Currently, 88 different Human Adenovirus (HAdV) types are grouped into seven HAdV species A to G. Most types (57) belong to species HAdV-D. Recombination between capsid genes (hexon, penton and fiber) is the main factor contributing to the diversity in species HAdV-D. Noteworthy, species HAdV-C contains so far only five types, although species HAdV-C is highly prevalent and clinically significant in immunosuppressed patients. Therefore, the evolution of species HAdV-C was studied by generating 51 complete genome sequences from circulating strains. Clustering of the whole genome HAdV-C sequences confirmed classical typing results (fifteen HAdV-C1, thirty HAdV-C2, four HAdV-C5, two HAdV-C6). However, two HAdV-C2 strains had a novel penton base sequence and thus were re-labeled as the novel type HAdV-C89. Fiber and early gene region 3 (E3) sequences clustered always with the corresponding prototype sequence but clustering of the E4 region indicated recombination events in 26 out of the 51 sequenced specimens. Recombination of the E1 gene region was detected in 16 circulating strains. As early gene region sequences are not considered in the type definition of HAdVs, evolution of HAdV-C remains on the subtype level. Nonetheless, recombination of the E1 and E4 gene regions may influence the virulence of HAdV-C strains.

The products of the UL16 and UL21 genes represent tegument proteins which are conserved throughout the mammalian herpesviruses. To identify and functionally characterize the respective proteins in the alphaherpesvirus pseudorabies virus, monospecific antisera against bacterially expressed fusion proteins were generated. In immunoblots the UL16 antiserum detected a ca. 40-kDa protein in infected cells and purified virion preparations, whereas the anti-UL21 serum recognized a protein of approximately 60 kDa. Interestingly, in immunoprecipitations using either antiserum, both proteins were coprecipitated, demonstrating the formation of a physical complex. To investigate protein function, viruses lacking either UL16, UL21, or both were constructed. Mutant viruses could be propagated on noncomplementing cells, indicating that these proteins, either alone or in combination, are not required for viral replication in cell culture. However, plaque sizes and viral titers were reduced. Electron microscopy showed only slight alterations in cytoplasmic virion morphogenesis, whereas intranuclear maturation stages were not affected. Similar results were obtained with a triple mutant simultaneously lacking the three conserved tegument proteins UL11, UL16, and UL21. In summary, our results uncover a novel interaction between conserved herpesvirus tegument proteins that increases the complexity of the intricate network of protein-protein interactions involved in herpesvirus morphogenesis.

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Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein.Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C 26 S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C 26 S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.Herpesviruses possess a large double-stranded DNA genome, comprising more than 70 open reading frames (ORFs) encoding viral proteins, of which at least 30 are incorporated into the mature virus particle. The most complex virion component is the tegument, which, in the alphaherpesviruses, contains more than 15 different viral proteins. Operationally, the tegument has been divided into an "inner," capsid-associated part and an "outer," envelope-juxtaposed portion (reviewed in references 42 and 43). The innermost part contains the largest herpesvirus tegument protein, which, in the prototypic alphaherpesvirus herpes simplex virus type 1 (HSV-1), is the product of the UL36 gene. Homologs of this protein, for clarity henceforth named pUL36, are present in all members of the Herpesviridae which have been analyzed in this respect to date.Previous studies revealed two conserved domains within the N termini of pUL36 homologs. One is required for interaction with pUL37, another conserved tegument protein (4,22,33,44,50), whereas the second specifies a ubiquitin-specific cysteine protease activity (20,24,27,47,48,51). This activity is not required for herpesvirus replication, as has been shown for human cytomegalovirus (HCMV) (51) and Marek's disease virus (MDV) (24). Nevertheless, abrogation of deubiquitinating activity resulted in impairment of replication in vitro (51) and in vivo (24).In pseudorabies virus (PrV), pUL36 is the only truly essential tegument protein (14), and its deletion completely blocks viral replication. Interestingly, recent data demonstrated that about one-third of PrV pUL36 located in the C-terminal half can be deleted without drastic impairment of viral replication. In contrast, the extreme C terminus of pUL36 is essential (6, 35), probably due to its association with the capsid-associated pUL25 (10). Moreover, deletion of an N-terminal domain of about 200 amino acids comprising the deubi...

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