MVM(p) NS-2 Protein Expression Is Required with NS-1 for Maximal Cytotoxicity in Human Transformed Cells

Late in infection, parvovirus minute virus of mice (MVMp) induces the lysis of mouse A9 fibroblasts. This effect depends on the large nonstructural phosphoprotein NS1, which plays in addition a major role in viral DNA replication and progeny particle production. Since the NS1 C-terminal region is subjected to late phosphorylation events and protein kinase C (PKC) family members regulate NS1 replicative activities, the present study was conducted to determine the impact of PKCs on NS1 cytotoxic functions. To this end, we performed site-directed mutagenesis, substituting alanine residues for two consensus PKC-phosphorylation sites located within the NS1 C-terminal region, T585 and S588. Although these substitutions had no detectable effect on virus multiplication in a single-round infection, the NS1-585A mutant virus was significantly less toxic to A9 cells than wild-type MVMp, whereas the NS1-588A mutant virus was endowed with a higher killing potential. These alterations correlated with specific changes in the late phosphorylation pattern of the mutant NS1 proteins compared to the wild-type polypeptide. Since the mutations introduced in this region of the viral genome also made changes in the minor nonstructural protein NS2, a contribution of this polypeptide to the above-mentioned phenotypes of mutant viruses cannot be excluded at present. However, the involvement of NS1 in these phenotypes was directly supported by the respective reduced and enhanced capacity of NS1-585A and NS1-588A recombinant proteins for inducing morphological alterations and cell detachment in transfected A9 cultures. Altogether, these data suggest that late-occurring phosphorylation of NS1 specifically regulates the cytotoxic functions of the viral product and that residues T585 and S588 contribute to this control in an antagonistic way.The autonomous parvovirus minute virus of mice (MVMp) is a small icosahedral particle with a linear single-stranded DNA (ssDNA) of 5.1 kb as a genome. MVMp replication depends on host cell factors specifically present in S phase, resulting in a restriction of its propagation in proliferating tissues. This feature is likely to contribute to the oncotropism displayed by this virus, although additional factors appear to be required (51). The viral DNA comprises two overlapping transcription units. The right-hand unit encodes two capsid proteins, VP1 and VP2, which are expressed under the control of the P38 promoter, while the left-hand unit, driven by the P4 promoter, produces the two nonstructural proteins NS1 and NS2. The large nonstructural protein NS1 is endowed with multiple functions necessary for progeny virus production, while the small nonstructural proteins NS2, expressed in three distinct isoforms according to their unique C termini, are dispensable for MVMp propagation in nonmurine cell types (15,35).The functions and regulation of the NS1 protein have been investigated in great detail. This protein combines multiple enzymatic and nonenzymatic functions, such as ATP binding and hydrolysis (6, 56)...

Section: Resultsmentioning

confidence: 99%

Modulation of Minute Virus of Mice Cytotoxic Activities through Site-Directed Mutagenesis within the NS Coding Region

Daeffler

Hörlein

Rommelaere

et al. 2003

“…The samples were fractionated by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide) and electrophoretically transferred to a nitrocellulose membrane in 20 mM Tris-HCl, 150 mM glycine, and 20% methanol (pH 8). The nitrocellulose membranes were blocked in PBS-0.1% Tween 20-5% nonfat dry milk for NS1, p34 cdc2 , p21 cip1 , p27 kip1 , and cyclins A, B, and E. The blots were incubated with a polyclonal rabbit antiserum raised against a bacterial fusion protein containing an MVMp-NS1-specific carboxy-terminal amino acid sequence (31) or with rabbit polyclonal antibody raised against cyclin A (H-432) (1:1,000 dilution, sc-741; Santa Cruz) or cyclin E (M-20) (1:1,000 dilution, sc-481; Santa Cruz). Mouse monoclonal antibodies were used to detect p34 cdc2 (IgG2a 17; 1:1,000 dilution; Santa Cruz sc-54), p33 cdk2 (polyclonal serum kindly provided by F. Hall), cyclin B1 (IgG1 GNS1; 1:1,000 dilution; Santa Cruz sc-245), and p27 kip1 (IgG1 K25020; 1:1,000 dilution; Transduction Laboratories; 1:1,000 dilution).…”

Section: Methodsmentioning

confidence: 99%

NS1- and Minute Virus of Mice-Induced Cell Cycle Arrest: Involvement of p53 and p21 ^cip1

Beeck

Sobczak-Thépot

Sirma

et al. 2001

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 cip1 . 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.

“…NS2 has more elusive functions and appears to be particularly important in certain cells for capsid assembly and release of progeny viruses (11,16). The viral effector of cytotoxicity has been assigned mainly to the nonstructural protein NS1 (6,31), though other viral products may also be involved (4,32). Completion of the viral life cycle requires the assistance of various cellular molecules; some of these have been identified, including transcription factors, cofactors, and other nuclear proteins.…”

mentioning

confidence: 99%

Expression Profiling of Human Hepatoma Cells Reveals Global Repression of Genes Involved in Cell Proliferation, Growth, and Apoptosis upon Infection with Parvovirus H-1

Werner

Hergenhahn

et al. 2005

Autonomous parvoviruses are small, nonenveloped, linear single-strand DNA viruses. Their 5-kb-long genome consists of two overlapping transcription units. An early promoter, P4, directs the expression of nonstructural proteins NS1 and NS2, and a late promoter, P38, controls the expression of capsid proteins VP1 and VP2 (12). Owing to their low genetic complexity, parvoviruses are tightly dependent on cellular factors that are expressed as a function of proliferation and differentiation in order to complete their life cycle (53, 60). Parvoviruses are incapable of inducing quiescent cells to enter S phase (66), and infection remains cryptic until host cells start a round of genomic DNA replication on their own. Once the appropriate cellular conditions are met, the virus starts its replication at the G 1 /S transition and a lytic or even productive infection can ensue (12,22), during which the infected cells get blocked in S/G 2 phase and eventually die (48, 53). The tight dependence of parvovirus replication on S-phase-associated factors accounts, in part, for the tissue specificity, oncotropism, and oncolytic activity of parvoviruses (55). In particular, MVMp and the closely related H-1 virus were found to replicate and exert cytopathic effects in a variety of transformed or tumorderived cells while sparing their normal counterparts in vitro. In vivo, these viruses may prevent tumors from appearing or cause the repression of established tumors, making them candidates for vectors in cancer therapy (9).The molecular mechanisms underlying parvovirus-host cell interactions were the objects of a number of recent studies. On the viral side, the nonstructural proteins NS1 and NS2 are key regulators of the virus life cycle. NS1 is a multifunctional protein that is endowed with a transcriptional function targeted to parvoviral but also heterologous promoters (18,31,54,67), and with enzymatic (ATPase, helicase [69], and site-specific nickase [44]) and site-specific DNA binding properties (10). NS1 thus plays critical roles during parvovirus replication and gene expression, starting from the earlier stages of the viral life cycle. NS2 has more elusive functions and appears to be particularly important in certain cells for capsid assembly and release of progeny viruses (11,16). The viral effector of cytotoxicity has been assigned mainly to the nonstructural protein NS1 (6, 31), though other viral products may also be involved (4, 32). Completion of the viral life cycle requires the assistance of various cellular molecules; some of these have been identified, including transcription factors, cofactors, and other nuclear proteins. CyclinA (2), parvovirus initiation factor (PIF) (8), and two members of the protein kinase C (PKC/) family play distinct roles in virus replication (28,45). The transcription factors E2F (15) and SP1 (27), the transcription coactivator CBP (47), and the basal transcriptional factors TBP and TFIIA(␣/␤) (36) help NS1 to trans-regulate target promoters * Corresponding author. Mailing address: Applied Tu...