ArticlePDF Available

Oncolytic H-1 parvovirus NS1 protein : identifying and characterizing new transcriptional and posttranslational regulatory elements

Authors:
  • The Scripps Research Institute, United States, Jupiter

Abstract and Figures

H 1 parvovirus (H-1PV) is a little single stranded DNA virus that preferentially replicates in a lytic manner in transformed cells due to their expression profile that meets the requirements for the activation of H¬ 1 PV life cycle unlike normal cells. This feature is known as oncotropism. H 1PV genome is constituted by two transcriptional units. The first one is driven by the proliferation and transformation dependent P4 promoter and allows the expression of both non structural proteins NS1 and NS2, and the second one controls the expression of both capsid proteins VP1 and VP2 through the activation of P38 promoter. H-1PV life cycle tightly depends on NS1 protein that is involved in crucial events, including viral DNA replication, P38 promoter activation as well as cytotoxicity.NS1 protein is regulated at both transcriptional and post translational levels.My thesis aimed at identifying new determining elements for both of these regulations and characterizing their involvement in both H-1PV life cycle and oncotropism.
No caption available
… 
No caption available
… 
No caption available
… 
No caption available
… 
Content may be subject to copyright.
A preview of the PDF is not available
ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
Regulated gene expression is essential for a proper progression through the cell cycle. The transcription factor NF-Y has a fundamental function in transcriptional regulation of cell cycle genes, particularly of G2/M genes. In order to investigate common and distinct functions of NF-Y subunits in cell cycle regulation, NF-YA, NF-YB and NF-YC have been silenced by shRNAs in HCT116 cells. NF-YA loss led to a delay in S-phase progression, DNA damage and apoptosis: we showed the activation of the replication checkpoint, through the recruitment of Δp53 and of the replication proteins PCNA and Mcm7 to chromatin. Differently, NF-YB depletion impaired cells from exiting G2/M, but did not interfere with S-phase progression. Gene expression analysis of NF-YA and NF-YB inactivated cells highlighted a common set of hit genes, as well as a plethora of uncommon genes, unveiling a different effect of NF-Y subunits loss on NF-Y binding to its target genes. Chromatin extracts and ChIP analysis showed that NF-YA depletion was more effective than NF-YB in hitting NF-Y recruitment to CCAAT-promoters. Our data suggest a critical role of NF-Y expression, highlighting that the lack of the single subunits are differently perceived by the cells, which activate diverse cell cycle blocks and signaling pathways.
Article
Full-text available
H-1 parvovirus (H-1 PV), a rodent autonomous oncolytic parvovirus, has emerged as a novel class of promising anticancer agents, because of its ability to selectively find and destroy malignant cells. However, to probe H-1 PV multimodal antitumor potential one of the major prerequisites is to decipher H-1 PV direct interplay with human immune system, and so prevent any risk of impairment. Non activated peripheral blood mononuclear cells (PBMCs) are not sensitive to H-1 PV cytotoxic effect. However, the virus impairs both activated PBMC proliferation ability and viability. This effect is related to H-1 PV infection as evidenced by Western blotting detection of H-1 PV main protein NS1. However, TCID50 experiments did not allow newly generated virions to be detected. Moreover, flow cytometry has shown that H-1 PV preferentially targets B lymphocytes. Despite seeming harmful at first sight, H-1 PV seems to affect very few NK cells and CD8+ T lymphocytes and, above all, clearly does not affect human neutrophils and one of the major CD4+ T lymphocyte subpopulation. Very interestingly, flow cytometry analysis and ELISA assays proved that it even activates human CD4+ T cells by increasing activation marker expression (CD69 and CD30) and both effective Th1 and Th2 cytokine secretion (IL-2, IFN-γ and IL-4). In addition, H-1 PV action does not come with any sign of immunosuppressive side effect. Finally, we have shown the efficiency of H-1 PV on xenotransplanted human nasopharyngeal carcinoma, in a SCID mouse model reconstituted with human PBMC. Our results show for the first time that a wild-type oncolytic virus impairs some immune cell subpopulations while directly activating a Helper CD4+ T cell response. Thus, our data open numerous gripping perspectives of investigation and strongly argue for the use of H-1 PV as an anticancer treatment.
Article
The nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVMp) is cytolytic when expressed in transformed cells. Before causing extensive cell lysis, NS1 induces a multistep cell cycle arrest in G(1) S, and G(2) well reproducing the arrest in S and G(2) observed upon MVMp infection. In this work we investigated the molecular mechanisms of growth inhibition mediated by NS1 and MVMp. We show that NS1-mediated cell cycle arrest correlates with the accumulation of the cyclin-dependent kinase (Cdk) inhibitor p21(cip1) associated with both the cyclin A/Cdk and cyclin E/Cdk2 complexes but in the absence of accumulation of p53, a potent transcriptional activator of p21(cip1). By comparison, MVMp infection induced the accumulation of both p53 and p21(cip). We demonstrate that p53 plays an essential role in the MVMp-induced cell cycle arrest in both S and G(2) by using p53 wild-type (+/+) and null (-/-) cells. Furthermore, only the G(2) arrest was abrogated in p21(cip1) null (-/-) cells. Together these results show that the MVMp-induced cell cycle arrest in S is p53 dependent but p21(cip1) independent, whereas the arrest in G(2) depends on both p53 and its downstream effector p21(cip1). They also suggest that induction of p21(cip1) by the viral protein NS1 arrests cells in G(2) through inhibition of cyclin A-dependent kinase activity.
Chapter
During the past ten years parvoviruses have attracted considerable interest. This is in part due to their unique molecular organization as well as to the fact that they provide an excellent experimental tool to study the replication of a small, single-stranded viral DNA genome and to probe into the synthesis of eucaryotic cell DNA. On the other hand, parvoviruses were shown to be associated with various economically important diseases of animals. At lease since the very recent world-wide epidemic of canine parvovirus enteritis they are no longer regarded as constituting a mere laboratory problem.
Article
Viral proteins interact with one another during viral replication, assembly, and maturation. Systematic interaction assays of the hepatitis C virus (HCV) proteins using the yeast two-hybrid method have uncovered a novel interaction between core and NS5A. This interaction was confirmed by in vitro binding assays, and coimmunoprecipitation in mammalian cells. Core and NS5A are also colocalized in COS-7 cells. Interestingly, NS5A is cleaved to give specific-size fragments, when core is coexpressed in mammalian cells. Overexpression of core produced many dying and rounded cells and effects such as DNA laddering and the truncation of poly(ADP-ribose) polymerase 1 (PARP1), both indicators of apoptosis. These observations led us to investigate the link between the induction of apoptosis by core and the cleavage of NS5A. The proteolysis of NS5A and these apoptotic events can be inhibited by caspase inhibitor, Z-VAD, indicating that core induces apoptosis and the cleavage of NS5A by caspases. In cells infected by the HCV, core may provide the intrinsic apoptotic signal, which produces truncated forms of NS5A. The biological function of core–NS5A interaction and the downstream effect of NS5A cleavage are discussed.