Role of herpes simplex virus 1 protein ICP47 in antigen presentation and pathogenesis

The herpes simplex virus (HSV) immunomodulatory protein, ICP47, conceals infected cells from CD8+ T cells by inhibiting the presentation of peptides on MHC class I. The mechanism by which ICP47 exerts this function is by binding to the transporter associated with antigen processing (TAP) protein, blocking peptide transport and loading onto MHC I molecules in the ER. The earliest studies of ICP47 supported by biochemical and in vitro observations noted marked species specificity with human but not mouse TAP being inhibited by this protein. However, later work demonstrated that ICP47 can contribute to HSV neurovirulence in mice. The discordance between biochemical and in vivo data leaves our understanding of ICP47 and its role in evading CD8+ T cells incomplete. Data from our laboratory suggested that ICP47 is likely to be expressed during the establishment and maintenance of HSV-1 latency, however, its exact function during these stages of infection is unknown. Therefore, in this study, we sought to re-visit the discrepancies discussed above and investigate the role of ICP47 during HSV-1 infection. We utilised different strains of HSV and mice, as well as an alternate infection model and unique methods to quantify the effect of ICP47 on levels of antigen presentation. In our mouse model, where HSV is confined to the peripheral nervous system, deletion of ICP47 from HSV-1 KOS did not alter lesion development, virus load, spread or reactivation. Likewise, latency was unaffected by ICP47 deficiency as determined using a sensitive Cre-marking mouse model. Further observations from the Cre-marking mouse model revealed that unlike the ICP47 promoter inserted in an ectopic locus, native promoters did not induce additional neuronal marking by Cre beyond lytic infection. We evaluated the reasons behind the difference in marking using newly generated recombinant viruses. Subsequent flank infection of ROSA26R mice with these viruses showed that the local genomic context is also important for regulation of gene expression. By contrast to our in vivo pathogenesis data, we were able to show that ICP47 does inhibit antigen presentation significantly on HSV-infected mouse cells using in vitro antigen presentation assays. However, in mouse cells, antigen presentation was ablated by 44%, compared to an 85% reduction in human cells. As CD8+ T cells have been shown to recognize very few peptide-MHC I complexes on the surface of target cells, it is important to consider the efficiency at which ICP47 inhibits human and mouse TAP. Therefore, we used mass spectrometry to identify and quantify MHC I bound peptides derived from HSV-1 during viral infection. We found that more peptide sequences were presented on mouse cells infected with ICP47 null virus compared to those infected with wild-type virus. We quantified the presentation of 14 of these peptides and the contribution of ICP47 to this process in human and mouse cells. We found that ICP47 almost entirely blocks human TAP-mediated peptide presentation, though the degree of inhibition was somewhat peptide-specific. Conversely, the effect of ICP47 on mouse TAP was far less profound, resulting in only up to five-fold reduction in MHC-peptide abundance. In conclusion, this study shows that despite significant inhibition of antigen presentation in mouse cells, ICP47 may not be an effective immune modulator in mice and suggests a need for re-evaluation of suitable mouse models

Multi-targeted loss of the antigen presentation molecule MR1 during HSV-1 and HSV-2 infection

Summary: The major histocompatibility complex (MHC), Class-I-related (MR1) molecule presents microbiome-synthesized metabolites to Mucosal-associated invariant T (MAIT) cells, present at sites of herpes simplex virus (HSV) infection. During HSV type 1 (HSV-1) infection there is a profound and rapid loss of MR1, in part due to expression of unique short 3 protein. Here we show that virion host shutoff RNase protein downregulates MR1 protein, through loss of MR1 transcripts. Furthermore, a third viral protein, infected cell protein 22, also downregulates MR1, but not classical MHC-I molecules. This occurs early in the MR1 trafficking pathway through proteasomal degradation. Finally, HSV-2 infection results in the loss of MR1 transcripts, and intracellular and surface MR1 protein, comparable to that seen during HSV-1 infection. Thus HSV coordinates a multifaceted attack on the MR1 antigen presentation pathway, potentially protecting infected cells from MAIT cell T cell receptor-mediated detection at sites of primary infection and reactivation

Increasing antigen presentation on HSV-1-infected cells increases lesion size but does not alter neural infection or latency

CD8+ T cells have a role in the control of acute herpes simplex virus (HSV) infection and may also be important in the maintenance of latency. In this study we have explored the consequences of boosting the efficacy of CD8+ T cells against HSV by increasing the amount of an MHC I-presented epitope on the surface of infected cells. To do this we used HSVs engineered to express an extra copy of the immunodominant CD8+ T cell epitope in C57Bl/6 mice, namely gB498 (SSIEFARL). Despite greater presentation of gB498 on infected cells, CD8+ T cell responses to these viruses in mice were similar to those elicited by a control virus. Further, the expression of extra gB498 did not significantly alter the extent or stability of latency in our mouse model, and virus loads in skin and sensory ganglia of infected mice were not affected. Surprisingly, mice infected with these viruses developed significantly larger skin lesions than those infected with control viruses and notably, this phenotype was dependent on MHC haplotype. Therefore increasing the visibility of HSV-infected cells to CD8+ T cell attack did not impact neural infection or latency, but rather enhanced pathology in the skin