Fig 1.
Experimental Design of VOC detection by GC-MS.
HSV-1 infected cells were collected and stored in capped vials, with a SPME device inserted into the headspace of the vial to adsorb the VOC molecules emitted from the infected samples. The adsorbed VOCs were released under high temperature at 240°C at the GC inlet for subsequent GC-MS analyses.
Fig 2.
Overview of VOC characterization.
The chromatograms were recorded using a 2D GC-GC configuration (heartcut) to attain better VOC separation prior to identification using a MS. The heartcut time periods logged are A. 0–5 min; B. 5–10 min; C. 10–15 min; D. 15–20 min; and E. 20–25 min.
Fig 3.
Examination of GBL release profile.
A. The GBL peak was identified from the chromatograms taken using the 5–10 min heartcut. A close-up view shows the peak intensity was very weak among all samples and the controls except for the 24 hpi one, for which the GBL peak exhibited a very large increase. B. Measurement of integrated TIC during different infection times of 0, 1, 5, 10, 24 hpi, A significant increase was detected at 24 hpi. C. The enlarged GBL peak (labelled with retention time) where the measured mass spectra were compared with the GBL standard mass spectrum as shown in D. D. Comparison of mass spectra collected at different retention times with the GBL standard mass spectrum from the NIST library.
Fig 4.
Characterization of GBL induction during stress condition and differentiated LNCaP cells.
A. Relative viability of cells upon infections, scraping, and CHX treatment was evaluated in comparison to control. They were measured at 48 h post infection or post treatment. B. A GBL standard was run as control for GC/MS analysis. The spike was detected approximately at the retention time of 24.4 min. C. GBL was identified only in infected Vero cells but not from CHX, scraping, mock control, and culture media. The CHX concentration was 250 μg/ml. D. The GBL detection was not different in the infected LNCaP cells in comparison to the control. Infection of LNCaP was performed with the same moi of Vero infection.
Fig 5.
GBL altered RMP of differentiated LNCaP.
A. Stimulation of differentiated LNCaP cells with 40 mM GBL caused a significant depolarization of the RMP. B. Treatment with 40 mM GBL for 2–3 min resulted in a significant depolarization of the RMP (p≤0.05 using single tail, unequal variance, unpaired student's t-test). In this and subsequent bar figures, N represents the number of recorded cells in each test group. C. Stimulation of differentiated LNCaP cells with HSV-1 did not result in any change in RMP by Student’s t-test (NS denotes No significance, p>0.05). D. Stimulation of differentiated LNCaP cells with 30 mM extracellular KCl resulted in a significant depolarization of the RMP. Exposure to GBL did not generate any further change in the RMP following membrane depolarization with 30 mM KCl. E. Summary of the changes in the RMP evoked by 30 mM KCl and GBL treatment in differentiated LNCaP cells. Bars labeled with an asterisk (*) were shown to be statistically different compared to controls with a p≤0.05 using an ANOVA multiple comparison test. NS indicates Not Significant. F. Stimulation of Vero cells with GBL (40 mM) has no significant effect on the RMP. Statistical study was calculated by the Student’s t-tests.
Fig 6.
Concentration-dependent effects of GBL on viral gene expression.
A. Fully differentiated LNCaP cells infected by recombinant HSV-1 emitting GFP. The image was captured with an exposure time of 1/6 sec. BF: Bright field; FL: Fluorescent microscopy. B. The concentration-dependent effects of GBL on infection of differentiated LNCaP was determined at 48 hpi by q-RT-PCR measuring HSV-1 TK standardized by the housekeeping gene PPIA. A statistical study was presented using ANOVA with Dunnett’s post-hoc test. Bars labeled with an asterisk (*) were found to be statistically different in comparison to a vehicle with a p<0.05. NS: Not Significant. C. Vero cells were infected by recombinant GFP expressing HSV-1. The exposure time was 1/6 sec when the image was captured. BF: Bright field; FL: Fluorescent microscopy. D. The concentration-dependent effects of GBL on infection of Vero was determined by q-RT-PCR using the same method described in Fig 6B. A statistical study was determined by ANOVA. NS: Not Significant with p>0.05.
Fig 7.
Concentration-dependent effects of GBL on viral replication.
A. The replication was determined by plaque assays (PFU/ml) using media to measure the release of infectious virus particles. Different concentrations of GBL ranging from 5-30mM were used. A statistical examination was done by ANOVA with Dunnett’s post-hoc test. Bars marked with an asterisk (*) were shown to be statistically significant in comparison to no GBL control with a p<0.05. NS: not significant with p>0.05. B. Total DNA was isolated followed by a quantitative PCR measuring virus genome via the method of Normalized Relative Quantity (NRQ). A statistical investigation was executed using Student's t-tests. Bars marked with an asterisk (*) were shown to be statistically significant in comparison to the infection control with a p<0.05.
Fig 8.
Proposed model of latency establishment by GBL.
The working hypothesis is that GBL production is induced upon HSV-1 primary infection and this modulated the membrane potential of sensory neurons adjacent to the infected epithelial cells. This event can potentially alter the signaling pathway within the neurons, inhibiting viral gene expression and replication, thereby promoting latency establishment.