Human cytomegalovirus (HCMV) genome encapsidation requires several essential viral proteins, among them pUL56, pUL89, and the recently described pUL51, which constitute the viral terminase. To gain insight into terminase complex assembly, we investigated interactions between the individual subunits. For analysis in the viral context, HCMV bacterial artificial chromosomes carrying deletions in the open reading frames encoding the terminase proteins were used. These experiments were complemented by transient-transfection assays with plasmids expressing the terminase components. We found that if one terminase protein was missing, the levels of the other terminase proteins were markedly diminished, which could be overcome by proteasome inhibition or providing the missing subunit in trans. These data imply that sequestration of the individual subunits within the terminase complex protects them from proteasomal turnover. The finding that efficient interactions among the terminase proteins occurred only when all three were present together is reminiscent of a folding-upon-binding principle leading to cooperative stability. Furthermore, whereas pUL56 was translocated into the nucleus on its own, correct nuclear localization of pUL51 and pUL89 again required all three terminase constituents. Altogether, these features point to a model of the HCMV terminase as a multiprotein complex in which the three players regulate each other concerning stability, subcellular localization, and assembly into the functional tripartite holoenzyme.IMPORTANCE HCMV is a major risk factor in immunocompromised individuals, and congenital CMV infection is the leading viral cause for long-term sequelae, including deafness and mental retardation. The current treatment of CMV disease is based on drugs sharing the same mechanism, namely, inhibiting viral DNA replication, and often results in adverse side effects and the appearance of resistant virus strains. Recently, the HCMV terminase has emerged as an auspicious target for novel antiviral drugs. A new drug candidate inhibiting the HCMV terminase, Letermovir, displayed excellent potency in clinical trials; however, its precise mode of action is not understood yet. Here, we describe the mutual dependence of the HCMV terminase constituents for their assembly into a functional terminase complex. Besides providing new basic insights into terminase formation, these results will be valuable when studying the mechanism of action for drugs targeting the HCMV terminase and developing additional substances interfering with viral genome encapsidation.

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Type 41 of human adenovirus species F (HAdV-F41) is a frequent aetiology of gastroenteritis in children, and nosocomial as well as kindergarten outbreaks have been frequently described. In contrast to other HAdV types, HAdV-F41 was not associated with life-threatening disseminated disease in allogeneic haematopoietic stem cell transplant (HSCT) recipients or any severe organ infections so far. Due to the limited clinical significance, the evolution of HAdV-F41 has not been studied in detail. Recently, HAdVF41 has been associated with severe hepatitis in young children, and interest in HAdV-F41 has skyrocketed, although the aetiology of the hepatitis has not been resolved. Complete genomic HAdV-F41 sequences from 32 diagnostic specimens of the past 11 years (20112022) were generated, all originating from gastroenteritis patients. Additionally, 33 complete HAdVF41 genomes from GenBank were added to our phylogenetic analysis. Phylogenetic analysis of 65 genomes indicated that HAdV-F41 evolved with three lineages cocirculating. Lineage 1 included the prototype 'Tak' from 1973 and six isolates from 2007 to 2017 with an average nucleotide identity of 99.3 %. Lineage 2 included 53 isolates from 2000 to 2022, had an average nucleotide identity of 99.8 %, and split into two sublineages. Lineage 3, probably described for the first time in 2009, had a 45nt deletion in the long fiber gene and had evolved significantly in the short fiber and E3 region. Moreover, a recent lineage 3 isolate from 2022 had a recombinant phylogeny of the short fiber gene. Fibers interact with cellular receptors and determine cellular tropism, whereas E3 gene products interfere with the immune recognition of HAdV infected cells. This in-depth study on the phylogeny of HAdV-F41 discovered significant evolution of recently described lineage 3 of HAdV-F41, possibly resulting in altered cellular tropism, virulence and pathophysiology.

Healthy individuals infected with human cytomegalovirus (HCMV) are largely asymptomatic, but HCMV infection can result in high morbidity and death in immunosuppressed patients (12,38,48). HCMV establishes lifelong persistence, which is thought to require manipulation of the host's immune system by the coordinated function of many viral immunomodulatory genes (34).As adaptive immune responses develop during infection, interactions occur between mature antigen-presenting cells (APC) and their cognate T cells. The quality and quantity of the resulting T cell responses are regulated in large part by the affinity of peptide/major histocompatibility complex-T cell receptor (MHC-TCR) interactions and commensurate cosignals mediated by members of the tumor necrosis factor receptor (TNFR) and CD28 families. CD40 is a costimulatory TNFR superfamily member that is upregulated upon APC maturation and plays a role in promoting both CD4 and CD8 T cell responses (reviewed in references 14 and 29). The infection of dendritic cells with mouse CMV (MCMV) or HCMV restricts antigen presentation, inhibits costimulatory molecule expression, and enhances the expression of negative cosignaling ligands, resulting in reduced priming, expansion, and survival of antigen-specific T cells that encounter these APC (3,7,8,16,17,19,22,35). Consequently, it is quite possible that these mechanisms contribute to the immune suppression that is observed for cases of clinical HCMV disease (10,15,44).MCMV infection results in reduced cell surface expression of CD40 in dendritic cells (DC) (3,8). Two cell lines were utilized to investigate this effect; the RAW264.7 monocytic macrophage cell line and the DC2.4 bone marrow-derived DC line (kindly provided by K. Rock) (41). Throughout the study, fluorescence-activated cell sorter (FACS) and immunofluorescence experiments with MCMV that expresses green fluorescent protein (MCMV-GFP) (33) were performed using multiplicities of infection (MOI) of 0.4 and centrifugal enhancement, resulting in approximately 30% infected RAW264.7 and 20% infected DC2.4 cells, respectively. Immunoblot and RNA analysis of infected cells used MOI of 1.2 and centrifugal enhancement. Microscopy of infected and unin-