# HSV-1 UL56 protein recruits cellular NEDD4-family ubiquitin ligases to suppress CD1d expression and NKT cell function

## TL;DR

This study reveals how the HSV-1 UL56 protein suppresses NKT cell function by recruiting cellular NEDD4-family ubiquitin ligases to reduce CD1d expression.

## Contribution

The novel finding is that UL56 suppresses CD1d via collaboration with NEDD4L, not through direct ubiquitination.

## Key findings

- UL56 suppresses CD1d expression via proteasome- and lysosome-dependent degradation.
- NEDD4L, a member of the NEDD4 family, is recruited by UL56 to downregulate CD1d.
- UL56-deficient virus is less virulent in NKT-sufficient mice but more virulent in NKT-deficient mice.

## Abstract

Herpesviruses, including α-herpesvirus and herpes simplex virus (HSV-1), are masters of immune evasion. Previously we demonstrated that CD1d-restricted NKT cells are required for optimal anti-HSV-1 immune responses and HSV-1 efficiently downregulates CD1d to suppress NKT cell function. To delineate how the virus evades NKT cell function and establishes infection in vivo, we screened an HSV-1 expression library to identify the viral gene(s) downregulating CD1d and discovered that a leaky late gene, UL56, most efficiently suppresses CD1d expression by degrading the protein, apparently via both proteasome- and lysosome-dependent pathways. To investigate the molecular mechanism of UL56 suppression of CD1d expression, we purified and identified UL56-associated proteins by mass spectrometry. The most abundant associated proteins were members of NEDD4 E3 ubiquitin ligase family. Interestingly overexpression of one member, NEDD4L is sufficient to downregulate CD1d expression. However, different from the K5 protein from Kaposi sarcoma’s herpesvirus (KSHV), UL56 and NEDD4L did not directly ubiquitinate CD1d. CD1d protein lacking the key lysine residue in its cytoplasmic tail is similarly downregulated by UL56 and NEDD4L protein. Co-expression of UL56 and NEDD4L synergistically reduced the CD1d expression, suggesting that UL56 collaborates with NEDD4L to downregulate CD1d. During in vivo infection, UL56-deficient mutant virus showed significantly weaker virulence in NKT-sufficient mice but demonstrated higher virulence in mutant mice lacking NKT cells. All our results supported that at least one of the pathogenesis functions of UL56 is its suppression of NKT cell function during infection.

In the large DNA genomes of herpeviruses, there are many genes encoding associate proteins. Most of these proteins are not essential for viral replication but play key roles in viral pathogenesis, in particular, modulating the host immune system to allow efficient viral replication in vivo and latency. The HSV-1 UL56 gene is one of such genes, and its exact pathogenic roles have remain elusive. After we demonstrated the essential roles of CD1d-restricted NKT cells in anti-HSV-1 immunity during HSV-1 ocular infection (P. Rao, X. Wen, J. H. Lo, S. Kim, X. Li, et al., J Virol 92:e01490-18, 2018, https://doi.org/10.1128/jvi.01490-18), we now screened the HSV-1 expression library and identified UL56 is a key factor downregulating CD1d and suppressing NKT cell function. In this manuscript, we are reporting our molecular mechanism study of how UL56 evades CD1d antigen presentation and NKT cell function.