Binding of herpesvirus entry mediator (HVEM) and HSV-1 gD affect reactivation but not latency levels

Abstract

Previously we reported that the HSV-1 latency associated transcript (LAT) specifically upregulates the cellular herpesvirus entry mediator (HVEM) but no other known HSV-1 receptors. HSV-1 glycoprotein D (gD) binds to HVEM but the effect of this interaction on latency-reactivation is not known. We found that the levels of latent viral genomes were not affected by the absence of gD binding to HVEM. However, reactivation of latent virus in trigeminal ganglia explant cultures was blocked in the absence of gD binding to HVEM. Neither differential HSV-1 replication and spread in the eye nor levels of latency influenced reactivation. Despite similar levels of latency, reactivation in the absence of gD binding to HVEM correlated with reduced T cell exhaustion. Our results indicate that HVEM-gD signaling plays a significant role in HSV-1 reactivation but not in ocular virus replication or levels of latency. The results presented here identify gD binding to HVEM as an important target that influences reactivation and survival of ganglion resident T cells but not levels of latency. This concept may also apply to other herpesviruses that engages HVEM.

Fig 1. Virus titers and latency-reactivation in KOS-Rid1 infected mice.

A) Virus titers in the eyes of infected mice. Twenty WT C57BL/6 mice were infected ocularly with KOS-Rid1 or KOS virus and the amount of infectious HSV-1 in tear films was determined daily by standard plaque assays as described in Materials and Methods. For each time point, the virus titer (Y-axis) represents the average of the titers from 20 eyes ± SEM; B and C) LAT and gB expression in TG of latently-infected mice. On day 28 PI, mice described above were euthanized and TG were harvested from latently-infected mice. QRT-PCR or qPCR was performed on RNA or DNA from individual mice TG. In each experiment, the estimated relative copy number of gB or LAT was calculated using standard curves generated from pGem-gB1 or pGEM-LAT5317, respectively. Briefly, DNA template was serially diluted 10-fold such that 5 μl contained from 10³ to 10¹¹ copies of gB, or LAT then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH expression was used to normalize the relative expression of gB DNA and LAT RNA in the TG. Each point represents the mean ± SEM from 20 TG for each virus; and D) Effect of gD binding on kinetics of induced reactivation in explanted TG from latently-infected mice. Twenty WT C57BL/6 mice were ocularly infected with each virus as above and 28 days PI individual TG were harvested from each group. Each TG was incubated in tissue culture media and a 10 μl aliquot was removed from each culture daily and used to infect RS cell monolayers for 10 days as described in Materials and Methods. RS cells were monitored daily for the appearance of CPE for up to 5 days to determine the time of first appearance of reactivated virus from each TG. Results are plotted as the number of TG that reactivated daily. Numbers indicate the average time that the TG from each group first showed CPE ± SEM. For each group 40 TG from 20 mice were used.

Fig 2. KOS-Rid1 infection affects T cell exhaustion in TG of latently-infected mice.

Total TG RNA from latently-infected mice used to measure LAT expression as described in Fig 1B, were used to measure CD8 and PD-L1 expression by qRT-PCR. CD8 and PD-L1 expression in naive mice were used as a control to estimate relative expression of each transcript in TG of latently-infected mice. GAPDH expression was used to normalize the relative expression of each transcript. Each point represents the mean ± SEM from 20 TG. Panels: A) CD8 transcript; and B) PD-L1 transcript.

Fig 3. Virus titers and reactivation in KOS-Rid2 infected mice.

A) Virus titers in the eyes of infected mice. WT C57BL/6 mice were infected ocularly with KOS-Rid2 or KOS control virus and the amount of infectious HSV-1 in tear films was determined daily by standard plaque assays as Fig 1. For each time point, the virus titer (Y-axis) represents the average of the titers from 20 eyes ± SEM; and B) Kinetics of induced reactivation in explanted TG from latently-infected mice. On day 28 PI, mice described above were euthanized and TG were harvested for explant reactivation as described in Fig 1D. Results are plotted as the number of TG that reactivated daily. Numbers indicate the average time that the TG from each group first showed CPE ± SEM. For each group 20 TG from ten mice were used.

Fig 4. Absence of HVEM affects kinetics of induced reactivation in explanted TG from latently-infected mice.

HVEM^-/- mice were ocularly infected with 2X10⁵ pfu/eye of KOS-Rid1, KOS-Rid2, or control KOS after corneal scarification. Twenty-eight days PI, individual TGs were isolated and incubated in tissue culture media as in Fig 1 above. Results are plotted as the number of TG that reactivated daily. Numbers indicate average time that TG from each group first showed CPE ± SEM. For each group 20 TG from ten mice were used.

Fig 5. Effect of ANG and ANGpath viruses on latency-reactivation in infected mice.

C57BL/6 mice were ocularly infected with HSV-1 strains ANG and ANGpath as well as KOS-Rid1, KOS-Rid2, KOS, and RE strains of HSV-1. TG from 20 mice per virus were isolated individually on day 28 PI. qPCR was performed to detect gB DNA (Panel A) and explant reactivation was used to detect reactivation (Panel B) as in Fig 1 above. TG from KOS-Rid2 infected mice was used for measuring gB DNA, while TG from KOS-Rid1 mice was used as a control for reactivation.

Fig 6. Absence of gD binding to HVEM affects levels of viral RNA but not DNA in infected RS cells.

RS cells were infected with 0.04 pfu/cell of KOS, KOS-Rid1, or KOS-Rid2 viruses. Infected cells were isolated 24 and 48 hr PI and total RNA and DNA from infected cells were isolated. qPCR were performed to detect LAT, gB, and gD DNA, while qRT-PCR were performed to detect LAT, gB, and gD RNA isolated from infected cells. In each experiment, the estimated relative copy number of LAT, gB or gD was calculated using standard curves generated from pGEM-LAT5317, pGem-gB1 or, pGem-gD1, respectively as described in Fig 1 (above). Each point represents the mean ± SEM from 3 experiments.