, sc-21719; VP5, HA018-100) (red) and exosome proteins (CD63, sc-15363; Alix, sc-99010) (green) within the gradients. For secondary detection, IRDye antibodies 680 and 800 (926-68070 and 926-32211, respectively) from Li-Cor Biotechnology were used. Blots were visualized and quantified with Odyssey FC Imager and Image Studio Lite v4.0 programs, and the fluorescence intensity of CD63 is shown in panel C. In all Western blot assays, 5 μg of protein was used per lane and an exposure time for each channel was 2 min. gD, 61 kDa; VP5, 155 kDa; Alix, 95 kDa; CD63, 30 to 60 kDa. Chameleon Duo from Li-Cor was used as a molecular mass standard (std).

FIG 3.

Us3 deletion does not affect the exosome secretion. The Us3 deletion mutant R7041 and its rescue virus, R7306, were produced in HaCaT cells and purified in an Optiprep 10 to 40% density gradient. Fractions were collected in 0.5- to 1-ml batches, and virus titer (A) and the total protein concentration (B) in each fraction were determined. Exosome marker proteins (CD63 and Alix; green) and viral proteins (gD and VP5; red) were analyzed by Western blotting (C and D) from five selected fractions. Five micrograms of protein was loaded on the gel. Blots were visualized and quantified with Odyssey FC Imager and Image Studio Lite v4.0 programs, and the fluorescence intensity of CD63 is shown in panel C. An exposure time for each channel was 2 min. gD, 61 kDa; VP5, 155 kDa; Alix, 95 kDa; CD63, 30 to 60 kDa. Chameleon Duo from Li-Cor was used as a molecular mass standard (std).

Because the lower yields of the γ₁34.5 mutant HSV could contribute to the lack of detectable exosome proteins, we determined semiquantitatively the ratio of CD63 exosome marker to the viral VP5 protein in fractions to reduce the effects of differing virus yields. The CD63/VP5 ratios, determined using Odyssey Image Studio software, of all γ₁34.5 mutants were low (range, 1.1 × 10⁻³ to 2.7 × 10⁻³), whereas those of the other viruses were 10- to 200-fold higher (wt or Us3 deletion viruses). Our data underline the need for characterization of all HSV stocks for purity. The cell-derived exosomes, if present in the viral stocks, could modify the effects of HSV vectors intended for gene therapy. We propose that the stocks of γ₁34.5 deletion HSV-1, purified using the current protocol, contain only small amounts of contaminating exosomes, if any, whereas the wt virus preparations are rich in exosomes.