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The genome of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D), which is the most extensively used virus pesticide in the world, was completely sequenced and shown to have 132 239 bp (G+C content 44?5 mol%) and to be capable of encoding 152 non-overlapping open reading frames (ORFs). Three ORFs were unique to AgMNPV-2D, one of which (ag31) had similarity to eukaryotic poly(ADP-ribose) polymerases. The lack of chiA and v-cath may explain some of the success and growth of the AgMNPV biological control programme, as it may explain the high recovery of polyhedra sequestered inside dead larvae in the field, which are collected and used for further application as biological pesticides in soybean fields. The genome organization was similar to that of the Choristoneura fumiferana defective MNPV (CfDefNPV). Most of the variation between the two genomes took place near highly repetitive regions, which were also closely associated with bro-coding regions. The separation of the NPVs into groups I and II was supported by: (i) a phenogram of the complete genomes of 28 baculovirus and Heliothis zea virus 1, (ii) the most parsimonious reconstruction of gene content along the phenograms and (iii) comparisons of genomic features. Moreover, these data also reinforced the notion that group I of the NPVs can be split further into the AgMNPV lineage (AgMNPV, CfDefNPV, Epiphyas postvittana NPV, Orgyia pseudotsugata MNPV and C. fumiferana MNPV), sharing eight defining genes, and the Autographa californica MNPV (AcMNPV) lineage (AcMNPV, Rachiplusia ou NPV and Bombyx mori NPV), sharing nine defining genes.

Ectopic expression of conditional murine p53 (p53val135) and oncogenic ras is enough to induce a senescent-like growth arrest at the restrictive temperature. We took advantage of this cellular system to identify new key players in the ras/p53-induced senescence. Applying a retroviral-based genetic screen, we obtained an antisense RNA fragment against PPP1CA, the catalytic subunit of protein phosphatase 1alpha, whose loss of function bypasses ras/p53-induced growth arrest and senescence. Expression of a specific short hairpin (sh)RNA against PPP1CA impairs the p53-dependent induction of p21 after DNA damage and blocks the subsequent pRb dephosphorylation, thus bypassing p53-induced arrest. We found that oncogenic ras promotes an increase in the intracellular level of ceramides together with an increase in the PPP1CA protein levels. Addition of soluble ceramide to the cells induced a senescence phenotype that is blocked through PPP1CA downregulation by specific shRNA. Analysis of human tumors suggests that one of the PPP1CA alleles might be lost in a high percentage of carcinomas such as kidney and colorectal. The overexpression of two out of five PPP1CA alternative spliced variants reduced tumor cell growth and the downregulation of the protein to hemizygosity increased the anchorage-independent growth. We propose that oncogenic stress induced by ras causes ceramide accumulation, therefore, increasing PPP1CA activity, pRb dephosphorylation and onset of the p53-induced arrest, contributing to tumor suppression.

Senescence is a mechanism that limits cellular lifespan and constitutes a barrier against cellular immortalization. To identify new senescence regulatory genes that might play a role in tumorigenesis, we have designed and performed a large-scale antisense-based genetic screen in primary mouse embryo fibroblasts (MEFs). Out of this screen, we have identified five different genes through which loss of function partially bypasses senescence. These genes belong to very different biochemical families: csn2 (component of the Cop9 signalosome), aldose reductase (a metabolic enzyme) and brf1 (subunit of the RNA polymerase II complex), S-adenosyl homocysteine hydrolase and Bub1. Inactivation, at least partial, of these genes confers resistance to both p53-and p16INK4a-induced proliferation arrest. Furthermore, such inactivation inhibits p53 but not E2F1 transcriptional activity and impairs DNA-damage-induced transcription of p21. Since the aim of the screen was to identify new regulators of tumorigenesis, we have tested their inactivation in human tumors. We have found, either by northern blot or quantitative reverse transcriptase-PCR analysis, that the expression of three genes, Csn2, Aldose reductase and Brf1, is lost at different ratios in tumors of different origins. These genes are located at common positions of loss of heterogeneity (15q21.2, 7q35 and 14q32.33); therefore,we have measured genomic losses of these specific genes in different tumors. We have found that Csn2 and Brf1 also show genomic losses of one allele in different tumors. Our data suggest that the three genes identified in the genome-wide loss-of-function genetic screen are putative tumor suppressors located at 15q21.2; 7q35 and 14q32.33.

The caterpillar Pseudoplusia includens (Walker, 1857) (Lepidoptera, Noctuidae), known as soybean looper, is a pest that has recently assumed greater importance in soybean in Brazil. Isolates of nucleopolyhedroviruses (NPVs) of this pest have been identified from cotton in Guatemala and soybean farms in Brazil, providing an interesting perspective of potential use of viral insecticide against the insect in lieu to chemical insecticides. With the objective to contribute to the characterization studies of this virus, morphological and molecular analyses and biological activity were carried out with seven P. includens viral isolates (I-A to I-G). Electron microscopy of viral samples, purified from macerated infected larvae, showed particles with typical morphology of the Baculoviridae family, genus Alphabaculovirus (Nucleopolyhedrovirus - NPV) presenting virions with only a single nucleocapsid per envelope (SNPV) occluded in a protein matrix, forming occlusion bodies (OB). This virus was then classified as P. includens single nucleopolyhedrovirus (PsinSNPV). OB particles analyzed in SDS-polyacrylamide gel showed an intense band corresponding in size to NPV polyhedrin protein. DNA restriction profiles of the PsinSNPV isolates showed differences in the fragment size and number suggesting the existence of genotypic variants, except between I-E and I-F profiles that were similar. Among the isolates tested for infectivity against P. includens, I-A, I-E and I-F were the most virulent. Survival times (ST(50)) varied according to viral concentration, with significant differences among isolates for the three higher concentrations.

Although activated macrophages destroy cancer cells more effectively than normal cells, the ability to escape activated macrophages is a characteristic of tumor cells. One of the mechanisms responsible for the specific killing of tumor cells by macrophages is the production of the cytokine tumor necrosis factor (TNF) alpha. Therefore, resistance to TNF may provide such cancer cells a selective advantage against host elimination. With the aim of identifying genes with these properties we undertook a large scale genetic screen to identify genes able to bypass TNF-induced G1 arrest. We identified MAP17, a small 17 kDa nonglycosylated membrane protein that localizes to the plasma membrane and the Golgi apparatus. Ectopic expression of MAP17 in tumor cells prevents TNF-induced G1 arrest by impairing p21waf1 induction. However, expression of MAP17 does not inhibit TNF-induced apoptosis in Me180-sensitive tumor cells. The inhibition of TNF is specific since MAP17 does not alter the response to other cytokines such as IFNgamma. As described in the Xenopus oocyte system, MAP17 increases the uptake of mannose in some cells, but this effect is not responsible for TNF bypass. We have also analyzed the expression of MAP17 mRNA in a panel of cell lines. MAP17 is expressed in 30% of cell lines of different origin. However, MAP17 mRNA expression did not correlate with TNF resistance. Our data indicates that although MAP17 expression might bypass TNF-induced growth arrest, it is not the only determinant of this response.

Programmed cell death or apoptosis is one of the defense mechanisms used by insect cells in response to baculovirus infection. Baculoviruses harbour antiapoptotic genes to prevent apoptosis and to maintain the normal course of infection. In this work, we showed that, like other baculoviruses, Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) has a functional inhibitor of apoptosis gene (iap-3). The iap-3 gene was cloned, sequenced and its transcription confirmed by RT-PCR. The putative iap-3 gene of the baculovirus AgMNPV has 864 nucleotides and codes an ORF of 287 amino acids. We have found two BIR motifs (baculoviral iap repeats) at the amino-terminal region and a carboxi-terminal RING finger motif. The IAP-3 protein of AgMNPV is closely related to IAP-3 proteins of baculoviruses and lepidopteran IAPs, with most amino acid identity (75%) with the IAP-3 protein of CfDefNPV (Choristoneura fumiferana DEF nucleopolyhedrovirus). Transcriptional analysis of the AgMNPV iap-3 gene showed that iap-3-specific transcripts could be detected early and late in the infection. The iap-3 gene of AgMNPV was shown to encode a functional IAP since insect cells transfected with increasing amounts of a plasmid containing the iap-3 of AgMNPV showed increased resistance to apoptosis induced by a AgMNPV mutant virus.

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