The parvoviruses are among the smallest DNA viruses, which infects the vertebrates and insects. They are divided into dependoviruses and autonomous parvoviruses. The dependoviruses depend on helper adenovirus or herpes virus to provide functions needed for their replication, whereas the autonomous parvoviruses, Parvovirus and Erythrovirus, do not need any helper virus for their DNA multiplication.
Parvoviruses (PV) are non-enveloped, single stranded DNA viruses consisting of the structural proteins viral protein 1 (VP1) and VP2 and non-structural protein 1 (NS1). VP1 is the largest protein (83 kDa) contains NLS and a PLA2 activity that is not exposed on viral capsids. NS1 protein acts as a transactivator, which is covalently bound to viral DNA, and which is exposed on capsids surface. It is believed to be cleaved from the capsid upon uptake into the cells. Parvoviruses enter via clathrin-mediated endocytosis and need a low pH for productive infection. Parvoviruses exhibit 18-24 nm diameters, which is small enough to pass the nuclear pore complex (NPC) without any disassembled form.
Accordingly some authors propose that NLS on VP1 that becomes exposed upon endosomal acidification mediates entry of the capsid into the nucleus. However microinjections, by passing the endosomes, generate progeny capsids being contradictory to this model. Other proposed that the PLA2 actively allows the capsids to pass the nuclear membrane, while others suggest that this activity is required for endosomal release.
As the before mentioned models were based on the analysis of the autonomous parvoviruses MVM and CPV, we first evaluated whether our analysis done on parvovirus H-1 showed similar phenomenon. In fact we showed that acidification resulted in a structural change of the H-1 capsid resulting in the exposure of a NLS without leading to capsid disintegration. However nuclear entry did not occur upon infection but was shown to be caused by nuclear accumulation of progeny capsid or capsid subunits.
Analysing the early steps of infection, we found that H-1 caused a local nuclear envelope breakdown (NEBD). This phenomenon was reproduced in digitonin-permeabilized cells even in the absence of cytosolic proteins. Corresponding experiments with CPV confirmed that this phenomenon was not H-1 specific. Further investigation showed that parvovirus-mediated NEBD was an enzymatically driven process involving PKC, cdk-2 and caspase-3 that are the executing enzymes in physiological NEBD upon mitosis and apoptosis.
Inhibition experiments revealed that H-1 triggered NEBD by an interaction with the nuclear pores not requiring nuclear transport receptor that facilitate attachment. Infection experiments in the presence of PKC, cdk2 and caspase-3 inhibitors confirmed that all these enzymes were needed for PV-mediated NEBD upon infection. Further their inhibition blocked the appearance of progeny nuclear capsids. Further investigations revealed that PKC was however required for nuclear translocation of the progeny capsid subunits, cdk-2 for their synthesis while only caspase-3 did not affect these two steps.
Indirectly these finding thus indicate that NEBD is essential for the entry of the parvoviral capsids upon infection, which is a new import principle for viruses.
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