ISG15 is an interferon-induced and anti-viral ubiquitin-like protein (Ubl). Ube1L, UbcH8, and Herc5 have been identified as the E1-E2-E3 enzymes for ISG15 conjugation, and, like ISG15, their expression is induced by type I interferons. Although Herc5 is the major E3 for ISG15, over 300 proteins have been identified as ISG15 target proteins in interferon-stimulated cells. In this work, I address two aspects of the human ISG15 conjugation system: 1) the specificity of the Ube1L-UbcH8 interaction and 2), the basis of substrate recognition by Herc5. Regarding the selection of UbcH8 by Ube1L, my experiments show that although UbcH8 had been reported to function as an E2 for both Ub and ISG15, UbcH8 is preferentially activated by Ube1L compared to Ube1 (E1[superscript Ub]). The basis of this preference is a result of specific interactions between the ubiquitin-fold domain (UFD) of Ube1L and the amino-terminal [alpha]1 helix and [beta]1 [beta]2 region within UbcH8. Examination of the interferon-induced and transfected expression levels of UbcH8, combined with the kinetic constants, suggest that UbcH8 is unlikely to function as a Ub E2 in most cell lines. In examining the selection of target proteins by Herc5, I show that the range of substrates extends far beyond the proteins identified in proteomics studies and includes many exogenously expressed foreign proteins. Furthermore, I show that ISG15 conjugation is restricted to newly synthesized pools of proteins and Herc5 is associated with polyribosomes. I propose a model for ISGylation in which Herc5 broadly modifies newly synthesized proteins in a co-translational manner and suggest that, in the context of an interferon-stimulated cell, newly translated viral proteins may be primary targets of ISG15. Consistent with this, I show that ISGylation of human papillomavirus (HPV) L1 capsid protein has a dominant-inhibitory effect on the infectivity of HPV16 pseudoviruses. These discoveries have greatly increased our understanding of the mechanism of ISG15 pathway and provide a framework for establishing an in vitro ISG15 conjugation system and further examination of the anti-viral function of ISG15. / text

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that establishes life-long latent infection in sensory neurones. This makes it potentially useful as a gene therapy vector to target neuronal cells. HSV-1 enters cells by membrane fusion, the viral envelope and most tegument proteins dissociate, and the capsid is transported to the cell nucleus to establish infection. There is increasing evidence that the retrograde transport of HSV-1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. It is a large complex molecule, with heavy chains providing motility, while intermediate and light chains are involved in specific cargo binding. A library of HSV-1 capsid and tegument structural genes was constructed and tested for interaction with dynein subunits in a yeast two-hybrid system. A strong interaction was demonstrated between the HSV-1 outer capsid protein VP26 (UL35), as well as the tegument protein VP11/12 (UL46), with the homologous 14 kDa dynein light chains rp3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to rp3, Tctex1 and cytoplasmic dynein complexes. Recombinant HSV-1 capsids +/- VP26 were used in similar pull-down assays. Only VP26+ capsids bound to rp3. Recombinant HSV-1 capsids were microinjected into living cells and incubated at 37�C. After 1 h capsids were observed to co-localise with rp3, Tctex1 and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, while VP26- capsids remained in a random distribution. Our results suggest that the HSV-1 outer capsid protein VP26 mediates binding of incoming capsids to the retrograde motor cytoplasmic dynein during cellular infection, through interactions with dynein light chains. It is hoped that these findings will help in the development of a synthetic viral vector, which may allow targeted gene therapy in patients with neurological diseases.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Sydney, 2005. / Title from title screen (viewed 20 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Medicine. Degree awarded 2005; thesis originally submitted 2004, corrected version submitted April 2005. Includes bibliographical references. Also available in print form.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 198-225). Online version available via ProQuest Digital Dissertations.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb 10, 2009). Includes bibliographical references.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Molecular Virology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains iv, 66, [6] p. : ill. Includes abstract. Includes bibliographical references.