Selective killing of transformed rat cells by minute virus of mice does not require infectious virus production.

Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice [(parvovirus MVM(p)], became sensitive to MVM when transformed by polyomavirus. This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus. The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection. The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones. Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used.     

Nuclear Export Factor CRM1 Interacts with Nonstructural Proteins NS2 from Parvovirus Minute Virus of Mice

The nonstructural NS2 proteins of autonomous parvoviruses are known to act in a host cell-dependent manner and to play a role in viral DNA replication, efficient translation of viral mRNA, and/or encapsidation. Their exact function during the parvovirus life cycle remains, however, still obscure. We report here the characterization of the interaction with the NS2 proteins from the parvovirus minute virus of mice (MVM) and rat as well as mouse homologues of the human CRM1 protein, a member of the importin-beta family recently identified as an essential nuclear export factor. Using the two-hybrid system, we could detect the interaction between the carboxy-terminal region of rat CRM1 and each of the three isoforms of NS2 (P [or major], Y [or minor], and L [or rare]). NS2 proteins were further shown to interact with the full-length CRM1 by coimmunoprecipitation experiments using extracts from both mouse and rat cell lines. Our data show that CRM1 preferentially binds to the nonphosphorylated isoforms of NS2. Moreover, we observed that the treatment of MVM-infected cells with leptomycin B, a drug that specifically inhibits the CRM1-dependent nuclear export pathway, leads to a drastic accumulation of NS2 proteins in the nucleus. Both NS2 interaction with CRM1 and nuclear accumulation upon leptomycin B treatment strongly suggest that these nonstructural viral proteins are actively exported out of the nuclei of infected cells via a CRM1-mediated nuclear export pathway.     

Sendai Virus Infection Induces Apoptosis through Activation of Caspase-8 (FLICE) and Caspase-3 (CPP32)

Sendai virus (SV) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. We now show that SV infection triggers an apoptotic program in target cells. Incubation of infected cells with the peptide inhibitor z-VAD-fmk abrogated SV-induced apoptosis, indicating that proteases of the caspase family were involved. Moreover, proteolytic activation of two distinct caspases, CPP32/caspase-3 and, as shown for the first time in virus-infected cells, FLICE/caspase-8, could be detected. So far, activation of FLICE/caspase-8 has been described in apoptosis triggered by death receptors, including CD95 and tumor necrosis factor (TNF)-R1. In contrast, we could show that SV-induced apoptosis did not require TNF or CD95 ligand. We further found that apoptosis of infected cells did not influence the maturation and budding of SV progeny. In conclusion, SV-induced cell injury is mediated by CD95- and TNF-R1-independent activation of caspases, leading to the death of host cells without impairment of the viral life cycle.     

Interactions of minute virus of mice and adenovirus with host nucleoli.

Biochemical evidence is presented that both minute virus of mice (MVM) and adenovirus interact with the nucleolus during lytic growth and that MVM can also target specific changes involving nucleolar components in adenovirus-infected cells. These virus-nucleolus interactions were studied by analysis of intranuclear compartmentalization of both viral DNAs and host nucleolar proteins: (i) MVM in mouse cells (its normal host) replicates its DNA in the host nucleoli; (ii) specific nucleolar proteins as well as small nuclear ribonucleoprotein antigens are recompartmentalized to multiple intranuclear foci in adenovirus-infected HeLa cells; and (iii) when adenovirus helps MVM DNA replication in a nonpermissive human cell (HeLa), the MVM DNA is also recompartmentalized for synthesis. The data suggest mechanisms for disruption of nucleolar function common to oncogenic or oncolytic virus lytic growth and cell transformation.     

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues.     

Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways

Vesicular stomatitis virus (VSV) induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the recombinant M51R-M (rM51R-M) virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. Here, we determine the caspase pathways by which the rM51R-M virus induces apoptosis. An analysis of caspase activity, using fluorometric caspase assays and Western blots, indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. The overexpression of Bcl-2, an inhibitor of the mitochondrial pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, the inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.     

Vesicular egress of non-enveloped lytic parvoviruses depends on gelsolin functioning

The autonomous parvovirus Minute Virus of Mice (MVM) induces specific changes in the cytoskeleton filaments of infected permissive cells, causing in particular the degradation of actin fibers and the generation of "actin patches." This is attributed to a virus-induced imbalance between the polymerization factor N-WASP (Wiscott-Aldrich syndrome protein) and gelsolin, a multifunctional protein cleaving actin filaments. Here, the focus is on the involvement of gelsolin in parvovirus propagation and virus-induced actin processing. Gelsolin activity was knocked-down, and consequences thereof were determined for virus replication and egress and for actin network integrity. Though not required for virus replication or progeny particle assembly, gelsolin was found to control MVM (and related H1-PV) transport from the nucleus to the cell periphery and release into the culture medium. Gelsolin-dependent actin degradation and progeny virus release were both controlled by (NS1)/CKIIalpha, a recently identified complex between a cellular protein kinase and a MVM non-structural protein. Furthermore, the export of newly synthesized virions through the cytoplasm appeared to be mediated by (virus-modified) lysomal/late endosomal vesicles. By showing that MVM release, like entry, is guided by the cytoskeleton and mediated by vesicles, these results challenge the current view that egress of non-enveloped lytic viruses is a passive process.     

Correlation between nucleocytoplasmic transport and caspase-3-dependent dismantling of nuclear pores during apoptosis

During apoptosis (also called programmed cell death), the chromatin condenses and the DNA is cleaved into oligonucleosomal fragments. Caspases are believed to play a major role in nuclear apoptosis. However, the relation between dismantling of nuclear pores, disruption of the nucleocytoplasmic barrier, and nuclear entry of caspases is unclear. We have analyzed nuclear import of the green fluorescent protein fused to a nuclear localization signal (GFP-NLS) in tissue culture cells undergoing apoptosis. Decreased nuclear accumulation of GFP-NLS could be detected at the onset of nuclear apoptosis manifested as dramatic condensation and redistribution of chromatin toward the nuclear periphery. At this step, dismantling of nuclear pores was already evident as indicated by proteolysis of the nuclear pore membrane protein POM121. Thus, disruption of nuclear compartmentalization correlated with early signs of nuclear pore damage. Both these events clearly preceded massive DNA fragmentation, detected by TUNEL assay. Furthermore, we show that in apoptotic cells, POM121 is specifically cleaved at aspartate-531 in its large C-terminal portion by a caspase-3-dependent mechanism. Cleavage of the C-terminal portion of POM121, which is adjoining the nuclear pore complex, is likely to disrupt interactions with other nuclear pore proteins affecting the stability of the pore complex. A temporal correlation of apoptotic events supports a model where caspase-dependent disassembly of nuclear pores and disruption of the nucleocytoplasmic barrier paves the way for nuclear entry of caspases and subsequent activation of CAD-mediated DNA fragmentation.     

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.     

Complementary roles of multiple nuclear targeting signals in the capsid proteins of the parvovirus minute virus of mice during assembly and onset of infection

This report describes the distribution of conventional nuclear localization sequences (NLS) and of a beta-stranded so-called nuclear localization motif (NLM) in the two proteins (VP1, 82 kDa; VP2, 63 kDa) forming the T=1 icosahedral capsid of the parvovirus minute virus of mice (MVM) and their functions in viral biogenesis and the onset of infection. The approximately 10 VP1 molecules assembled in the MVM particle harbor in its 142-amino-acid (aa) N-terminal-specific region four clusters of basic amino acids, here called BC1 (aa 6 to 10), BC2 (aa 87 to 90), BC3 (aa 109 to 115), and BC4 (aa 126 to 130), that fit consensus NLS and an NLM placed toward the opposite end of the polypeptide (aa 670 to 680) found to be necessary for VP2 nuclear uptake. Deletions and site-directed mutations constructed in an infectious MVM plasmid showed that BC1, BC2, and NLM are cooperative nuclear transport sequences in singly expressed VP1 subunits and that they conferred nuclear targeting competence on the VP1/VP2 oligomers arising in normal infection, while BC3 and BC4 did not display nuclear transport activity. Notably, VP1 proteins mutated at BC1 and -2, and particularly with BC1 to -4 sequences deleted, induced nuclear and cytoplasmic foci of colocalizing conjugated ubiquitin that could be rescued from the ubiquitin-proteasome degradation pathway by the coexpression of VP2 and NS2 isoforms. These results suggest a role for VP2 in viral morphogenesis by assisting cytoplasmic folding of VP1/VP2 subviral complexes, which is further supported by the capacity of NLM-bearing transport-competent VP2 subunits to recruit VP1 into the nuclear capsid assembly pathway regardless of the BC composition. Instead, all four BC sequences, which are located in the interior of the capsid, were absolutely required by the incoming infectious MVM particle for the onset of infection, suggesting either an important conformational change or a disassembly of the coat for nuclear entry of a VP1-associated viral genome. Therefore, the evolutionarily conserved BC sequences and NLM domains provide complementary nuclear transport functions to distinct supramolecular complexes of capsid proteins during the autonomous parvovirus life cycle.     

How viruses access the nucleus

Many viruses depend on nuclear proteins for replication. Therefore, their viral genome must enter the nucleus of the host cell. In this review we briefly summarize the principles of nucleocytoplasmic transport, and then describe the diverse strategies used by viruses to deliver their genomes into the host nucleus. Some of the emerging mechanisms include: (1) nuclear entry during mitosis, when the nuclear envelope is disassembled, (2) viral genome release in the cytoplasm followed by entry of the genome through the nuclear pore complex (NPC), (3) capsid docking at the cytoplasmic side of the NPC, followed by genome release, (4) nuclear entry of intact capsids through the NPC, followed by genome release, and (5) nuclear entry via virus-induced disruption of the nuclear envelope. Which mechanism a particular virus uses depends on the size and structure of the virus, as well as the cellular cues used by the virus to trigger capsid disassembly and genome release. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.     

Parvovirus-Induced Depletion of Cyclin B1 Prevents Mitotic Entry of Infected Cells

Parvoviruses halt cell cycle progression following initiation of their replication during S-phase and continue to replicate their genomes for extended periods of time in arrested cells. The parvovirus minute virus of mice (MVM) induces a DNA damage response that is required for viral replication and induction of the S/G2 cell cycle block. However, p21 and Chk1, major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, are either down-regulated, or inactivated, respectively, during MVM infection. This suggested that parvoviruses can modulate cell cycle progression by another mechanism. In this work we show that the MVM-induced, p21- and Chk1-independent, cell cycle block proceeds via a two-step process unlike that seen in response to other DNA-damaging agents or virus infections. MVM infection induced Chk2 activation early in infection which led to a transient S-phase block associated with proteasome-mediated CDC25A degradation. This step was necessary for efficient viral replication; however, Chk2 activation and CDC25A loss were not sufficient to keep infected cells in the sustained G2-arrested state which characterizes this infection. Rather, although the phosphorylation of CDK1 that normally inhibits entry into mitosis was lost, the MVM induced DDR resulted first in a targeted mis-localization and then significant depletion of cyclin B1, thus directly inhibiting cyclin B1-CDK1 complex function and preventing mitotic entry. MVM infection thus uses a novel strategy to ensure a pseudo S-phase, pre-mitotic, nuclear environment for sustained viral replication.     

Caspases disrupt the nuclear-cytoplasmic barrier

During apoptosis, caspases, a family of proteases, disassemble a cell by cleaving a set of proteins. Caspase-3 plays a major role in the dissassembly of the nucleus by processing several nuclear substrates. The question is how caspase-3 which is usually cytoplasmic, gains access to its nuclear targets. It was suggested that caspase-3 is actively transported to the nucleus through the nuclear pores. We found that caspase-9, which is activated earlier than caspase-3, directly or indirectly inactivates nuclear transport and increases the diffusion limit of the nuclear pores. This increase allows caspase-3 and other molecules that could not pass through the nuclear pores in living cells to enter or leave the nucleus during apoptosis by diffusion. Hence, caspase-9 contributes to cell disassembly by disrupting the nuclear cytoplasmic barrier.     

Induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during Crimean-Congo hemorrhagic fever virus infection

Regulation of apoptosis during infection has been observed for several viral pathogens. Programmed cell death and regulation of apoptosis in response to a viral infection are important factors for host or virus survival. It is not known whether Crimean-Congo hemorrhagic fever virus (CCHFV) infection regulates the apoptosis process in vitro. This study for the first time suggests that CCHFV induces apoptosis, which may be dependent on caspase-3 activation. This study also shows that the coding sequence of the S segment of CCHFV contains a proteolytic cleavage site, DEVD, which is conserved in all CCHFV strains. By using different recombinant expression systems and site-directed mutagenesis, we demonstrated that this motif is subject to caspase cleavage. We also demonstrate that CCHFV nucleocapsid protein (NP) is cleaved into a 30-kDa fragment at the same time as caspase activity is induced during infection. Using caspase inhibitors and cells lacking caspase-3, we clearly demonstrate that the cleavage of NP is caspase-3-dependent. We also show that the inhibition of apoptosis induced progeny viral titers of ～80-90%. Thus, caspase-3-dependent cleavage of NP may represent a host defense mechanism against lytic CCHFV infection. Taken together, these data suggest that the most abundant protein of CCHFV, which has several essential functions such as protection of viral RNA and participation in various processes in the replication cycle, can be subjected to cleavage by host cell caspases.     

Recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death.

Influenza virus infection induces apoptosis in cultured cells with an augmented expression of Fas (APO-1/CD95). Caspases, a family of cysteine proteases structurally related to interleukin-1-beta-converting enzyme (ICE), play crucial roles in apoptosis induced by various stimuli, including Fas. However, activation of the caspase-cascade seems to be different in various pathways of apoptotic stimuli. We therefore examined the involvement of caspases in influenza virus-induced apoptosis using caspase inhibitors. We found that z-VAD-fmk and z-IETD-fmk effectively inhibited virus-induced apoptosis, whereas Ac-DEVD-CHO and Ac-YVAD-CHO showed partial and little effect on virus-induced cell death, respectively. Consistently, caspase-3-like activity, but not caspase-1-like activity, was increased in the virus-infected cells. The transfection of plasmids encoding viral inhibitors of caspase (v-FLIP or crmA) into HeLa cells inhibited apoptosis by virus infection. The peptide inhibitors of caspases used in this study did not inhibit viral replication. We conclude that influenza virus infection activates some caspases, and that this activation may be downstream of viral replication.     

Genome Replication and Postencapsidation Functions Mapping to the Nonstructural Gene Restrict the Host Range of a Murine Parvovirus in Human Cells

The infection outcome of the Parvoviridae largely relies on poorly characterized intracellular factors modulated by proliferation, differentiation, and transformation of host cells. We have studied the interactions displayed by the highly homologous p and i strains of the murine parvovirus minute virus of mice (MVM), with a series of transformed cells of rat (C6) and human (U373, U87, SW1088, SK-N-SH) nervous system origin, seeking for molecular mechanisms governing parvovirus host range. The MVMp infection of C6 and U373 cells was cytotoxic and productive, whereas the other nervous cells behaved essentially as resistant to this virus. In contrast, MVMi did not complete its life cycle in any of the human nervous cells, though it efficiently killed the astrocytic tumor cells by two types of nonproductive infections: (i) normal synthesis of all viral macromolecules with a late defect in infectious virion maturation and release to the medium in U373; and (ii) high levels of accumulation of the full set of viral messenger RNAs and of both nonstructural (NS-1) and structural (VP-1 and VP-2) proteins, under a very low viral DNA amplification, in U87 and SW1088 cells. Further analyses showed that U87 was permissive for nuclear transport of MVMi proteins, leading to efficient assembly of empty viral capsids with a normal phosphorylation and VP1-to-VP2 ratio. The DNA amplification blockade in U87 occurred after conversion of the incoming MVMi genome to the monomeric replicative form, and it operated independently of the delivery pathway used by the viral particle, since it could not be overcome by transfection with cloned infectious viral DNA. Significantly, a chimeric MVMi virus harboring the coding region of the nonstructural (NS) gene replaced with that of MVMp showed a similar pattern of restriction in U87 cells as the parental MVMi virus, and it attained in U373 cultures an infectious titer above 100-fold higher under equal levels of DNA amplification and genome encapsidation. The results suggest that the activity of complexes formed by the NS polypeptides and recruited cellular factors restrict parvovirus DNA amplification in a cell type-dependent manner and that NS functions may in addition determine MVM host range acting at postencapsidation steps of viral maturation. These data are relevant for understanding the increased multiplication of autonomous parvovirus in some transformed cells and the transduction efficacy of nonreplicative parvoviral vectors, as well as a general remark on the mechanisms by which NS genes may regulate viral tropism and pathogenesis.     

The small nonstructural protein (NS2) of the parvovirus minute virus of mice is required for efficient DNA replication and infectious virus production in a cell-type-specific manner.

Seven mutations which affect only the small nonstructural protein NS2 were introduced into the infectious clone of the autonomous parvovirus, minute virus of mice (MVM). The majority of these mutants were severely defective for replication following transfection of normal host murine A9 fibroblasts; however, all were found to replicate more efficiently and produce infectious virus in certain other cell types, including human NB324K. The isolation of viral stocks from NB324K cells permitted a more detailed analysis of the mutant defect on A9 cells. NS2 mutant NS2-2018 was shown to be approximately 10-fold deficient for viral monomer replicative-form DNA production within a single-burst cycle in infected A9 cells and produced a reduced amount of progeny single strand. Mutant NS2-2018 generated wild-type levels of monomer replicative-form DNA on NB324K cells but made reduced levels of progeny single strand and small plaques on these cells. The accumulation of NS1 is reduced late in NS2-2018 infection of A9 cells, but NS1 accumulates to wild-type levels late in NB324K cell infections. NS1 nuclear localization is not dependent on NS2 in A9 or NB324K cells. These results indicate that NS2 participates in MVM DNA replication and is required for efficient viral growth. The requirement for NS2 during MVM replication is also host cell specific. This requirement is significantly more pronounced in the normal host murine A9 cells than in certain other cell types, including NB324K.     

Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.     

Parvovirus minute virus of mice induces a DNA damage response that facilitates viral replication

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells.     

The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells

The chicken anemia virus protein Apoptin has been shown to induce apoptosis in a large number of transformed and tumor cell lines, but not in primary cells. Whereas many other apoptotic stimuli (e.g., many chemotherapeutic agents and radiation) require functional p53 and are inhibited by Bcl-2, Apoptin acts independently of p53, and its activity is enhanced by Bcl-2. Here we study the involvement of caspases, an important component of the apoptotic machinery present in mammalian cells. Using a specific antibody, active caspase-3 was detected in cells expressing Apoptin and undergoing apoptosis. Although Apoptin activity was not affected by CrmA, p35 did inhibit Apoptin-induced apoptosis, as determined by nuclear morphology. Cells expressing both Apoptin and p35 showed only a slight change in nuclear morphology. However, in most of these cells, cytochrome c is still released and the mitochondria are not stained by CMX-Ros, indicating a drop in mitochondrial membrane potential. These results imply that although the final apoptotic events are blocked by p35, parts of the upstream apoptotic pathway that affect mitochondria are already activated by Apoptin. Taken together, these data show that the viral protein Apoptin employs cellular apoptotic factors for induction of apoptosis. Although activation of upstream caspases is not required, activation of caspase-3 and possibly also other downstream caspases is essential for rapid Apoptin-induced apoptosis.