Indirect visualization of endogenous nuclear actin by correlative light and electron microscopy (CLEM) using an actin-directed chromobody.

Actin fulfills important cytoplasmic but also nuclear functions in eukaryotic cells. In the nucleus, actin modulates gene expression and chromatin remodeling. Monomeric (G-actin) and polymerized actin (F-actin) have been analyzed by fluorescence microscopy in the nucleus; however, the resolution at the ultrastructural level has not been investigated in great detail. We provide a first documentation of nuclear actin in mouse fibroblasts by electron microscopy (EM). For this, we employed correlative light and electron microscopy on the same section using actin-directed nanobodies recognizing endogenous monomeric and polymeric actin proteins (so-called nuclear Actin-chromobody-GFP; nAC-GFP). Indeed, using this strategy, we could identify actin proteins present in the nucleus. Here, immunogold-labeled actin proteins were spread throughout the entire nucleoplasm. Of note, nuclear actin was complementarily localized to DAPI-positive areas, the latter marking preferentially transcriptionally inactive heterochromatin. Since actin aggregates in rod structures upon cell stress including neurodegeneration, we analyzed nuclear actin at the ultrastructural level after DMSO or UV-mediated cell damage. In those cells the ratio between cytoplasmic and nuclear gold-labeled actin proteins was altered compared to untreated control cells. In summary, this EM analysis (i) confirmed the presence of endogenous nuclear actin at ultrastructural resolution, (ii) revealed the actin abundance in less chromatin-dense regions potentially reflecting more transcriptionally active euchromatin rather than transcriptionally inactive heterochromatin and (iii) showed an altered abundance of actin-associated gold particles upon cell stress.

Investigating HCMV entry into host cells by STEM tomography.

Human cytomegalovirus (HCMV) entry into susceptible cells is a fast intricate process that is not fully understood. Although, previous studies explored different aspects of this process by means of biochemical and inhibitors assays, a clear morphological characterization of its steps at the ultrastructural level is still lacking. We attempted to characterize those intermediates involved during HCMV entry by developing a methodological approach that resulted in optimal ultrastructure preservation and allowed for 3D imaging. It involves rapid freezing and cryosubstitution which ensure a clear visibility of membranous leaflets as well as retained membranous continuity. Likewise, it delivered a reproducible optimization of the growth and infection conditions that are pivotal towards maintaining biologically active enriched input virus particles. Data acquisition was achieved through STEM tomography in a 3D context. Indeed, several intermediates that characterize HCMV entry-related events were observed both extra- and intracellularly. Some of the cell-membrane associated viral particles that we referred to as "Pinocchio particles" were morphologically altered in comparison to the cell-free virions. We were also able to characterize intracellular fusion intermediates taking place between the viral envelope and the vesicular membranes. Furthermore, inhibiting actin polymerization by Latrunculin-A enabled us to spot fusion-like intermediates of the viral envelope with the host cell plasma membrane that we did not observe in the untreated infected cells. Our data also suggests that Dyngo-4a; a dynamin-2 inhibitor, does not interfere with the internalization of the HCMV into the host cells as previously deduced.

Importance of Highly Conserved Peptide Sites of Human Cytomegalovirus gO for Formation of the gH/gL/gO Complex.

The glycoprotein O (gO) is betaherpesvirus specific. Together with the viral glycoproteins H and L, gO forms a covalent trimeric complex that is part of the viral envelope. This trimer is crucial for cell-free infectivity of human cytomegalovirus (HCMV) but dispensable for cell-associated spread. We hypothesized that the amino acids that are conserved among gOs of different cytomegaloviruses are important for the formation of the trimeric complex and hence for efficient virus spread. In a mutational approach, nine peptide sites, containing all 13 highly conserved amino acids, were analyzed in the context of HCMV strain TB40-BAC4 with regard to infection efficiency and formation of the gH/gL/gO complex. Mutation of amino acids (aa) 181 to 186 or aa 193 to 198 resulted in the loss of the trimer and a complete small-plaque phenotype, whereas mutation of aa 108 or aa 249 to 254 caused an intermediate phenotype. While individual mutations of the five conserved cysteines had little impact, their relevance was revealed in a combined mutation, which abrogated both complex formation and cell-free infectivity. C343 was unique, as it was sufficient and necessary for covalent binding of gO to gH/gL. Remarkably, however, C218 together with C167 rescued infectivity in the absence of detectable covalent complex formation. We conclude that all highly conserved amino acids contribute to the function of gO to some extent but that aa 181 to 198 and cysteines 343, 218, and 167 are particularly relevant. Surprisingly, covalent binding of gO to gH/gL is required neither for its incorporation into virions nor for proper function in cell-free infection. IMPORTANCE: Like all herpesviruses, the widespread human pathogen HCMV depends on glycoproteins gB, gH, and gL for entry into target cells. Additionally, gH and gL have to bind gO in a trimeric complex for efficient cell-free infection. Homologs of gO are shared by all cytomegaloviruses, with 13 amino acids being highly conserved. In a mutational approach we analyzed these amino acids to elucidate their role in the function of gO. All conserved amino acids contributed either to formation of the trimeric complex or to cell-free infection. Notably, these two phenotypes were not inevitably linked as the mutation of a charged cluster in the center of gO abrogated cell-free infection while trimeric complexes were still being formed. Cysteine 343 was essential for covalent binding of gO to gH/gL; however, noncovalent complex formation in the absence of cysteine 343 also allowed for cell-free infectivity.