Abstract
For many applications it is necessary to detect target proteins in living cells. This is particularly the case when monitoring viral infections, in which the presence (or absence) of distinct target polypeptides potentially provides vital information about the pathology caused by the agent. To obtain suitable tools with which to monitor parvoviral infections, we thus generated monoclonal antibodies (mAbs) in order to detect the major non-structural protein NS1 in the intracellular environment and tested them for sensitivity and specificity, as well as for cross-reactivity towards related species. Using different immunogens and screening approaches based on indirect immunofluorescence, we describe here a panel of mAbs suitable for monitoring active infections with various parvovirus species by targeting the major non-structural protein NS1. In addition to mAbs detecting the NS1 of parvovirus H-1 (H-1PV) (belonging to the Rodent protoparvovirus 1 species, which is currently under validation as an anti-cancer agent), we generated tools with which to monitor infections by human cutavirus (CuV) and B19 virus (B19V) (belonging to the Primate protoparvovirus 3 and the Primate erythroparvovirus 1 species, respectively, which were both found to persistently infect human tissues). As well as mAbs able to detect NS1 from a broad range of parvoviruses, we obtained entities specific for either (distinct) members of the Rodent protoparvovirus 1 species, human CuV, or human B19V.
Highlights
Autonomous, vertebrate parvoviruses (PVs) are icosahedral, non-enveloped particles of approximately 24 nm in diameter, with a 5 kb linear single-stranded DNA as their genome
To obtain the monoclonal antibodies (mAbs) we considered distinct amino acid regions in NS1, which are exposed and accessible to interact with cell proteins and/or other reagents, such as antibodies
As only limited sequence identity was detected between the helicase domains of B19 virus (B19V) NS1 and H-1PV NS1 we assessed potential similarities of the secondary structures between the two polypeptides (Figure S1)
Summary
Autonomous, vertebrate parvoviruses (PVs) are icosahedral, non-enveloped particles of approximately 24 nm in diameter, with a 5 kb linear single-stranded DNA as their genome. Similar to rodent PV NS1, B19V NS1 contains a nuclear localization signal, has a DNA-binding/endonuclease domain and an NTP-binding motif in the central region, and has a putative trans-activation domain at the C-terminus It is responsible for multiple different activities and is necessary for progeny particle production and spreading, including viral DNA replication and the induction of cell disturbances, such as cell cycle arrest, DNA damage responses, and the induction of apoptosis. To discriminate silent from productive parvoviral infections, we were interested in generating such tools for various (potential) oncolytic PVs, including H-1PV, together with parvoviruses known to cause persistent infections in humans This is the case of B19V [8] and the recently identified primate protoparvovirus CuV [9,10]. We generated a number of mAbs using antigens for H-1PV, CuV, and B19V NS1, respectively, and characterized them for their specificity/cross-reactivity towards different parvovirus species and, their suitability to detect viral activity in human tissues through immunological assays
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