Visual Universitätsmedizin Mainz

Role of HCMV tegument proteins in viral replication and particle assembly

Impact of cellular location of the major tegument protein p65

The phosphoprotein 65 (pp65) is an abundant constituent of HCMV virions. It also interferes with the innate defense of the host cell, thus supporting viral replication. We could previously show that pp65 in non-essential for viral replication and that it shuttles between the cytoplasm and the nucleus (1-3). This project focusses on the question, how the subcellular distribution of pp65 affects its functions in the viral life cycle. A viral mutant that expressed a nuclear version of pp65 was markedly impaired in replication (4). Another mutant that expressed a version of pp65 with mutated nuclear localization signals (pp65nls) surprisingly showed higher levels of replication compared to wt-virus. Packaging of pp65nls, in addition, was impaired despite high expression levels of that protein in infected cells. Current work focusses on elucidating the molecular mechanisms underlying this phenotype. First results hint at an altered interaction of pp65nls with components of the innate defense mechanisms of the cell.

Interference of HCMV tegument proteins with the intrinsic and innate immunity

The tegument of HCMV virions contains proteins that interfere with both the intrinsic and the innate immunity. One protein with thus far unknown function is pUL25. The deletion of pUL25 in a viral mutant (Towne-ΔUL25) had no impact on the release of virions and subviral Dense Bodies or on virion morphogenesis. Proteomic analyses showed little alterations in the overall protein composition of extracellular particles. A surprising result, however, was the almost complete absence of pUL26 in virions and Dense Bodies of Towne-ΔUL25 and a reduction of the large isoform pUL26-p27 in mutant virus infected cells. The pUL26 was shown to inhibit protein conjugation with the interferon-stimulated-gene 15 protein (ISG15), thereby supporting HCMV replication. To test a functional relationship between pUL25 and pUL26, we addressed the steady-state levels of pUL26 and found them reduced in Towne-ΔUL25-infected cells. Co-immunoprecipitation experiments proved an interaction between pUL25 and pUL26. Surprisingly, the overall protein ISGylation was enhanced in Towne-ΔUL25-infected cells, thus mimicking the phenotype of a pUL26-deleted HCMV mutant. The functional relevance of this was confirmed by showing that the replication of Towne-ΔUL25 was more sensitive to IFN-β. The increase of protein ISGylation was also seen in cells infected with a mutant lacking the tegument protein pp65. Upon retesting, we found that pUL26 degradation was also increased when pp65 was unavailable. Our experiments show that both pUL25 and pp65 regulate pUL26-degradation and pUL26-dependent reduction of ISGylation and add pUL25 as another HCMV tegument protein that interferes with the intrinsic immunity of the host cell (5). Current work focusses on the question how the interaction of individual tegument proteins promotes the evasion mechanisms of HCMV from the innate response of the host cell.




Schmolke S, Drescher P, Jahn G, Plachter B. Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport. J Virol. 1995;69:1071-8.

Schmolke S, Kern HF, Drescher P, Jahn G, Plachter B. The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture. J Virol. 1995;69:5959-68.

Frankenberg N, Lischka P, Pepperl-Klindworth S, Stamminger T, Plachter B. Nucleocytoplasmic shuttling and CRM1-dependent MHC class I peptide presentation of human cytomegalovirus pp65. Med Microbiol Immunol. 2012;201(4):567-79.

Mersseman V, Besold K, Reddehase MJ, Wolfrum U, Strand D, Plachter B, et al. Exogenous introduction of an immunodominant peptide from the non-structural IE1 protein of human cytomegalovirus into the MHC class I presentation pathway by recombinant Dense Bodies. J Gen Virol. 2008;89(Pt 2):369-79.

Zimmermann C, Büscher N, Krauter S, Kramer N, Wolfrum U, Sehn E, et al. The Abundant Tegument Protein pUL25 of Human Cytomegalovirus Prevents Proteasomal Degradation of pUL26 and Supports Its Suppression of ISGylation. J Virol. 2018;92(24).