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

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.

The expression and function of HSV ICP47 and its promoter in mice.

IMPORTANCE: Immune evasion and latency are key mechanisms that underlie the success of herpesviruses. In each case, interactions between viral and host proteins are required and due to co-evolution, not all mechanisms are preserved across host species, even if infection is possible. This is highlighted by the herpes simplex virus (HSV) protein immediate early-infected cell protein (ICP)47, which inhibits the detection of infected cells by killer T cells and acts with high efficiency in humans, but poorly, if at all in mouse cells. Here, we show that ICP47 retains modest but detectable function in mouse cells, but in an in vivo model we found no role during acute infection or latency. We also explored the activity of the ICP47 promoter, finding that it could be active during latency, but this was dependent on genome location. These results are important to interpret HSV pathogenesis work done in mice.

CRISPR/Cas9-Based Genome Editing of HSV.

The CRISPR/Cas9 gene editing system is a robust and versatile technology that has revolutionized our capacity for genome engineering and is applicable in a wide range of organisms, including large dsDNA viruses. Here we provide an efficient methodology that can be used both for marker-based and for marker-free CRISPR/Cas9-mediated editing of the HSV-1 genome. In our method, Cas9, guide RNAs and a homology-directed repair template are provided to cells by cotransection of plasmids, followed by introduction of the HSV genome by infection. This method offers a great deal of flexibility, facilitating editing of the HSV genome that spans the range from individual nucleotide changes to large deletions and insertions.

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.

A mutational analysis of the cytosolic domain of the tomato Cf-9 disease-resistance protein shows that membrane-proximal residues are important for Avr9-dependent necrosis.

The tomato Cf-9 gene encodes a membrane-anchored glycoprotein that imparts race-specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N-terminal half of the extracellular leucine-rich repeat (eLRR) domain of the Cf-9 protein determines its specificity for Avr9, the C-terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf-9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9-dependent necrosis triggered by Cf-9. Our results indicate that the membrane-proximal region of the cytosolic domain of Cf-9 plays an important role in Cf-9-mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf-9 function. An alanine mutation of T835 had no effect on Cf-9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf-9 function. We therefore postulate that phosphorylation/de-phosphorylation of T835 could act as a molecular switch to determine whether Cf-9 is in a primed or inactive state. Yeast two-hybrid analysis was used to show that the cytosolic domain of Cf-9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline-induced kink in the membrane-proximal region of the cytosolic domain of Cf-9 may be important for interaction with VAP27, which may, in turn, be important for Cf-9 function.