BACKGROUND:Subspecies B1 human adenoviruses (HAdV-B1) are prevalent respiratory pathogens. Compared to their species C (HAdV-C) counterparts, relatively little work has been devoted to the characterization of their unique molecular biology. The early region 3 (E3) transcription unit is an interesting target for future efforts because of its species-specific diversity in genetic content among adenoviruses. This diversity is particularly significant for the subset of E3-encoded products that are membrane glycoproteins and may account for the distinct pathobiology of the different human adenovirus species. In order to understand the role of HAdV-B-specific genes in viral pathogenesis, we initiated the characterization of unique E3 genes. As a continuation of our efforts to define the function encoded in the highly polymorphic ORF E3-10.9K and testing the hypothesis that the E3-10.9K protein orthologs with a hydrophobic domain contribute to the efficient release of viral progeny, we generated HAdV-3 mutant viruses unable to express E3-10.9K ortholog E3-9K and examined their ability to grow, disseminate, and egress in cell culture.RESULTS:No differences were observed in the kinetics of infected cell death, and virus progeny release or in the plaque size and dissemination phenotypes between cells infected with HAdV-3 E3-9K mutants or the parental virus. The ectopic expression of E3-10.9K orthologs with a hydrophobic domain did not compromise cell viability.CONCLUSIONS:Our data show that despite the remarkable similarities with HAdV-C E3-11.6K, HAdV-B1 ORF E3-10.9K does not encode a product with a "death-like" biological activity.

The egress pathway of herpes simplex virus-1 (HSV-1) is a complicated process mediated by co-ordinated activity of several virus glycoproteins. The virions are first assembled and enveloped at trans-Golgi-network (TGN) or endosome membranes and then travel through a guided pathway that is directed towards the cell adherent points for secretion. Once secreted the vast majority of virions remain associated with the extracellular membrane of cells and very few free virions are released into the culture medium (< 1%). The mechanisms that mediate both the targeted secretion of newly assembled virions at cell contact points and post-secretion attachment of virions with the extracellular surface of cells are poorly understood, and were the topics of this research. In this thesis, an HSV-1 passage mutant of increased virion secretion phenotype had been studied. Genome sequencing of the mutant virus identified mutations in three viral envelope proteins. Study of recombinant viruses that were constructed based on those three mutations revealed that a single amino acid change in glycoprotein I (gI) of glycine to arginine at residue 39 is responsible for the increased release of virus. The result suggests the principal effect of this mutation is to modify the secretory pathway used by virions during their release from infected cells. Data also suggests a role of gC in the attachment of virions to the extracellular surface of cells after egress. In the context of HSV-1 envelopment and egress glycoprotein E (gE), which forms a heterodimeric complex with gI (gE/gI), is known to be important. The gE/gI complex has been shown to interact with many tegument proteins and have a redundant role in secondary envelopment. The gE/gI complex has been also proposed to colocalise with various cellular components and sort the nascent virions to cell contact points. However, there is little understanding of the cellular proteins that gE/gI interact with, or the mechanisms that mediate targeted secretion of virions. This research has identified a novel interactome of gE/gI by mass-spectrometric analysis utilising stable isotope labelling with amino acids in cell culture (SILAC) medium. Among the cellular interactome obtained, Nipsnap1 was validated by co-precipitation assays from both infected and transfected cells, and furthermore using cell free systems, suggesting gE and Nipsnap1 directly interact. Nipsnap1 and its homologue Nipsnap2 have been proposed to contribute in vesicle transport and membrane fusion in cells. Using CRISPR-Cas9 technology these proteins were knocked out in a keratinocyte cell line (HaCaT) to investigate their role in HSV-1 egress. However, little or no effect on HSV-1 egress could be observed upon loss of either or both of these proteins suggesting the biological significance of gE-Nipsnap1 interaction may not be directly linked to any egress function of gE/gI. Two further interesting 'hits' from the gE/gI interactome were interferon-induced transmembrane protein type-2 (IFITM2), a virus restriction factor, and Myoferlin that has a putative role in endocytic vesicle recycling. This study could validate gE-Myoferlin interaction and co-localisation in infected or transfected cells however, functional significance of this interaction remains to be determined. Overall, the research of this thesis has provided a better understanding of the role of the gE/gI complex in HSV-1 egress and investigated the role of some interesting cellular proteins in the context of virion egress.

Like all other viruses, a successful egress of functional particles from infected cells is a prerequisite for foamy virus (FV) spread within the host. The budding process of FVs involves steps, which are shared by other retroviruses, such as interaction of the capsid protein with components of cellular vacuolar protein sorting (Vps) machinery via late domains identified in some FV capsid proteins. Additionally, there are features of the FV budding strategy quite unique to the spumaretroviruses. This includes secretion of non-infectious subviral particles and a strict dependence on capsid-glycoprotein interaction for release of infectious virions from the cells. Virus-like particle release is not possible since FV capsid proteins lack a membrane-targeting signal. It is noteworthy that in experimental systems, the important capsid-glycoprotein interaction could be bypassed by fusing heterologous membrane-targeting signals to the capsid protein, thus enabling glycoprotein-independent egress. Aside from that, other systems have been developed to enable envelopment of FV capsids by heterologous Env proteins. In this review article, we will summarize the current knowledge on FV budding, the viral components and their domains involved as well as alternative and artificial ways to promote budding of FV particle structures, a feature important for alteration of target tissue tropism of FV-based gene transfer systems.

Like all other viruses, a successful egress of functional particles from infected cells is a prerequisite for foamy virus (FV) spread within the host. The budding process of FVs involves steps, which are shared by other retroviruses, such as interaction of the capsid protein with components of cellular vacuolar protein sorting (Vps) machinery via late domains identified in some FV capsid proteins. Additionally, there are features of the FV budding strategy quite unique to the spumaretroviruses. This includes secretion of non-infectious subviral particles and a strict dependence on capsid-glycoprotein interaction for release of infectious virions from the cells. Virus-like particle release is not possible since FV capsid proteins lack a membrane-targeting signal. It is noteworthy that in experimental systems, the important capsid-glycoprotein interaction could be bypassed by fusing heterologous membrane-targeting signals to the capsid protein, thus enabling glycoprotein-independent egress. Aside from that, other systems have been developed to enable envelopment of FV capsids by heterologous Env proteins. In this review article, we will summarize the current knowledge on FV budding, the viral components and their domains involved as well as alternative and artificial ways to promote budding of FV particle structures, a feature important for alteration of target tissue tropism of FV-based gene transfer systems.