The putative metal coordination motif in the endonuclease domain of human Parvovirus B19 NS1 is critical for NS1 induced S phase arrest and DNA damage.

The non-structural proteins (NS) of the parvovirus family are highly conserved multi-functional molecules that have been extensively characterized and shown to be integral to viral replication. Along with NTP-dependent helicase activity, these proteins carry within their sequences domains that allow them to bind DNA and act as nucleases in order to resolve the concatameric intermediates developed during viral replication. The parvovirus B19 NS1 protein contains sequence domains highly similar to those previously implicated in the above-described functions of NS proteins from adeno-associated virus (AAV), minute virus of mice (MVM) and other non-human parvoviruses. Previous studies have shown that transient transfection of B19 NS1 into human liver carcinoma (HepG2) cells initiates the intrinsic apoptotic cascade, ultimately resulting in cell death. In an effort to elucidate the mechanism of mammalian cell demise in the presence of B19 NS1, we undertook a mutagenesis analysis of the protein's endonuclease domain. Our studies have shown that, unlike wild-type NS1, which induces an accumulation of DNA damage, S phase arrest and apoptosis in HepG2 cells, disruptions in the metal coordination motif of the B19 NS1 protein reduce its ability to induce DNA damage and to trigger S phase arrest and subsequent apoptosis. These studies support our hypothesis that, in the absence of replicating B19 genomes, NS1-induced host cell DNA damage is responsible for apoptotic cell death observed in parvoviral infection of non-permissive mammalian cells.

Mechanisms of cell death in canine parvovirus-infected cells provide intuitive insights to developing nanotools for medicine.

Viruses have great potential as nanotools in medicine for gene transfer, targeted gene delivery, and oncolytic cancer virotherapy. Here we have studied cell death mechanisms of canine parvovirus (CPV) to increase the knowledge on the CPV life cycle in order to facilitate the development of better parvovirus vectors. Morphological studies of CPV-infected Norden laboratory feline kidney (NLFK) cells and canine fibroma cells (A72) displayed characteristic apoptotic events. Apoptosis was further confirmed by activation of caspases and cellular DNA damage. However, results from annexin V-propidium iodide (PI) labeling and membrane polarization assays indicated disruption of the plasma membrane uncommon to apoptosis. These results provide evidence that secondary necrosis followed apoptosis. In addition, two human cancer cell lines were found to be infected by CPV. This necrotic event over apoptotic cell death and infection in human cells provide insightful information when developing CPV as a nanotool for cancer treatments.

Parvovirus B19 genotype specific amino acid substitution in NS1 reduces the protein's cytotoxicity in culture.

A clinical association between idiopathic liver disease and parvovirus B19 infection has been observed. Fulminant liver failure, not associated with other liver-tropic viruses, has been attributed to B19 in numerous reports, suggesting a possible role for B19 components in the extensive hepatocyte cytotoxicity observed in this condition. A recent report by Abe and colleagues (Int J Med Sci. 2007;4:105-9) demonstrated a link between persistent parvovirus B19 genotype I and III infection and fulminant liver failure. The genetic analysis of isolates obtained from these patients demonstrated a conservation of key amino acids in the nonstructural protein 1 (NS1) of the disease-associated genotypes. In this report we examine a conserved residue identified by Abe and colleagues and show that substitution of isoleucine 181 for methionine, as occurs in B19 genotype II, results in the reduction of B19 NS1-induced cytotoxicity of liver cells. Our results support the hypothesis that in the setting of persistent B19 infection, direct B19 NS1-induced cytotoxicity may play a role in idiopathic fulminant liver failure.

Late steps of parvoviral infection induce changes in cell morphology.

Previously, virus-induced non-filopodial extensions have not been encountered in connection with viral infections. Here, we report emergence of long extensions protruding from Norden laboratory feline kidney (NLFK) and A72 (canine fibroma) cells infected with canine parvovirus for 72 h. These extensions significantly differ in length and number from those appearing in control cells. The most striking feature in the extensions is the length, reaching up to 130 microm, almost twice the average length of a healthy NLFK cell. In A72 cells, the extensions were even longer, up to 200 microm. The results presented here also suggest that the events leading to the growth of these extensions start earlier in infection and abnormal extension growth is detectable already at 24-h post-infection (p.i.). These extensions may have a vital role in the cell-to-cell transmission of the virus.

Diversity of bacteria contaminating paper machines.

Formation of microbial biofilms and slimes is a general and serious problem in the operation of paper machines. Studies of microbial populations in paper machine-derived biofilms have been conducted using standard microbiological procedures; however, the bacterial genera present in this type of samples as well as their diversity are quite poorly known. Here, the bacterial diversity of 38 process water and 22 biofilm samples from four different Finnish paper machines were analyzed by length heterogeneity analysis of PCR-amplified 16S ribosomal DNA (LH-PCR). In addition, sequencing of the amplified 16S rRNA gene from 69 clones was conducted for characterization of the bacterial genera present in biofilm and slime samples. The LH-PCR profiles of both the free-living (process waters) and immobilized (biofilms) bacteria were diverse at all stages of the papermaking process. Out of the 69 sequenced clones, 44 belonged to alpha-Proteobacteria, most of which were close to the nitrogen-fixing root nodule genera Sinorhizobium, Rhizobium and Azorhizobium. Other clones were assigned to beta- and gamma-Proteobacteria and the phylum Bacteroidetes. In addition, eight of the clones were assigned to a yet uncultivated phylum, TM7. Finally, epifluorescence microscopy revealed that Gram-negative bacteria were predominant in both the biofilm (65%) and process water (54%) samples and a small coccoid cell morphology was most common in all samples. Together, our results show that the analysis of microbial samples from paper machines using modern molecular biology techniques adds valuable information and should, therefore, be useful as a more specific and sensitive microbiological method for the paper industry. This information could further be applied, e.g., in the development of more specific and environmental friendly antimicrobial agents for paper mills.

Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles.

Although sharing a T=1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence correlation spectroscopy showed that an average of nine EGFP domains were associated with these virus-like structures. Atomic force microscopy and immunoprecipitation studies showed that EGFP was displayed on the surface of these fVLPs. Confocal imaging indicated that these chimeric complexes were targeted to late endosomes when expressed in insect cells. The fVLPs were able to efficiently enter cancer cells and traffic to the nucleus via the microtubulus network. Finally, immunoglobulins present in human parvovirus B19 acute and past-immunity serum samples were able to detect antigenic epitopes present in these fVLPs. In summary, we have developed fluorescent virus-like nanoparticles displaying a large heterologous entity that should be of help to elucidate the mechanisms of infection and pathogenesis of human parvovirus B19. In addition, these B19 nanoparticles serve as a model in the development of targetable vehicles designed for delivery of biomolecules.

Exploitation of microtubule cytoskeleton and dynein during parvoviral traffic toward the nucleus.

Canine parvovirus (CPV), a model virus for the study of parvoviral entry, enters host cells by receptor-mediated endocytosis, escapes from endosomal vesicles to the cytosol, and then replicates in the nucleus. We examined the role of the microtubule (MT)-mediated cytoplasmic trafficking of viral particles toward the nucleus. Immunofluorescence and immunoelectron microscopy showed that capsids were transported through the cytoplasm into the nucleus after cytoplasmic microinjection but that in the presence of MT-depolymerizing agents, viral capsids were unable to reach the nucleus. The nuclear accumulation of capsids was also reduced by microinjection of an anti-dynein antibody. Moreover, electron microscopy and light microscopy experiments demonstrated that viral capsids associate with tubulin and dynein in vitro. Coprecipitation studies indicated that viral capsids interact with dynein. When the cytoplasmic transport process was studied in living cells by microinjecting fluorescently labeled capsids into the cytoplasm of cells containing fluorescent tubulin, capsids were found in close contact with MTs. These results suggest that intact MTs and the motor protein dynein are required for the cytoplasmic transport of CPV capsids and contribute to the accumulation of the capsid in the nucleus.

Role of recycling endosomes and lysosomes in dynein-dependent entry of canine parvovirus.

Canine parvovirus (CPV) is a nonenveloped virus with a 5-kb single-stranded DNA genome. Lysosomotropic agents and low temperature are known to prevent CPV infection, indicating that the virus enters its host cells by endocytosis and requires an acidic intracellular compartment for penetration into the cytoplasm. After escape from the endocytotic vesicles, CPV is transported to the nucleus for replication. In the present study the intracellular entry pathway of the canine parvovirus in NLFK (Nordisk Laboratory feline kidney) cells was studied. After clustering in clathrin-coated pits and being taken up in coated vesicles, CPV colocalized with coendocytosed transferrin in endosomes resembling recycling endosomes. Later, CPV was found to enter, via late endosomes, a perinuclear vesicular compartment, where it colocalized with lysosomal markers. There was no indication of CPV entry into the trans-Golgi or the endoplasmic reticulum. Similar results were obtained both with full and with empty capsids. The data thus suggest that CPV or its DNA was released from the lysosomal compartment to the cytoplasm to be then transported to the nucleus. Electron microscopy analysis revealed endosomal vesicles containing CPV to be associated with microtubules. In the presence of nocodazole, a microtubule-disrupting drug, CPV entry was blocked and the virus was found in peripheral vesicles. Thus, some step(s) of the entry process were dependent on microtubules. Microinjection of antibodies to dynein caused CPV to remain in pericellular vesicles. This suggests an important role for the motor protein dynein in transporting vesicles containing CPV along the microtubule network.

Properties of baculovirus particles displaying GFP analyzed by fluorescence correlation spectroscopy.

Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89 +/- 0.74) x 10(-8) cm2s(-1) and an apparent hydrodynamic radius of 83.35 +/- 21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.57 x 10(-7) cm2s(-1)), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.