Proteome-wide production of monoclonal antibodies and study of intracellular localisation for Varicella-zoster virus (VZV)

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.