Expression Of P33 Research Articles

The Intriguing Conundrum of a Nonconserved Multifunctional Protein of Citrus Tristeza Virus That Interacts with a Viral Long Non-Coding RNA.

Citrus tristeza virus (CTV), the largest non-segmented plant RNA virus, has several peculiar features, among which is the production of a 5′-terminal long non-coding RNA (lncRNA) termed low-molecular-weight tristeza 1 (LMT1). In this study, we found that p33, a unique viral protein that performs multiple functions in the virus infection cycle, specifically binds LMT1, both in vivo and in vitro. These results were obtained through the expression of p33 under the context of the wild type virus infection or along with a mutant CTV variant that does not produce LMT1 as well as via ectopic co-expression of p33 with LMT1 in Nicotiana benthamiana leaves followed by RNA immunoprecipitation and rapid amplification of cDNA ends assays. Further experiments in which a recombinant p33 protein and an in vitro transcribed full-length LMT1 RNA or its truncated fragments were subjected to an electrophoretic mobility shift assay demonstrated that p33 binds to at least two distinct regions within LMT1. To the best of our knowledge, this is the first report of a plant virus protein binding to a lncRNA produced by the same virus. The biological significance of the interaction between these two viral factors is discussed.

Human inhibitor of growth 1 inhibits hepatoma cell growth and influences p53 stability in a variant-dependent manner #

Inhibitor of growth 1 (ING1) is a type II tumor suppressor that affects cell function by altering chromatin structure and regulating transcription. Recently, three ING1 splice variants have been cloned, but their roles in apoptosis and p53 regulation in human hepatocellular carcinoma (HCC) have not been fully elucidated. The present study found that ING1, in a variant-dependent manner, inhibited hepatoma cell proliferation and colony formation, induced apoptosis and cell cycle arrest at G(0)/G(1) phase, and postponed tumor formation in nude mice. Expression of p33(ING1b) and p24(ING1c) variants, but not p47(ING1a), was markedly reduced in HCC samples. Reverse transcription polymerase chain reaction and western blotting analysis revealed that ectopic overexpression of p33(ING1b) or p24(ING1c) variant increased the expression of p53 downstream genes such as p21(waf1) and bax, and repressed bcl-2 expression (P < 0.01), whereas p47(ING1a) inactivated p21(waf1) promoter (P < 0.01). Furthermore, we found that p33(ING1b) and p24(ING1c) repressed Mdm2 expression (P < 0.01) and competed with Mdm2 for binding to p53. Interestingly, p33(ING1b)and p24(ING1c) did not directly bind to Mdm2 protein but strongly increased p14(arf) expression (P < 0.01) and interacted with p14(arf) protein to stimulate p53. Moreover, we found that ectopic overexpression of p33(ING1b) or p24(ING1c) significantly induced p53 protein acetylation at Lys-373/Lys-382 residue, but did not alter the phosphorylation status of p53. ING1 variants p33(ING1b) and p24(ING1c) may modulate p53 activity and subsequently inhibit hepatoma cell growth by at least two possible mechanisms: interacting with Mdm2 and p14(arf) to stabilize and activate p53, or increasing p53 acetylation.

Cytological analysis of Saccharomyces cerevisiae cells supporting cymbidium ringspot virus defective interfering RNA replication

The replicase proteins p33 and p92 of Cymbidium ringspot virus (CymRSV) were found to support the replication of defective interfering (DI) RNA in Saccharomyces cerevisiae cells. Two yeast strains were used, differing in the biogenesis of peroxisomes, the organelles supplying the membranous vesicular environment in which CymRSV RNA replication takes place in infected plant cells. Double-labelled immunofluorescence showed that both p33 and p92 replicase proteins localized to peroxisomes, independently of one another and of the presence of the replication template. It is suggested that these proteins are sorted initially from the cytosol to the endoplasmic reticulum and then to peroxisomes. However, only the expression of p33, but not p92, increased the number of peroxisomes and induced membrane proliferation. DI RNA replication occurred in yeast cells, as demonstrated by the presence of monomers and dimers of positive and negative polarities. Labelling with BrUTP showed that peroxisomes were the sites of nascent viral synthesis, whereas in situ hybridization indicated that DI RNA progeny were diffused throughout the cytoplasm. DI RNA replication also took place in yeast cells devoid of peroxisomes. It is suggested that replication in these cells was targeted to the endoplasmic reticulum.

Expression and sequence analyses of p33(ING1) gene in myeloid leukemia.

p33(ING1) is a novel candidate tumor suppressor gene which is involved in the regulation of apoptosis. p33(ING1) interacts with p53 signaling pathway and regulates cellular growth. It has reported that the expression of p33(ING1) mRNA was decreased in lymphoid malignancies. We thus investigated the potential involvement of p33(ING1) abnormalities in myeloid leukemias. However, the levels of p33(ING1) transcript were almost equal in 3 AML cell lines and 10 fresh AML samples. In addition, neither point mutations nor deletions in p33(ING1) gene were found in myeloid leukemias. These results suggest that p33(ING1) may not be a major candidate tumor suppressor gene in myeloid leukemias.

The ORF1 products of tombusviruses play a crucial role in lethal necrosis of virus-infected plants.

Hybrids of cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. Surprisingly, symptoms of Nicotiana benthamiana infected with CymRSV/CIRV hybrids were distinctly different. It was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents CymRSV and CIRV did. We showed here that hybrids containing chimeric ORF1 were not able to induce lethal necrosis even if the viral replication of these constructs was not altered significantly. However, if a wt p33 (product of ORF1) of CymRSV was provided in trans in transgenic plants expressing p33 and its readthrough product p92, the lethal necrosis characteristic to tombusvirus infection was restored. In addition, the expression of p33 by a potato virus X viral vector in N. benthamiana caused severe chlorosis and occasionally necrosis, indicating the importance of p33 in wt symptoms of tombusviruses. Thus, our results provide evidence that elicitation of the necrotic phenotype requires the presence of the wt p33 in addition to the p19 protein of tombusviruses.

Expression of major piroplasm protein (p33) of Theileria sergenti (Korean isolate) and its immunogenicity in guinea pigs.

To investigate the development of a subunit vaccine against theileriosis in cattle, the DNA fragments encoding piroplasm surface protein (p33) of Theileria sergenti of a Korean isolate were expressed in baculoviruses. The expressed p33 was characterized by indirect fluorescent antibody (IFA) and western blotting analysis. The expression of p33 was mainly detected on the surface of infected Sf21 cells by IFA. The immunoblotting analysis revealed the presence of a same molecular weight protein band of p33. The antigenicity of expressed polypeptide was further examined through the inoculation of a guinea pig. The sera of guinea pigs immunized with p33 expressed cell lysate showed similar fluorescent antibody patterns and reacted with the same molecular weight protein of T. sergenti in immunoblotting analysis, thus indicating that this protein can be a promising candidate for a subunit vaccine in the future.