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Pothineni, Venkata Raveendra (2010): Proteome-wide production of monoclonal antibodies and study of intracellular localisation for Varicella-zoster virus (VZV). Dissertation, LMU München: Medizinische Fakultät



Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and with a genome encoding 70 proteins the smallest of all human herpesviruses. Upon primary infection it causes varicella also called chickenpox in children. As a consequence, it reaches sensory nerve ganglia where latency is established. Upon reactivation it causes a secondary disease called Herpes zoster mostly in adults. Todate, VZV is the least studied human herpesvirus due to the lack of cell-free virus in culture, of virus-specific tools and an effective animal model. Therefore, many aspects of the VZV infection cycle, of latency and reactivation are poorly characterized. Moreover, the function of many proteins specific to VZV has not been identified. The goal of this research was to generate hybridoma clones as a permanent source of VZV specific antibodies and to use the antibodies produced to study the localisation of VZV proteins in the viral context on a proteome-wide level. To this end, a VZV ORFeome entry library was constructed using the Gateway® recombinational cloning technology. For VZV protein expression in E. coli, the entry library was subcloned into four different pET derived expression vectors providing either an N-terminal His6, a C-terminal His6, an N-terminal MBP, or an N-terminal GST tag. Following purification of 64 VZV proteins, mice were immunised and subsequently used to generate antibody producing hybridoma clones. So far, our clone collection contains 218 mother clones producing antibodies to 61 (87%) VZV proteins. In this clone collection 190 clones were identified as positive in Western blotting covering 57 VZV ORFs while 123 antibodies were tested positive in immunofluorescence covering 52 VZV ORFs. Using this novel antibody collection, the localisation of 52 (74%) proteins could be determined in the context of VZV infection 22 of which were analysed for the first time. In total, 20 ORFs were localised in the nucleus, 16 ORFs were present in the cytoplasm and 16 ORFs were found in both the nucleus and cytoplasm. Comparison of 41 core proteins present in HSV-1, VZV, CMV, EBV as well as KSHV showed excellent agreement in localisation of conserved glycoproteins, capsid and tegument proteins. Several immunodominant regions on the viral glycoproteins gK, gB, gL, gI, gE and the membrane associated phosphoprotein ORF24 were identified using the pepscan technique. This precious antibody collection gives access to various experimental approaches and will allow to unveil biological secrets in the field of Herpesvirology.