Understanding the function and structure of herpes simplex virus type 1 pUL55, a novel immune evasion protein
Herpes simplex virus-1 (HSV-1) infects approximately 67% of the human population, causing disease ranging from asymptomatic to severe. The virus affects individuals around the world, and results in lifelong infection. This work focusses on pUL55, a HSV-1 tegument protein, which we hypothesised to be an innate immune modulator with similar functions to ICP0. ICP0 exhibits E3 ubiquitin ligase activity and is known to stimulate proteasomal degradation of several host proteins in HSV-1 infected cells. Multiple studies to date have used the *dl*1403 mutant strain of HSV-1, engineered to lack ICP0, which has demonstrated the importance of ICP0 activity in the establishment of viral infection in multiple model systems. However, a recently generated mutant strain of HSV-1 lacking ICP0 did not recapitulate many of the published characteristics of the well-established *dl*1403 strain. Comparative proteomic analysis had uncovered that the *dl*1403 mutant lacked expression of pUL55 in addition to ICP0. Accordingly, it was hypothesised that the additional loss of pUL55 may be required for some of the observed phenotypes in cells infected with the *dl*1403 mutant. The aim of this research was to identify whether pUL55 has a similar function to ICP0 and investigate the mechanism by which pUL55 can modulate the host proteome. Recombinant viruses lacking pUL55 or lacking both pUL55 and ICP0 were generated and examined for their ability to form plaques on monolayers of cells known to restrict *dl*1403 as well as their ability to degrade specific host proteins that are known to be ICP0 targets. These data demonstrated that viruses lacking either pUL55 or ICP0 individually are able to form plaques effectively on restrictive cells and degrade both PML and IFI16, known targets of ICP0-mediated degradation. Conversely, the newly generated virus lacking both pUL55 and ICP0 was significantly inhibited in its ability to form plaques on restrictive cell monolayers and was unable to degrade PML or IFI16 in a similar manner to *dl*1403. These data confirmed the hypothesis that pUL55 mediates similar host-modulatory effects as ICP0 in infected cells and loss of both is required for replication deficits previously ascribed just to ICP0 activity. Multiple immunoprecipitation-based experiments were conducted to identify and validate pUL55 interaction partners. These data uncovered two viral tegument proteins (pUL13 and pUS10) as well as a cellular ubiquitin ligase (HUWE1) as candidate binding partners. The viral proteins pUL55, pUL13, and pUS10 were found to form a tripartite complex with both pUL55 and pUS10 interacting with pUL13. Expression of both pUL13 and pUS10 was shown to be necessary for pUL55-dependent modulation of host cell functions, suggesting activity of all three viral proteins is required for a functional complex. Further data suggested the kinase activity of pUL13 was required for pUL55:pUL13:pUS10 complex activity. In addition, data suggested that HUWE1 activity is also necessary for pUL55:pUL13:pUS10 function. Recombinantly expressed pUL55 was purified at high yield and crystalised for structural studies. Using both crystal diffraction data and structural prediction data from AlphaFold2 allowed the structure of pUL55 to be solved using molecular replacement. The interaction between pUL55:pUL13 was modelled using AlphaFold2 Multimer. Point mutations predicted to disrupt the interaction from the model were incorporated expression plasmids and recombinant viruses. All tested mutations inhibited interaction between pUL55 and pUL13 and function of the pUL55:pUL13:pUS10 complex. These data have demonstrated HSV-1 expressed a previously unknown complex of three tegument proteins that modulates the host cell in an analogous way to ICP0, uncovering a novel mechanism of virus-host interactions.
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