Gender and BCR-ABL transcript type are correlated with molecular response to imatinib treatment in patients with chronic myeloid leukemia.

OBJECTIVES: Achieving a major molecular response (MMR) is the goal of imatinib therapy for chronic myeloid leukemia. However, the association between gender, BCR-ABL transcript type, and age with MMR is not well understood and often controversial. METHODS: We retrospectively analyzed 166 patients who have been treated with imatinib for up to 10 yr. RESULTS: Men had a lower MMR rate than women (63.3% vs. 81.6%, P = 0.006) and a shorter time to relapse (median 354 vs. 675 d, P = 0.049), while patients with b3a2 or with both b3a2 and b2a2 break point transcripts had higher MMR rate than those with b2a2 (81.8%, 77.1% vs. 60.7%, P = 0.023 for b3a2 vs. b2a2, P = 0.043 for both vs. b2a2). A striking difference was found between men with b2a2 and women with both b2a2 and b3a2 in terms of MMR rate (43.8% vs. 88.9%), MMR rate within 6 months (7.1% vs. 62.5%) and the time to MMR (median d 493 vs. 159, P = 0.036). CONCLUSIONS: Both gender and BCR-ABL transcript, but not age, were significantly associated with the molecular response. Men with b2a2 represent a less favorable group in their response to imatinib treatment and may need alternative therapy regimen and closer monitoring.

Expression of xCT and activity of system xc(-) are regulated by NRF2 in human breast cancer cells in response to oxidative stress.

Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate by activating key transcription factors. One such master regulator, the redox sensitive transcription factor NF E2 Related Factor 2 (NRF2), controls the expression of cellular defense genes including those encoding intracellular redox-balancing proteins involved in glutathione (GSH) synthesis. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) targets NRF2 for ubiquitination. In response to oxidative stress, NRF2 dissociates from KEAP1, entering the nucleus and binding to the antioxidant response element (ARE) in the promoter of its target genes. Elevated reactive oxygen species (ROS) production may deplete GSH levels within cancer cells. System xc(-), an antiporter that exports glutamate while importing cystine to be converted into cysteine for GSH synthesis, is upregulated in cancer cells in response to oxidative stress. Here, we provided evidence that the expression of xCT, the light chain subunit of system xc(-), is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H2O2) treatment increased nuclear translocation of NRF2, also increasing levels of xCT mRNA and protein and extracellular glutamate release. Overexpression of NRF2 up-regulated the activity of the xCT promoter, which contains a proximal ARE. In contrast, overexpression of KEAP1 repressed promoter activity and decreased xCT protein levels, while siRNA knockdown of KEAP1 up-regulated xCT protein levels and transporter activity. These results demonstrate the importance of the KEAP1/NRF2 pathway in balancing oxidative stress in breast cancer cells through system xc(-). We have previously shown that xCT is upregulated in various cancer cell lines under oxidative stress. In the current investigation, we focused on MCF-7 cells as a model for mechanistic studies.

[Sequence analysis of RNA3 of Rice stripe virus isolates found in China: evidence for reassortment in Tenuivirus].

The RNA3 segments of four isolates of Rice stripe virus (RSV), isolated from endemic sites at Panjin (PJ), Liaoning Province, Kunming (KM) and Yiliang (YL), Yunnan Province, as well as from outbreak sites at Hongze (HZ), Jiangsu Province, were determined. RNA3 of these four isolates were 2480 bp, 2509 bp, 2489 bp and 2497 bp in length, respectively. Compared with RNA3 of T and M isolates from Japan and Y isolate from Yunnan Province of China, that had been previously reported, these seven isolates could be divided into two groups. KM and YL isolates formed group one, and PJ, HZ, Y, T and M isolates belonged to another group. The two groups shared 97%-98% and 93%-94% sequence homology in viral RNA3 (vRNA3) and viral complementary RNA3 (vcRNA3) at the nucleotide levels, respectively, and there was no significant difference between the two groups at the amino acid levels. In the first group, Y isolate was significantly different from HZ,PJ and two Japan isolates in their RNA4 segment. These results show that there were two subgroups in RSV natural population related with geographical location, and reassorment may be the main factor leading to different segments of Y isolate belonging to different subgroups. The results may provide another evidence for reassortment variation in Tenuivirus.

Characterization of the spike protein of human coronavirus NL63 in receptor binding and pseudotype virus entry.

The spike (S) protein of human coronavirus NL63 (HCoV-NL63) mediates both cell attachment by binding to its receptor hACE2 and membrane fusion during virus entry. We have previously identified the receptor-binding domain (RBD) and residues important for RBD-hACE2 association. Here, we further characterized the S protein by investigating the roles of the cytoplasmic tail and 19 residues located in the RBD in protein accumulation, receptor binding, and pseudotype virus entry. For these purposes, we first identified an entry-efficient S gene template from a pool of gene variants and used it as a backbone to generate a series of cytoplasmic tail deletion and single residue substitution mutants. Our results showed that: (i) deletion of 18aa from the C-terminus enhanced the S protein accumulation and virus entry, which might be due to the deletion of intracellular retention signals; (ii) further deletion to residue 29 also enhanced the amount of S protein on the cell surface and in virion, but reduced virus entry by 25%, suggesting that residues 19-29 contributes to membrane fusion; (iii) a 29aa-deletion mutant had a defect in anchoring on the plasma membrane, which led to a dramatic decrease of S protein in virion and virus entry; (iv) a total of 15 residues (Y498, V499, V531, G534, G537, D538, S540, G575, S576, E582, W585, Y590, T591, V593 and G594) within RBD were important for receptor binding and virus entry. They probably form three receptor binding motifs, and the third motif is conserved between NL63 and SARS-CoV.

Genetic diversity and population structure of rice stripe virus in China.

Rice stripe virus (RSV) is one of the most economically important pathogens of rice and is repeatedly epidemic in China, Japan and Korea. The most recent outbreak of RSV in eastern China in 2000 caused significant losses and raised serious concerns. In this paper, we provide a genotyping profile of RSV field isolates and describe the population structure of RSV in China, based on the nucleotide sequences of isolates collected from different geographical regions during 1997-2004. RSV isolates could be divided into two or three subtypes, depending on which gene was analysed. The genetic distances between subtypes range from 0.050 to 0.067. The population from eastern China is composed only of subtype I/IB isolates. In contrast, the population from Yunnan province (southwest China) is composed mainly of subtype II isolates, but also contains a small proportion of subtype I/IB isolates and subtype IA isolates. However, subpopulations collected from different districts in eastern China or Yunnan province are not genetically differentiated and show frequent gene flow. RSV genes were found to be under strong negative selection. Our data suggest that the most recent outbreak of RSV in eastern China was not due to the invasion of new RSV subtype(s). The evolutionary processes contributing to the observed genetic diversity and population structure are discussed.

Identification of residues in the receptor-binding domain (RBD) of the spike protein of human coronavirus NL63 that are critical for the RBD-ACE2 receptor interaction.

Human coronavirus NL63 (NL63), a member of the group I coronaviruses, may cause acute respiratory diseases in young children and immunocompromised adults. Like severe acute respiratory syndrome coronavirus (SARS-CoV), NL63 also employs the human angiotensin-converting enzyme 2 (hACE2) receptor for cellular entry. To identify residues in the spike protein of NL63 that are important for hACE2 binding, this study first generated a series of S1-truncated variants, examined their associations with the hACE2 receptor and subsequently mapped a minimal receptor-binding domain (RBD) that consisted of 141 residues (aa 476-616) towards the C terminus of the S1 domain. The data also demonstrated that the NL63 RBD bound to hACE2 more efficiently than its full-length counterpart and had a binding efficiency comparable to the S1 or RBD of SARS-CoV. A further series of RBD variants was generated using site-directed mutagenesis and random mutant library screening assays, and identified 15 residues (C497, Y498, V499, C500, K501, R518, R530, V531, G534, G537, D538, S540, E582, W585 and T591) that appeared to be critical for the RBD-hACE2 association. These critical residues clustered in three separate regions (designated RI, RII and RIII) inside the RBD, which may represent three receptor-binding sites. These results may help to delineate the molecular interactions between the S protein of NL63 and the hACE2 receptor, and may also enhance our understanding of the pathogenesis of NL63 and SARS-CoV.

A multicomponent RNA-based control system regulates subgenomic mRNA transcription in a tombusvirus.

During infections, positive-strand RNA tombusviruses transcribe two subgenomic (sg) mRNAs that allow for the expression of a subset of their genes. This process is thought to involve an unconventional mechanism involving the premature termination of the virally encoded RNA-dependent RNA polymerase while it is copying the virus genome. The 3' truncated minus strands generated by termination are then used as templates for sg mRNA transcription. In addition to requiring an extensive network of long-distance RNA-RNA interactions (H.-X. Lin and K. A. White, EMBO J. 23:3365-3374, 2004), the transcription of tombusvirus sg mRNAs also involves several additional RNA structures. In vivo analysis of these diverse RNA elements revealed that they function at distinct steps in the process by facilitating the formation or stabilization of the long-distance interactions, modulating minus-strand template production, or promoting the initiation of sg mRNA transcription. All of the RNA elements characterized could be readily incorporated into a premature termination model for sg mRNA transcription. Overall, the analyses revealed a complex system that displays a high level of structural integration and functional coordination. This multicomponent RNA-based control system may serve as a useful paradigm for understanding related transcriptional processes in other positive-sense RNA viruses.

The p92 polymerase coding region contains an internal RNA element required at an early step in Tombusvirus genome replication.

The replication of positive-strand RNA viral genomes involves various cis-acting RNA sequences. Generally, regulatory RNA sequences are present at or near genomic termini; however, internal replication elements (IREs) also exist. Here we report the structural and functional characterization of an IRE present in the readthrough portion of the p92 polymerase gene of Tomato bushy stunt virus. Analysis of this element in the context of a noncoding defective interfering RNA revealed a functional core structure composed of two noncontiguous segments of sequence that interact with each other to form an extended helical conformation. IRE activity required maintenance of several base-paired sections as well as two distinct structural features: (i) a short, highly conserved segment that can potentially form two different and mutually exclusive structures and (ii) an internal loop that contains a critical CC mismatch. The IRE was also shown to play an essential role within the context of the viral genome. In vivo analysis with novel RNA-based temperature-sensitive genomic mutants and translationally active subgenomic viral replicons revealed the following about the IRE: (i) it is active in the positive strand, (ii) it is dispensable late in the viral RNA replication process, and (iii) it is functionally inhibited by active translation over its sequence. Together, these results suggest that IRE activity is required in the cytosol at an early step in the viral replication process, such as template recruitment and/or replicase complex assembly.

A complex network of RNA-RNA interactions controls subgenomic mRNA transcription in a tombusvirus.

Eukaryotic (+)-strand RNA viruses utilize a wide variety of gene expression strategies to achieve regulated production of their viral proteins. A common mechanism used by many is to transcribe viral subgenomic (sg) mRNAs. Transcription of sg mRNA2 in tombusviruses allows for expression of the p19 suppressor of gene silencing and p22 movement proteins. We have investigated the mechanism of transcription of this sg mRNA in Tomato bushy stunt virus and have determined that this process is facilitated by no less than three different RNA modules that are located throughout the viral genome. These RNA units perform distinct tasks and function via long-distance RNA-RNA interactions. Systematic deconstruction of the RNA network and analysis of related RNA promoter elements allowed us to identify fundamental properties necessary for productive sg mRNA2 transcription. Collectively, our results (i) establish specific roles for the different RNA components of a multipartite RNA-based control system, (ii) support a premature termination mechanism for tombusvirus sg mRNA transcription and (iii) reveal a close mechanistic relationship between sg mRNA transcription, viral RNA replication and RNA recombination.

Genetic diversity and biological variation among California isolates of Cucumber mosaic virus.

Genetic diversity and biological variation were compared for California isolates of Cucumber mosaic virus (CMV). These fell into five pathotypes based on their reactions on three cucurbits including a susceptible squash, a melon with conventional resistance and a commercial CMV-resistant transgenic squash. Thirty-three isolates infected and caused symptoms on CMV-resistant transgenic squash. Forty-two isolates infected the CMV-resistant melon, but only 25 isolates infected both. Single-strand conformation polymorphism (SSCP) analysis was used to differentiate 81 California isolates into 14 groups, and the coat protein (CP) genes of 27 isolates with distinct and indistinguishable SSCP patterns were sequenced. Fourteen isolates corresponding to the different SSCP patterns were also used for phylogenetic analysis. Seventy-nine isolates belonged to CMV subgroup IA, but two belonged to CMV subgroup IB. This is the first report of subgroup IB isolates in the Americas. All CMV isolates had a nucleotide identity greater than or equal to 93.24 %. There was no correlation between CP gene variation and geographical origin, collection year, original host plant, or between the degree of CP amino acid sequence identity and the capacity to overcome transgenic and/or conventional resistance. SSCP and sequence analyses were used to compare 33 CMV isolates on CMV-resistant transgenic squash and susceptible pumpkin plants. One isolate showed sequence differences between these two hosts, but this was not due to recombination or selection pressure of transgenic resistance. CMV isolates capable of infecting cucurbits with conventional and transgenic CMV resistance were present in California, even before CMV transgenic material was available.

Molecular population genetics of Cucumber mosaic virus in California: evidence for founder effects and reassortment.

The structure and genetic diversity of a California Cucumber mosaic virus (CMV) population was assessed by single-strand conformation polymorphism and nucleotide sequence analyses of genomic regions 2b, CP, MP, and the 3' nontranslated region of RNA3. The California CMV population exhibited low genetic diversity and was composed of one to three predominant haplotypes and a large number of minor haplotypes for specific genomic regions. Extremely low diversity and close evolutionary relationships among isolates in a subpopulation suggested that founder effects might play a role in shaping the genetic structure. Phylogenetic analysis indicated a naturally occurring reassortant between subgroup IA and IB isolates and potential reassortants between subgroup IA isolates, suggesting that genetic exchange by reassortment contributed to the evolution of the California CMV population. Analysis of various population genetics parameters and distribution of synonymous and nonsynonymous mutations revealed that different coding regions and even different parts of coding regions were under different evolutionary constraints, including a short region of the 2b gene for which evidence suggests possible positive selection.

Geographically distant isolates of the crinivirus Cucurbit yellow stunting disorder virus show very low genetic diversity in the coat protein gene.

The population structure and genetic variation of Cucurbit yellow stunting disorder virus (CYSDV) isolates were estimated by single-strand conformation polymorphism and nucleotide sequence analyses of the CYSDV coat protein gene. Analysis of 71 isolates collected from Spain, Jordan, Turkey, Lebanon, Saudi Arabia and North America showed that, from a genetic viewpoint, these isolates could be divided into two diverged subpopulations: an Eastern subpopulation composed of Saudi Arabian isolates and a Western subpopulation containing the rest of the CYSDV isolates. The genetic variation within the Western subpopulation was very small (nucleotide identity >99%) in spite of the extensive and discontinuous geographical distribution and different years of collection. We also estimated the within-isolate genetic structure and variation of three CYSDV isolates by analysing 30 clones per isolate. Our results showed that these CYSDV isolates had a quasispecies structure.