The
(PtPV) genus of the
family of viruses includes important animal pathogens and reference molecular models for the entire family. Some virus members of the PtPV genus have arisen as promising tools ...to treat tumoral processes, as they exhibit marked oncotropism and oncolytic activities while being nonpathogenic for humans. The PtPVs invade and replicate within the nucleus making extensive use of the transport, transcription and replication machineries of the host cells. In order to reach the nucleus, PtPVs need to cross over several intracellular barriers and traffic through different cell compartments, which limit their infection efficiency. In this review we summarize molecular interactions, capsid structural transitions and hijacking of cellular processes, by which the PtPVs enter and deliver their single-stranded DNA genome into the host cell nucleus. Understanding mechanisms that govern the complex PtPV entry will be instrumental in developing approaches to boost their anticancer therapeutic potential and improving their safety profile.
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.
Parvoviruses are promising anticancer and gene therapy agents, but a deep knowledge of the entry process is crucial to exploit their therapeutic potential. We addressed this issue while attempting to ...retarget the oncolytic parvovirus minute virus of mice (MVMp) to the tumor vasculature. Residues at three functional domains of the icosahedral capsid were substituted by rational design with peptides competing with the vascular endothelial growth factor. Most substitutions impaired virus maturation, though some yielded infectious chimeric virions, and substitutions in a dimple at the twofold axis that allocates sialic acid (SIA) receptors altered viral tropism. One dimple-modified chimeric virion was efficiently attached as MVMp to α2-linked SIA moieties, but the infection was impaired by the binding to some inhibitory α2-3,-6,-8 SIA pseudoreceptors, which hampers intracellular virus traffic to the endosome in a cell type-dependent manner. Infectious from nonproductive traffic could be mechanistically discriminated by an endosomal drastic capsid structural transition comprising the cleavage of some VP2-Nt sequences and its associated VP1-Nt exposure. Correspondingly, neuraminidase removal of inhibitory SIA moieties enhanced the infection quantitatively, correlating to the restored virus traffic to the endosome and the extent of VP2-Nt cleavage/VP1-Nt exposure. This study illustrates (i) structural constraints to retarget parvoviruses with evolutionary adopted narrow grooves allocating small SIA receptors, (ii) the possibility to enhance parvovirus oncolysis by relaxing the glycan network on the cancer cell surface, and (iii) the major role played by the attachment to cell type-specific SIAs in the intracellular virus traffic to the endosome, which may determine parvovirus tropism and host range.
Cancer therapy urges targeting of malignant subsets within self-renewing heterogeneous stem cell populations. We dissect the genetic and functional heterogeneity of human glioblastoma stem cells ...(GSCs) within patients by their innate responses to non-pathogenic mouse parvoviruses that are tightly restrained by cellular physiology. GSC neurospheres accumulate assembled capsids but restrict viral NS1 cytotoxic protein expression by an innate PKR/eIF2α-P response counteractable by electric pulses. NS1 triggers a comprehensive DNA damage response involving cell-cycle arrest, neurosphere disorganization, and bystander disruption of GSC-derived brain tumor architecture in rodent models. GSCs and cancer cell lines permissive to parvovirus genome replication require p53-Ser15 phosphorylation (Pp53S15). NS1 expression is enhanced by exogeneous Pp53S15 induction but repressed by wtp53. Consistently, patient-specific GSC subpopulations harboring p53 gain-of-function mutants and/or Pp53S15 are selective viral targets. This study provides a molecular foundation for personalized biosafe viral therapies against devastating glioblastoma and other cancers with deregulated p53 signaling.
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•Human GSCs mount heterogeneous patient-specific innate responses to parvoviruses•Parvovirus infection induces DDR and bystander damage in GSC-derived brain tumors•p53-Ser15 phosphorylation enhances parvovirus genome replication in cancer cells•GSCs with p53 gain-of-function mutations are main targets of parvovirus infection
Oncolytic viruses are being developed for cancer therapy, but their biosafety and specificity are major concerns. Gil-Ranedo et al. describe that human glioblastoma stem cells (GSCs) with patient-specific p53 mutants and p53-Ser15 phosphorylation are selective targets for two strains of the mouse parvovirus minute virus of mice (MVM) that are non-pathogenic for humans. The MVM strains disrupt GSC-derived brain tumors in orthotopic rodent models, showing promise for biosafe personalized therapy against human cancers.
The T1 parvovirus Minute Virus of Mice (MVM) was used to study the roles that phosphorylation and N-terminal domains (Nt) configuration of capsid subunits may play in icosahedral nuclear viruses ...assembly. In synchronous MVM infection, capsid subunits newly assembled as two types of cytoplasmic trimeric intermediates (3VP2, and 1VP1:2VP2) harbored a VP1 phosphorylation level fivefold higher than that of VP2, and hidden Nt. Upon nuclear translocation at S phase, VP1-Nt became exposed in the heterotrimer and subsequent subviral assembly intermediates. Empty capsid subunits showed a phosphorylation level restored to VP1:VP2 stoichiometry, and the Nt concealed in their interior. However ssDNA-filled virus maturing at S/G2 lacked VP1 phosphorylation and one major VP2 phosphopeptide, and exposed VP2-Nt. Endosomal VP2-Nt cleavage resulted in VP3 subunits devoid of any phospholabel, implying that incoming viral particles specifically harbor a low phosphorylation status. Phosphorylation provides a mechanistic coupling of parvovirus nuclear assembly to the cell cycle.
•This manuscript provides new insights into the role that capsid proteins phosphorylation and configuration play in viral assembly and traffic.•Using the icosahedral (T1) nuclear parvovirus Minute Virus of Mice (MVM) as a model, we show changes of N-terminal sequences configuration and differential phosphorylation of capsid subunits, along the cell-cycle coupled nuclear translocation of viral assembly intermediates.•Furthermore, a subsequent drastic though specific dephosphorylation of some capsid subunits is demonstrated as the viral genome becomes packaged.•These findings highlight the complex regulations of life cycle steps even for small nuclear viruses, and increase our understanding of crucial virus-cell cycle interactions.
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