Comparative functional analyses of conserved virus genes in the replication cycle of PrV and HSV-1
Deeper insight in fundamental mechanisms of herpesvirus replication requires unambiguous elucidation of the functions of those genes, which are conserved within the entire virus family. For unique properties of single subfamilies, like the neurotropism of most alphaherpesviruses, subgroup-specific genes might be also relevant. However, in the past distinct functions have been attributed to several structurally conserved genes in different herpesviruses. To verify, whether such contradictory results are evident, or only suggested by different experimental approaches, we have started to construct and to analyze isogenic deletion mutants of typical species of two alphaherpesvirus-genera, the human herpes simplex virus type 1 (HSV-1, genus Simplexvirus), and the animal pathogen pseudorabies virus (PrV, genus Varicellovirus) in parallel. These investigations are facilitated by the possibility to propagate both viruses in the same mammalian cell lines, and to study them in the same rodent animal models. Furthermore, the infectious full-length genomes of different PrV and HSV-1 strains have been cloned as bacterial artificial chromosomes (BAC) permitting fast and precise mutagenesis in E. coli. Comparative investigations of the generated PrV and HSV-1 mutants are performed in collaboration with the laboratories for herpesvirus host cell interactions (Dr. Klupp), for electron microscopy (Dr. Granzow), and for pathology and bacteriology (Prof Dr. Teifke). Besides replication in epithelial cell lines and in primary neuron cultures, virulence is studied in experimentally infected mice, and for evaluation conventional virological and histopathological methods are used, as well as electron microscopy and confocal fluorescence- and video microscopy.
Up to now, the functions of several conserved herpesvirus proteins were investigated in parallel, which are either involved in DNA packaging and capsid maturation in the cell nucleus (pUL25, pUL32), or in tegumentation and envelopment of virus particles in the cytoplasm (pUL37, pUL11, gM). All isogenic deletion mutants of PrV and HSV-1 exhibited similar phenotypes, and in several cases heterologous trans-complementation of PrV mutants by the homologous HSV-1 protein (pUL25, pUL32), or of HSV-1 mutants by the homologous PrV protein (pUL11) could be demonstrated (Fuchs et al., 2009; Kuhn et al., 2008; Leege et al., 2009). At present we focus on comparison of the still controversially discussed mechanisms of virion morphogenesis and intra-axonal transport of PrV und HSV-1 in neurons, during which the roles of the alphaherpesvirus-specific membrane proteins gE and pUS9 are of particular interest.