Aleutian Mink Disease Parvovirus Replication Research Articles

Caspase cleavage of the nonstructural protein NS1 mediates replication of Aleutian mink disease parvovirus.

Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian mink disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD downward arrow S) and NS1:285 (DQTD downward arrow S). Replication of ADV containing either of these mutations was reduced 10(3)- to 10(4)-fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.

Caspase Activation Is Required for Permissive Replication of Aleutian Mink Disease Parvovirus in Vitro

Aleutian mink disease parvovirus (ADV) is distinct among the parvoviruses as infection in vivo is persistent, restricted, and noncytopathic. In contrast, infections with other more prototypic parvoviruses, like mink enteritis virus (MEV), are acute, cytopathic, and characterized by permissive replication in vivo. Although apoptosis results in the death of cells acutely infected by parvoviruses, the role of apoptosis in ADV infections is unknown. Permissive infection of ADV resulted in apoptosis of Crandell feline kidney (CrFK) cells as indicated by TUNEL staining, Annexin-V staining, and characteristic changes in cell morphology. Pretreatment of infected cells with caspase 3 or broad-spectrum caspase inhibitors prevented apoptosis. In addition, treatment of infected cells with these inhibitors caused a 2 log10 reduction in the yield of infectious virus compared to untreated cultures. This block in replication preceded substantial viral DNA amplification and gene expression. However, inhibitors of caspases 1, 6, and 8 did not have this effect. MEV also induced caspase-dependent apoptosis following infection of CrFK cells, although production of infectious progeny was not affected by inhibition of apoptosis. Thus, permissive replication of ADV in vitro depended upon activation of specific caspases. If ADV infection of cells in vivo fails to initiate caspase activation, the requirement of caspase activity for replication may not be met, thus providing a possible mechanism for persistent, restricted infection.

A comparison between permissive and restricted infections with Aleutian mink disease parvovirus (ADV): characterization of the viral protein composition at nuclear sites of virus replication

We used three-color fluorescent labeling and confocal microscopy to compare the permissive and the antibody-mediated, restricted replication of Aleutian mink disease parvovirus (ADV). In both permissive (CRFK cells) and restricted (K562 cells) situations, both ADV non-structural proteins (NS1 and NS2) concentrated at focal sites in the nucleus, which also contained viral DNA. Bromodeoxyuridine labeling demonstrated that these sites also supported active ADV single-strand DNA synthesis, indicating that they were replication compartments. ADV capsid proteins were located in intranuclear shells surrounding the replication compartments. At later time points, NS2 was readily detected in the cytoplasm of permissively infected CRFK cells, whereas the cytoplasmic presence of NS2 was much less pronounced in the K562 cells. These results showed that both permissive and restricted ADV replication are associated with a tight nuclear subcompartmentalization of viral products. Furthermore, differences between the permissive and restricted virus–cell interactions were noted, suggesting that there may be a morphological basis for examining the outcome of ADV infection.

S-phase-dependent cell cycle disturbances caused by Aleutian mink disease parvovirus.

We examined replication of the autonomous parvovirus Aleutian mink disease parvovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cycle arrest occurred exclusively in cells containing de novo-synthesized viral nonstructural (NS) proteins. Production of ADV NS proteins, indicative of ADV replication, was triggered during S-phase traverse. The NS+ cells that were generated during later parts of S phase did not undergo cytokinesis and formed a distinct population, termed population A. Formation of population A was not prevented by VM-26, indicating that these cells were arrested in late S or G2 phase. Cells in population A continued to support high-level ADV DNA replication and production of infectious virus after the normal S phase had ceased. A second, postmitotic, NS+ population (termed population B) arose in G0/G1, downstream of population A. Population B cells were unable to traverse S phase but did exhibit low-level DNA synthesis. Since the nature of this DNA synthesis was not examined, we cannot at present differentiate between G1 and early S arrest in population B. Cells that became NS+ during S phase entered population A, whereas population B cells apparently remained NS- during S phase and expressed high NS levels postmitosis in G0/G1. This suggested that population B resulted from leakage of cells with subthreshold levels of ADV products through the late S/G2 block and, consequently, that the binary pattern of ADV-induced cell cycle arrest may be governed merely by viral replication levels within a single S phase. Flow cytometric analysis of propidium iodide fluorescence and bromodeoxyuridine uptake showed that population A cells sustained significantly higher levels of DNA replication than population B cells during the ADV-induced cell cycle arrest. Therefore, the type of ADV-induced cell cycle arrest was not trivial and could have implications for subsequent viral replication in the target cell.

Subcellular localization of Aleutian mink disease parvovirus proteins and DNA during permissive infection of Crandell feline kidney cells

Confocal microscopy allowed us to localize viral nonstructural (NS) and capsid (VP) proteins and DNA simultaneously in cells permissively infected with Aleutian mink disease parvovirus (ADV). Early after infection, NS proteins colocalized with viral DNA to form intranuclear inclusions, whereas VP proteins formed hollow intranuclear shells around the inclusions. Later, nuclei had irregular outlines and were virtually free of ADV products. In these cells, inclusions of viral DNA with or without associated NS protein were embedded in cytoplasmic VP protein. These findings implied that ADV replication within an infected cell is regulated spatially as well as temporally.

Purification and characterization of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus

We have previously described the expression of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus (ADV) in insect cells by using a baculovirus vector (J. Christensen, T. Storgaard, B. Bloch, S. Alexandersen, and B. Aasted, J. Virol. 67:229-238, 1993). To study its biochemical properties, ADV NS-1 was expressed in Sf9 insect cells and purified to apparent homogeneity with a combination of nuclear extraction, Zn2+ ion chromatography, and immunoaffinity chromatography on monoclonal antibodies. The purified protein showed ATP binding and ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg2+ or Mn2+ as a cofactor. The ATPase activity of NS-1 was efficiently stimulated by single-stranded DNA and, to a lesser extent, double-stranded DNA. We also describe the expression, purification, and characterization of a mutant NS-1 protein, in which a lysine in the putative nucleotide binding consensus sequence of the molecule was replaced with serine. The mutated NS-1 was expressed at 10-fold higher levels than wild-type NS-1, but it exhibited no ATP binding. ATPase, or helicase activity. The availability of large amounts of purified functional NS-1 protein will facilitate studies of the biochemistry of ADV replication and gene regulation leading to disease in mink.

Role of alveolar type II cells and of surfactant-associated protein C mRNA levels in the pathogenesis of respiratory distress in mink kits infected with Aleutian mink disease parvovirus.

Neonatal mink kits infected with Aleutian mink disease parvovirus (ADV) develop an acute interstitial pneumonia with clinical symptoms and pathological lesions that resemble those seen in preterm human infants with respiratory distress syndrome and in human adults with adult respiratory distress syndrome. We have previously suggested that ADV replicates in the alveolar type II epithelial cells of the lung. By using double in situ hybridization, with the simultaneous use of a probe to detect ADV replication and a probe to demonstrate alveolar type II cells, we now confirm this hypothesis. Furthermore, Northern (RNA) blot hybridization showed that the infection caused a significant decrease of surfactant-associated protein C mRNA produced by the alveolar type II cells. We therefore suggest that the severe clinical symptoms and pathological changes characterized by hyaline membrane formation observed in ADV-infected mink kits are caused by a dysfunction of alveolar surfactant similar to that observed in respiratory distress syndrome in preterm infants. However, in the infected mink kits the dysfunction is due to the replication of ADV in the lungs, whereas the dysfunction of surfactant in preterm infants is due to lung immaturity.

Characterization of chimeric full-length molecular clones of Aleutian mink disease parvovirus (ADV): identification of a determinant governing replication of ADV in cell culture

The ADV-G strain of Aleutian mink disease parvovirus (ADV) is nonpathogenic for mink but replicates permissively in cell culture, whereas the ADV-Utah 1 strain is highly pathogenic for mink but replicates poorly in cell culture. In order to relate these phenotypic differences to primary genomic features, we constructed a series of chimeric plasmids between a full-length replication-competent molecular clone of ADV-G and subgenomic clones of ADV-Utah 1 representing map units (MU) 15 to 88. After transfection of the plasmids into cell culture and serial passage of cell lysates, we determined that substitution of several segments of the ADV-Utah 1 genome (MU 15 to 54 and 65 to 73) within an infectious ADV-G plasmid did not impair the ability of these constructs to yield infectious virus in vitro. Like ADV-G, the viruses derived from these replication-competent clones caused neither detectable viremia 10 days after inoculation nor any evidence of Aleutian disease in adult mink. On the other hand, other chimeric plasmids were incapable of yielding infectious virus and were therefore replication defective in vitro. The MU 54 to 65 EcoRI-EcoRV fragment of ADV-Utah 1 was the minimal segment capable of rendering ADV-G replication defective. Substitution of the ADV-G EcoRI-EcoRV fragment into a replication-defective clone restored replication competence, indicating that this 0.53-kb portion of the genome, wholly located within shared coding sequences for the capsid proteins VP1 and VP2, contained a determinant that governs replication in cell culture. When cultures of cells were studied 5 days after transfection with replication-defective clones, rescue of dimeric replicative form DNA and single-stranded progeny DNA could not be demonstrated. This defect could not be complemented by cotransfection with a replication-competent construction.

Aleutian mink disease parvovirus infection of mink peritoneal macrophages and human macrophage cell lines.

Aleutian mink disease parvovirus (ADV) mRNAs are found in macrophages in lymph nodes and peritoneal exudate cells from ADV-infected mink. Therefore, we developed an in vitro infection system for ADV by using primary cultures of mink macrophages or macrophage cell lines. In peritoneal macrophage cultures from adult mink, virulent ADV-Utah I strain showed nuclear expression of viral antigens with fluorescein isothiocyanate-labeled ADV-infected mink serum, but delineation of specific viral proteins could not be confirmed by immunoblot analysis. Amplification of ADV DNA and production of replicative-form DNA were observed in mink macrophages by Southern blot analysis; however, virus could not be serially propagated. The human macrophage cell line U937 exhibited clear nuclear expression of viral antigens after infection with ADV-Utah I but not with tissue culture-adapted ADV-G. In U937 cells, ADV-Utah I produced mRNA, replicative-form DNA, virion DNA, and structural and nonstructural proteins; however, virus could not be serially passaged nor could [3H]thymidine-labeled virions be observed by density gradient analysis. These findings indicated that ADV-Utah I infection in U937 cells was not fully permissive and that there is another restricted step between gene amplification and/or viral protein expression and production of infectious virions. Treatment with the macrophage activator phorbol 12-myristate 13-acetate after adsorption of virus reduced the frequency of ADV-positive U937 cells but clearly increased that of human macrophage line THP-1 cells. These results suggested that ADV replication may depend on conditions influenced by the differentiation state of macrophages. U937 cells may be useful as an in vitro model system for the analysis of the immune disorder caused by ADV infection of macrophages.

Simultaneous identification of viral proteins and nucleic acids in cells infected with Aleutian mink disease parvovirus

A method combining in situ hybridization and immunohistochemistry was used to characterize cells infected with Aleutian mink disease parvovirus (ADV). Single-stranded RNA hybridization probes specific for obligate replicative intermediates and antisera specific for virion or nonstructural proteins were employed. Crandell feline kidney cells in which the ADV-G strain of ADV was permissively replicating contained virion and non-structural proteins, large amounts of single stranded virion DNA, duplex replicative form (RF) DNA, and mRNA. Late in the infectious cycle, however, cells containing non-structural proteins but little nucleic acid were observed, probably representing cells in the end stage of viral cytopathology. Sections of lung prepared from mink kits infected with the ADV-Utah 1 strain were then examined. Alveolar type II cells permissively replicating ADV contained viral nucleic acids and proteins in patterns nearly identical to CRFK cells, suggesting that permissive ADV replication was similar in vitro and in vivo. Another population of ADV containing cells that had cytoplasmic virion antigen, but undetectable levels of non-structural protein was found in vivo. Furthermore, although virion DNA was present in the cytoplasm of these cells, RF DNA or mRNA could not be detected. These cells may have been alveolar macrophages sequestering viral particles.