Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

Abstract

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.

Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein

“…The apparent size of all proteins was larger than their theoretical size of ϳ104 kDa (ϳ97 kDa for dC67-deYFP). This was in good agreement with Western blot results obtained for wild-type (wt) CPV NS1 and for previously characterized NS1 fluorescent fusion proteins, which were detected ϳ20-kDa larger than their theoretical size (15,35). Fluorescence microscopy analysis of transfected cells showed that all NS1-deYFP fusion proteins were predominantly nuclear in both infected and noninfected cells (results not shown).…”

“…All mutations were made to NS1-deYFP, which contains NS1 cloned to the multiple cloning site of the pEYFP-N3 plasmid (Clontech), conserved mutations at the P38 promoter area of the NS1 gene, and an ATG-to-ACG mutation at the start codon of the enhanced yellow fluorescent protein (EYFP) gene. This construct has been characterized (15,35). To study P38 promoter activity and replication efficacy, some of the mutations were also made to NS1-EYFP, in which the P38 promoter is intact (15), and to infectious clone pIC (pBI265 in reference36).…”

“…The diffusion coefficient for monomeric NS1-deYFP in simulations was 18.8 m 2 /s. This estimate was based on mass scaling from the diffusion coefficient of freely diffusing EYFP (15,35). The binding reaction k on and k off rates were changed in the modeling process until the recovery in the simulation fit the measured data.…”

“…To inhibit the P38 promoterdriven expression of the free EYFP, we introduced conserved mutations to the TATA box area of the P38 promoter and mutated the start codon of EYFP (deYFP constructs in Fig. 1D) (35). Moreover, constructs that combined two or three of the above mutations in NS1 were made.…”

Clathrin-Associated Endocytosis as a Route of Entry into Cells for Parvoviruses

“…Once in the nucleus, AAV appears to undergo subnuclear mobilization accumulating in the nucleolus , uncoating and genome replication. Although the purpose of this review is to only take the entering virus to the intranuclear environment, it is interesting to note that in CPV infection the nuclear replication compartment expands and is accompanied by chromatin marginalization to the vicinity of the nuclear membrane, virus capsids move by passive diffusion, intranuclear structure and dynamics are extensively affected enlarging the interchromosomal domain which contains viral proteins, genomes, and capsids (Ihalainen et al, 2009). After parvovirus assembly and maturation within the nucleus of infected cells, they egress by processes that include apoptosis (Poole et al, 2004;Poole et al, 2006) and cell necrosis (Abdel-Latif et al, 2006).…”