Protein Myristoylation Plays a Role in the Nuclear Entry of the Parvovirus Minute Virus of Mice.

Being nonpathogenic to humans, rodent parvoviruses (PVs) are naturally oncolytic viruses with great potential as anti-cancer agents. As these viruses replicate in the host cell nucleus, they must gain access to the nucleus during infection. The PV minute virus of mice (MVM) and several other PVs transiently disrupt the nuclear envelope (NE) and enter the nucleus through the resulting breaks. However, the molecular basis of this unique nuclear entry pathway remains uncharacterized. In this study, we used MVM as a model to investigate the molecular mechanism by which PVs induce NE disruption during viral nuclear entry. By combining bioinformatics analyses, metabolic labeling assays, mutagenesis, and pharmacological inhibition, we identified a functional myristoylation site at the sequence 78GGKVGH83 of the unique portion of the capsid protein VP1 (VP1u) of MVM. Performing proteolytic cleavage studies with a peptide containing this myristoylation site or with purified virions, we found tryptophan at position 77 of MVM VP1u is susceptible to chymotrypsin cleavage, implying this cleavage exposes G (glycine) 78 at the N-terminus of VP1u for myristoylation. Subsequent experiments using inhibitors of myristoylation and cellular proteases with MVM-infected cells, or an imaging-based quantitative NE permeabilization assay, further indicate protein myristoylation and a chymotrypsin-like activity are essential for MVM to locally disrupt the NE during viral nuclear entry. We thus propose a model for the nuclear entry of MVM in which NE disruption is mediated by VP1u myristoylation after the intact capsid undergoes proteolytic processing to expose the required N-terminal G for myristoylation. IMPORTANCE Rodent parvoviruses (PVs), including minute virus of mice (MVM), have the ability to infect and kill cancer cells and thereby possess great potential in anti-cancer therapy. In fact, some of these viruses are currently being investigated in both preclinical studies and clinical trials to treat a wide variety of cancers. However, the detailed mechanism of how PVs enter the cell nucleus remains unknown. In this study, we for the first time demonstrated a chemical modification called "myristoylation" of a MVM protein plays an essential role in the nuclear entry of the virus. We also showed, in addition to protein myristoylation, a chymotrypsin-like activity, which may come from cellular proteasomes, is required for MVM to get myristoylated and enter the nucleus. These findings deepen our understanding on how MVM and other related PVs infect host cells and provide new insights for the development of PV-based anti-cancer therapies.

Use of a small DNA virus model to investigate mechanisms of CpG dinucleotide-induced attenuation of virus replication.

Suppression of the CpG dinucleotide is widespread in RNA viruses infecting vertebrates and plants, and in the genomes of retroviruses and small mammalian DNA viruses. The functional basis for CpG suppression in the latter was investigated through the construction of mutants of the parvovirus, minute virus of mice (MVM) with increased CpG or TpA dinucleotides in the VP gene. CpG-high mutants displayed extraordinary attenuation in A9 cells compared to wild-type MVM (>six logs), while TpA elevation showed no replication effect. Attenuation was independent of Toll-like receptor 9 and STING-mediated DNA recognition pathways and unrelated to effects on translation efficiency. While translation from codon-optimized VP RNA was enhanced in a cell-free assay, MVM containing this sequence was highly attenuated. Further mutational analysis indicated that this arose through its increased numbers of CpG dinucleotides (7→70) and separately from its increased G+C content (42.3→57.4 %), which independently attenuated replication. CpG-high viruses showed impaired NS mRNA expression by qPCR and reduced NS and particularly VP protein expression detected by immunofluorescence and replication in A549 cells, effects reversed in zinc antiviral protein (ZAP) knockout cells, even though nuclear relocalization of VP remained defective. The demonstrated functional basis for CpG suppression in MVM and potentially other small DNA viruses and the observed intolerance of CpGs in coding sequences, even after codon optimization, has implications for the use of small DNA virus vectors in gene therapy and immunization.

Measuring the effectiveness of gaseous virus disinfectants.

The efficacy of gaseous disinfection is critical for prevention and treatment of microbial contamination in biotechnological facilities. For an evaluation of gaseous disinfection efficacy, a down-scaled laboratory model was established, using currently available carrier tests and a custom-made dry fog box. A mixture of peroxyacetic acid and hydrogen peroxide (PAA/HP) was investigated as example, at concentrations between 0.4 and 2.9 mL/m(3) for up to 3 h for inactivation of a panel of lipid-enveloped and non-lipid-enveloped viruses. The influenza viruses were most sensitive to PAA/HP treatment and minute virus of mice was most resistant. Bovine viral diarrhea virus and reovirus III showed intermediate stability and similar inactivation kinetics. Use of the dry fog box circumvents dedicating an entire lab for the investigation, which renders the generation of data more cost-effective and allows for production of highly reproducible kinetic data.

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/ß1 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.

The minute virus of mice exploits different endocytic pathways for cellular uptake.

The minute virus of mice, prototype strain (MVMp), is a non-enveloped, single-stranded DNA virus of the family Parvoviridae. Unlike other parvoviruses, the mechanism of cellular uptake of MVMp has not been studied in detail. We analyzed MVMp endocytosis in mouse LA9 fibroblasts and a tumor cell line derived from epithelial-mesenchymal transition through polyomavirus middle T antigen transformation in transgenic mice. By a combination of immunofluorescence and electron microscopy, we found that MVMp endocytosis occurs at the leading edge of migrating cells in proximity to focal adhesion sites. By using drug inhibitors of various endocytic pathways together with immunofluorescence microscopy and flow cytometry analysis, we discovered that MVMp can use a number of endocytic pathways, depending on the host cell type. At least three different mechanisms were identified: clathrin-, caveolin-, and clathrin-independent carrier-mediated endocytosis, with the latter occurring in transformed cells but not in LA9 fibroblasts.

Galectin-3 plays a role in minute virus of mice infection.

Galectin-3 has previously been found to be required by the parvovirus minute virus of mice prototype strain (MVMp) for infection of mouse fibroblast cells. Since MVMp is an oncotropic virus, and galectin-3 is a multifunctional protein implicated in cancer metastasis, we hypothesized that galectin-3 and Mgat5, the Golgi enzyme that synthesizes high-affinity glycan ligands of galectin-3, might play a role in MVMp infection. Using siRNA-mediated knockdown of galectin-3 in mouse cells transformed with polyomavirus middle T antigen and Mgat5(-/-) mouse mammary tumor cells, we found that galectin-3 and Mgat5 are both necessary for efficient MVMp cell entry and infection, but not for cell binding. Moreover, we found that human cancer cells expressing higher levels of galectin-3 were more efficiently infected with MVMp than cell lines expressing lower galectin-3 levels. We conclude that galectin-3 and Mgat5 are involved in MVMp infection, and propose that galectin-3 is a determinant of MVMp oncotropism.

Cell migration is another player of the minute virus of mice infection.

The parvovirus minute virus of mice, prototype strain (MVMp), preferentially infects and kills cancer cells. This intrinsic MVMp oncotropism may depend in part on the early stages of MVMp infection. To test this hypothesis, we investigated the early events of MVMp infection in mouse LA9 fibroblasts and a highly invasive mouse mammary tumor cell line derived from polyomavirus middle T antigen-mediated transformation. Using a combination of fluorescence and electron microscopy, we found that various parameters of the cell migration process affect MVMp infection. We show that, after binding to the plasma membrane, MVMp particles rapidly cluster at the leading edge of migrating cells, which exhibit higher levels of MVMp uptake than non-motile cells. Moreover, promoting cell migration on a fibronectin matrix increased MVMp infection, and induction of epithelial-mesenchymal transition allowed MVMp replication in non-permissive epithelial cells. Hence, we propose that cell migration influences the early stages of MVMp infection.

TLR-9 contributes to the antiviral innate immune sensing of rodent parvoviruses MVMp and H-1PV by normal human immune cells.

The oncotropism of Minute Virus of Mice (MVMp) is partially related to the stimulation of an antiviral response mediated by type-I interferons (IFNs) in normal but not in transformed mouse cells. The present work was undertaken to assess whether the oncotropism displayed against human cells by MVMp and its rat homolog H-1PV also depends on antiviral mechanisms and to identify the pattern recognition receptor (PRR) involved. Despite their low proliferation rate which represents a drawback for parvovirus multiplication, we used human peripheral blood mononuclear cells (hPBMCs) as normal model specifically because all known PRRs are functional in this mixed cell population and moreover because some of its subsets are among the main IFN producers upon infections in mammals. Human transformed models consisted in lines and tumor cells more or less permissive to both parvoviruses. Our results show that irrespective of their permissiveness, transformed cells do not produce IFNs nor develop an antiviral response upon parvovirus infection. However, MVMp- or H-1PV-infected hPBMCs trigger such defense mechanisms despite an absence of parvovirus replication and protein expression, pointing to the viral genome as the activating element. Substantial reduction of an inhibitory oligodeoxynucleotide (iODN) of the latter IFN production identified TLR-9 as a potential PRR for parvoviruses in hPBMCs. However, neither the iODN treatment nor an antibody-induced neutralization of the IFN-triggered effects restored parvovirus multiplication in these cells as expected by their weak proliferation in culture. Finally, given that a TLR-9 activation could also not be observed in parvovirus-infected human lines reported to be endowed with a functional TLR-9 pathway (Namalwa, Raji, and HEK293-TLR9(+/+)), our data suggest that transformed human cells do not sense MVMp or H-1PV either because of an absence of PRR expression or an intrinsic, or virus-driven defect in the endosomal sensing of the parvovirus genomes by TLR-9.

Replication of minute virus of mice in murine cells is facilitated by virally induced depletion of p21.

The DNA damage response to infection with minute virus of mice (MVM) leads to activated p53; however, p21 levels are reduced via a proteasome-mediated mechanism. This loss was sustained, as virus replicated in infected cells held at the G(2)/M border. Addition of the cyclin-dependent kinase (CDK) inhibitor roscovitine after S-phase entry reduced MVM replication, suggesting that CDK activity was critical for continued viral replication and virus-induced reduction of p21 may thus be necessary to prevent inhibition of CDK.

Management strategies for controlling endemic and seasonal mouse parvovirus infection in a barrier facility.

Despite improved diagnostic and rederivation capabilities, research facilities still struggle to manage parvovirus infections (e.g., mouse parvovirus (MPV) and minute virus of mice) in mouse colonies. Multi-faceted approaches are needed to prevent adventitious organisms such as MPV from breaching a barrier facility. In this article, the authors document recent changes to the Salk Institute's animal care program that were intended to help manage mouse parvovirus in the barrier facility. Specifically, the Institute started to use a new disinfectant and to give mice irradiated feed. The authors found an association between these modifications and a reduction in MPV incidence and prevalence in endemically infected colonies. These data suggest that using irradiated feed and appropriate disinfectants with contemporary management practices can be an effective plan for eradicating or controlling MPV infection in a research facility. The authors recommend further study of the environmental risk factors for parvovirus infection and of potential biological interactions associated with the use of irradiated feed.

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.

Viral oncolysis that targets Raf-1 signaling control of nuclear transport.

The central role of Raf protein kinase isoforms in human cancer demands specific anti-Raf therapeutic inhibitors. Parvoviruses are currently used in experimental cancer therapy due to their natural oncotropism and lytic life cycle. In searching for mechanisms underlying parvovirus oncolysis, we found that trimers of the major structural protein (VP) of the parvovirus minute virus of mice (MVM), which have to be imported into the nucleus for capsid assembly, undergo phosphorylation by the Raf-1 kinase. Purified Raf-1 phosphorylated the capsid subunits in vitro to the two-dimensional pattern found in natural MVM infections. VP trimers isolated from mammalian cells translocated into the nucleus of digitonin-permeabilized human cells. In contrast, VP trimers isolated from insect cells, which are devoid of Raf-1, were neither phosphorylated nor imported into the mammalian nucleus. However, the coexpression of a constitutively active Raf-1 kinase in insect cells restored VP trimer phosphorylation and nuclear transport competence. In MVM-infected normal and transformed cells, Raf-1 inhibition resulted in cytoplasmic retention of capsid proteins, preventing their nuclear assembly and progeny virus maturation. The level of Raf-1 activity in cancer cells was consistent with the extent of VP specific phosphorylation and with the permissiveness to MVM infection. Thus, Raf-1 control of nuclear translocation of MVM capsid assembly intermediates provides a novel target for viral oncolysis. MVM may reinforce specific therapies against frequent human cancers with deregulated Raf signaling.

Deaminase-independent inhibition of parvoviruses by the APOBEC3A cytidine deaminase.

The APOBEC3 proteins form a multigene family of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G (A3G), APOBEC3A (A3A) has no effect on lentiviruses but dramatically inhibits replication of the parvovirus adeno-associated virus (AAV). To study the contribution of deaminase activity to the antiviral activity of A3A, we performed a comprehensive mutational analysis of A3A. By mutation of non-conserved residues, we found that regions outside of the catalytic active site contribute to both deaminase and antiviral activities. Using A3A point mutants and A3A/A3G chimeras, we show that deaminase activity is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutants block replication of both wild-type AAV and the autonomous parvovirus minute virus of mice (MVM). In addition, we identify specific residues of A3A that confer activity against AAV when substituted into A3G. In summary, our results demonstrate that deaminase activity is not necessary for the antiviral activity of A3A against parvoviruses.

TNF-alpha and the IFN-gamma-inducible protein 10 (IP-10/CXCL-10) delivered by parvoviral vectors act in synergy to induce antitumor effects in mouse glioblastoma.

Interferon-gamma-inducible protein 10 is a potent chemoattractant for natural killer cells and activated T lymphocytes. It also displays angiostatic properties and some antitumor activity. Tumor necrosis factor-alpha (TNF-alpha) is a powerful immunomodulating cytokine with demonstrated tumoricidal activity in various tumor models and the ability to induce strong immune responses. This prompted us to evaluate the antitumor effects of recombinant parvoviruses designed to deliver IP-10 or TNF-alpha into a glioblastoma. When Gl261 murine glioma cells were infected in vitro with an IP-10- or TNF-alpha-transducing parvoviral vector and were subcutaneously implanted in mice, tumor growth was significantly delayed. Complete tumor regression was observed when the glioma cells were coinfected with both the vectors, demonstrating synergistic antitumor activity. In an established in vivo glioma model, however, repeated simultaneous peritumoral injection of the IP-10- and TNF-alpha-delivering parvoviruses failed to improve the therapeutic effect as compared with the use of a single cytokine-delivering vector. In this tumor model, cytokine-mediated immunostimulation, rather than inhibition of vascularization, is likely responsible for the therapeutic efficacy.

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.

Replication initiator protein NS1 of the parvovirus minute virus of mice binds to modular divergent sites distributed throughout duplex viral DNA.

To initiate DNA synthesis, the NS1 protein of minute virus of mice (MVM) first binds to a simple cognate recognition sequence in the viral origins, comprising two to three tandem copies of the tetranucleotide TGGT. However, this motif is also widely dispersed throughout the viral genome. Using an immunoselection procedure, we show that NS1 specifically binds to many internal sites, so that all viral fragments of more than approximately 170 nucleotides effectively compete for NS1, often binding with higher affinity to these internal sites than to sites in the origins. We explore the diversity of the internal sites using competitive binding and DNase I protection assays and show that they vary between two extreme forms. Simple sites with three somewhat degenerate, tandem TGGT reiterations bind effectively but are minimally responsive to ATP, while complex sites, containing multiple variably spaced TGGT elements arranged as opposing clusters, bind NS1 with an affinity that can be enhanced approximately 10-fold by ATP. Using immuno-selection procedures with randomized sequences embedded within specific regions of the genome, we explore possible binding configurations in these two types of site. We conclude that binding is modular, combinatorial, and highly flexible. NS1 recognizes two to six variably spaced, more-or-less degenerate forms of the 5'-TGGT-3' motif, so that it binds efficiently to a wide variety of sequences. Thus, despite complex coding constraints, binding sites are configured at frequent intervals throughout duplex forms of viral DNA, suggesting that NS1 may serve as a form of chromatin to protect and tailor the environment of replicating genomes.

A viral adaptor protein modulating casein kinase II activity induces cytopathic effects in permissive cells.

Autonomous parvoviruses induce severe morphological and physiological alterations in permissive host cells, eventually leading to cell lysis and release of progeny virions. Viral cytopathic effects (CPE) result from specific rearrangements and destruction of cytoskeletal micro- and intermediate filaments. We recently reported that inhibition of endogenous casein kinase II (CKII) protects target cells from parvovirus minute virus of mice (MVM)-induced CPE, pointing to this kinase as an effector of MVM toxicity. The present work shows that the parvoviral NS1 protein mediates CKII-dependent cytoskeletal alterations and cell death. NS1 can act as an adaptor molecule, linking the cellular protein kinase CKIIalpha to tropomyosin and thus modulating the substrate specificity of the kinase. This action results in an altered tropomyosin phosphorylation pattern both in vitro and in living cells. The capacity of NS1 to induce CPE was impaired by mutations abolishing binding with either CKIIalpha or tropomyosin. The cytotoxic adaptor function of NS1 was confirmed with fusion peptides, where the tropomyosin-binding domain of NS1 and CKIIalpha are physically linked. These adaptor peptides were able to mimic NS1 in its ability to induce death of transformed MVM-permissive cells.

Exploring the contribution of distal P4 promoter elements to the oncoselectivity of Minute Virus of Mice.

Minute Virus of Mice (MVM) shares inherent oncotropic properties with other members of the genus Parvovirus. Two elements responsible, at least in part, for this oncoselectivity have been mapped to an Ets1 binding site adjacent to the P4 TATA box of the initiating promoter, P4, and to a more distal cyclic AMP responsive element (CRE), located within the telomeric hairpin stem. Here the CRE overlaps one half-site for the binding of parvoviral initiation factor (PIF), which is essential for viral DNA replication. We used a degenerate oligonucleotide selection approach to show that CRE binding protein (CREB) selects the sequence ACGTCAC within this context, rather than its more generally accepted palindromic TGACGTCA recognition site. We have developed strategies for manipulating these sequences directly within the left-end palindrome of the MVM infectious clone and used them to clone mutants whose CRE either matches the symmetric consensus sequence or is scrambled, or in which the PIF binding site is incrementally weakened with respect to the CRE. The panel of mutants were tested for fitness relative to wildtype in normal murine fibroblasts A9 or transformed human fibroblasts 324 K, through multiple rounds of growth in co-infected cultures, using a differential real-time quantitative PCR assay. We confirmed that inactivating the CRE substantially abrogates oncoselectivity, but found that improving its fit to the palindromic consensus is somewhat debilitating in either cell type. We also confirmed that reducing the PIF half-site spacing by one basepair enhances oncoselectivity, but found that a further basepair deletion significantly reduces this effect.

Humoral immune responses against minute virus of mice vectors.

BACKGROUND: Owing to their oncolytic properties, autonomous rodent parvoviruses and derived vectors constitute potential anti-tumor agents. METHODS: Humoral immune responses to minute virus of mice (MVMp) were characterized. In particular, the generation of neutralizing antibodies on subsequent therapeutic virus applications was evaluated in a mouse melanoma model. Mice bearing subcutaneous melanomas were injected intratumorally with virus and re-injected 10 days later in a second tumor on the other flank. Four days after the first or second injection, the tumors and lymph nodes were analyzed by RT-PCR for gene expression. RESULTS: Injection of MVMp in tumor-bearing B6 mice resulted in viral gene expression in tumors and draining lymph nodes. A repeated virus administration did not lead to detectable viral transcription if it was preceded by a virus infection 10 days earlier. This protection correlated with the induction of virus-neutralizing antibodies following the first virus application. The restrictions on viral gene expression after a consecutive MVMp injection could be alleviated in subsequent applications by the use of viruses consisting of MVMp genomes packaged into capsids of a related parvovirus. Neutralizing antibody induction was irrespective of the route of administration and of the presence of a tumor and persisted at significant levels at least up to 26 weeks after the viral infection. MVMp infection of B6 mice stimulated the generation of IgM and IgG anti-viral antibodies, the latter mainly of the T-helper (Th) 1-dependent IgG2, and the T-cell-independent IgG3 subclasses. CONCLUSIONS: Neutralizing antibodies impede the effectiveness of a subsequent virus administration, but can be overcome by pseudotyping.

Low pH-dependent endosomal processing of the incoming parvovirus minute virus of mice virion leads to externalization of the VP1 N-terminal sequence (N-VP1), N-VP2 cleavage, and uncoating of the full-length genome.

Minute virus of mice (MVM) enters the host cell via receptor-mediated endocytosis. Although endosomal processing is required, its role remains uncertain. In particular, the effect of low endosomal pH on capsid configuration and nuclear delivery of the viral genome is unclear. We have followed the progression and structural transitions of DNA full-virus capsids (FC) and empty capsids (EC) containing the VP1 and VP2 structural proteins and of VP2-only virus-like particles (VLP) during the endosomal trafficking. Three capsid rearrangements were detected in FC: externalization of the VP1 N-terminal sequence (N-VP1), cleavage of the exposed VP2 N-terminal sequence (N-VP2), and uncoating of the full-length genome. All three capsid modifications occurred simultaneously, starting as early as 30 min after internalization, and all of them were blocked by raising the endosomal pH. In particles lacking viral single-stranded DNA (EC and VLP), the N-VP2 was not exposed and thus it was not cleaved. However, the EC did externalize N-VP1 with kinetics similar to those of FC. The bulk of all the incoming particles (FC, EC, and VLP) accumulated in lysosomes without signs of lysosomal membrane destabilization. Inside lysosomes, capsid degradation was not detected, although the uncoated DNA of FC was slowly degraded. Interestingly, at any time postinfection, the amount of structural proteins of the incoming virions accumulating in the nuclear fraction was negligible. These results indicate that during the early endosomal trafficking, the MVM particles are structurally modified by low-pH-dependent mechanisms. Regardless of the structural transitions and protein composition, the majority of the entering viral particles and genomes end in lysosomes, limiting the efficiency of MVM nuclear translocation.