Mechanism of LAT inhibition of apoptosis during latent HSV-1 infection

Abstract

Purpose : Herpes Simplex Virus type 1 (HSV-1) latency associated transcript (LAT) has been shown to inhibit apoptosis via inhibiting activation of pro-apoptotic caspases. However, the mechanism of LAT control of apoptosis is unclear, because LAT is not known to encode a functional protein, and the LAT transcript is found largely in the nucleus. We hypothesized that LAT inhibits apoptosis via a transcriptional mechanism and we sought to establish the molecular mechanism of LAT control of apoptosis at a transcriptional level during latent HSV-1 ocular infection in mice.

Methods : C57BL/6 mice were infected ocularly with 2x10⁵ PFU/eye of wild type (WT) HSV-1, LAT-deficient HSV-1 (dLAT2903) or HSV-1 with LAT replaced by baculovirus antiapoptotic gene (dLATcpIAP). Mice were euthanized 30 days post infection, and expression of various genes involved in apoptosis in trigeminal ganglia (TG) were determined by qRT-PCR.

Results : Expression of Bcl2, caspase 3, 8 and 9 were elevated in WT but not dLAT2903 infected TG (p<0.01). No difference in expression levels of TLR2, 4, 7, or NF-κB were seen in mice infected with WT or dLAT2903 virus (p>0.05). However, a significant decrease in JAK-1 and JAK-2 expression was seen in mice infected with WT HSV-1 but not dLAT2903 (p<0.0001). ISG15, ISG56, IRF-1, Mx-1, CIITA, components of the Type I IFN pathway, were downregulated in a LAT dependent manner (p<0.0001). Replacement of LAT with the cpIAP gene (dLATcpIAP) resulted in reduced expression of Caspase-8, Bcl2, BID, and FADD when compared to WT virus despite the two viruses having similar level of latency (p<0.0001). Markers of cell exhaustion were elevated in mice infected with WT but not with either the LAT2903 or dLATcpIAP virus (p<0.0001).

Conclusions : Our results suggest that: 1) LAT likely inhibits apoptosis via upregulation of several components of the Type I IFN pathway; 2) LAT does not inhibit apoptosis via the caspase cascade at a transcriptional level or via downregulating TLRs; 3) The mechanism of LAT antiapoptotic effect is distinct from the baculovirus cpIAP because replacement of LAT with cpIAP resulted in a different gene expression pattern than either WT or dLAT2903; and 4) Replacement of LAT with cpIAP does not cause upregulation of CD8, or markers of T cell exhaustion despite having similar levels of latency, further supporting that LAT and cpIAP function via distinct mechanisms.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.