Ultrastructural investigation of the C-terminal region of human cytomegalovirus tegument protein UL71 and its role in secondary envelopment

Abstract text (incl. figure legends and references)

Human cytomegalovirus (HCMV) is an important human pathogen which can cause serious diseases in immunocompromised patients and complications during pregnancy (Griffith et al., 2015). However, the molecular mechanisms underlying HCMV virion morphogenesis are still not well understood. In this work, an important step of HCMV morphogenesis en route to infectious virions, referred to as secondary envelopment, was investigated by electron microscopy (EM). Understanding the underlying mechanism could lead to the identification of new targets for antiviral therapy. pUL71 is a conserved herpesvirus tegument protein with 361 amino acids and plays an important role in secondary envelopment (Schauflinger et al., 2011). Several functional motifs, mainly localized in the N-terminus of pUL71, have been characterized in detail (Read et al., 2019; Fischer 2012; Metzner, 2022), but the role of the C-terminus of pUL71 is not well studied.

The role of the C-terminus of pUL71 was investigated by generating and characterizing a mutant virus (UL71insHA-299) expressing a truncated version lacking the last 60 amino acids and fused to an HA epitope. A first phenotypic characterization of UL71insHA-299 after infection of human fibroblasts was performed by immunofluorescence microscopy. In contrast to wild-type virus infected cells, virus particles accumulated at the periphery of the cytoplasmic viral assembly complex at large circular structures in UL71insHA-299 infected cells. For direct analysis of secondary envelopment and these capsid accumulations and circular structures, human fibroblasts were again infected and high-pressure frozen at five days post infection, freeze substituted and embedded in epoxy resin. Thereafter the cells were ultrathin sectioned, analyzed by transmission electron microscopy (TEM) and compared with wild-type infected cells.

The ultrastructural phenotype of the UL71insHA-299 mutant was very similar to that of a virus that is unable to express pUL71 (Schauflinger et al., 2011). Thus, our data show that the C-terminus of HCMV pUL71 contains functions that are necessary for sufficient virion morphogenesis and secondary envelopment.

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Schauflinger M, Fischer D, Schreiber A, Chevillotte M, Walther P, Mertens T, von Einem J. The tegument protein UL71 of human cytomegalovirus is involved in late envelopment and affects multivesicular bodies. J Virol. 2011 Apr;85(8):3821-32. doi: 10.1128/JVI.01540-10. Epub 2011 Feb 2. PMID: 21289123; PMCID: PMC3126135.

Fischer D, 2012. Dissecting Functional Motifs of the Human Cytomegalovirus Tegument Protein pUL71. Doctoral thesis, Ulm University.

Read C, Schauflinger M, Nikolaenko D, Walther P, von Einem J. Regulation of Human Cytomegalovirus Secondary Envelopment by a C-Terminal Tetralysine Motif in pUL71. J Virol. 2019 Jun 14;93(13):e02244-18. doi: 10.1128/JVI.02244-18. PMID: 30996102; PMCID: PMC6580969.

Annika Metzner. Role of the non-conserved C-terminus of pUL71 for human cytomegalovirus infection. 2022, Bachelor thesis, Ulm University, unpublished.