Predicting Statistical Properties of Open Reading Frames in Bacterial Genomes

An analytical model based on the statistical properties of Open Reading Frames (ORFs) of eubacterial genomes such as codon composition and sequence length of all reading frames was developed. This new model predicts the average length, maximum length as well as the length distribution of the ORFs of 70 species with GC contents varying between 21% and 74%. Furthermore, the number of annotated genes is predicted with high accordance. However, the ORF length distribution in the five alternative reading frames shows interesting deviations from the predicted distribution. In particular, long ORFs appear more often than expected statistically. The unexpected depletion of stop codons in these alternative open reading frames cannot completely be explained by a biased codon usage in the +1 frame. While it is unknown if the stop codon depletion has a biological function, it could be due to a protein coding capacity of alternative ORFs exerting a selection pressure which prevents the fixation of stop codon mutations. The comparison of the analytical model with bacterial genomes, therefore, leads to a hypothesis suggesting novel gene candidates which can now be investigated in subsequent wet lab experiments.     

Varicella-Zoster Virus Open Reading Frame 48 Encodes an Active Nuclease

Based on a DNA sequence and relative genomic position similar to those other herpesviruses, varicella-zoster virus (VZV) open reading frame 48 (ORF48) is predicted to encode an alkaline nuclease. Here we report the cloning, expression, purification, and characterization of recombinant VZV ORF48 protein and a VZV ORF48 point mutation (T172P). Protein encoded by wild-type ORF48, but not mutant protein, displayed both endo- and exonuclease activity, identifying ORF48 as a potential therapeutic target in VZV disease since efficient viral replication requires viral nuclease activity.     

The UL2 Open Reading Frame of Bovine Herpesvirus 1 Encodes a Uracil-DNA Glycosylase

Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV-1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil-DNA glycosylase (UDGase) signature sequence. To determine whether the BHV-1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence downstream of the T7 promoter on the vector pET-28b(+) and expressed it in Escherichia coli. Upon induction with isopropyl β-D -thiogalactopyranoside these cells produced a 23-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using nickel-chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay. These results provide evidence that the BHV-1 UL2 gene does encode a UDGase.     

Long Open Reading Frame Transcripts Escape Nonsense-Mediated mRNA Decay in Yeast

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我国两个不同地区猪繁殖和呼吸综合征病毒分离株ORF5基因序列比例

猪繁殖和呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)为动脉炎病毒科成员之一,可引起感染母猪流产、死胎及断奶仔猪呼吸困难和死亡.该病毒呈球形,有囊膜,大小45nm～83nm,基因组为单股RNA,大小约15kb,有8个阅读框(ORF),分别编码2种非结构蛋白和6种结构蛋白,其中ORF5编码病毒糖基化膜蛋白(GP5)[1,2].GP5蛋白为该病毒主要结构蛋白之一,含有病毒线性中和抗原表位.该蛋白可诱导感染细胞发生细胞凋亡[3,4].目前,PRRSV有欧洲型和美州型两个血清型,其结构蛋白基因同源性为54%～70%[5,6].不同美洲型PRRSV野毒株基因也有一定差异.由ORF5基因推导的氨基酸序列有4个相对保守区,但其N端和C端氨基酸残基可变性较大.由该基因构建的重组质粒具有良好免疫原性[7,8].尽管一些欧美国家已普遍使用Resp PRRS弱毒疫苗,但该病仍时有发生[9,10].我国于1996年亦已证实存在该病,并有不断蔓延趋势,已造成我国养猪业严重经济损失.     

Posttranscriptional Regulation by the Upstream Open Reading Frame of the Phosphoethanolamine N-Methyltransferase Gene

Phosphoethanolamine N-methyltransferase (PEAMT) is involved in choline biosynthesis in plants. The 5′ untranslated region (UTR) of several PEAMT genes was found to contain an upstream open reading frame (uORF). We generated transgenic Arabidopsis calli that expressed a chimeric gene constructed by fusing the 5′ UTR of the Arabidopsis PEAMT gene (AtNMT1) upstream of the β-glucuronidase gene. The AtNMT1 uORF was found to be involved in declining levels of the chimeric gene mRNA and repression of downstream β-glucuronidase gene translation in the calli when the cells were treated with choline. Further, we discuss the role of the uORF.     

人乳头瘤病毒16型晚期蛋白质基因L1的克隆及表达

人乳头瘤病毒16型(HPV16)含有两个晚期开放读码框(L_1ORF和L_2ORF),L_1ORF编码主要衣壳蛋白,我们用质粒pHPV16、p16L_2BX_5和pATH,采用基因重组技术,制备了含有HPV16个长L_1ORF序列的基因克隆p16L_1BN(5071—253),它能在大肠杆菌中有效表达,产生分子量约90KD的含有E. coli trpE的融合蛋白。Western blot检测,该蛋白可被抗牛乳头瘤病毒的抗体识别,这说明克隆p16L_1BN能有效表达,基因产物具有乳头瘤病毒型的共同抗原的性质。     

Open Reading Frame III of Borna Disease Virus Encodes a Nonglycosylated Matrix Protein

The open reading frame III of Borna disease virus (BDV) codes for a protein with a mass of 16 kDa, named p16 or BDV-M. p16 was described as an N-glycosylated protein in several previous publications and therefore was termed gp18, although the amino acid sequence of p16 does not contain any regular consensus sequence for N glycosylation. We examined glycosylation of p16 and studied its membrane topology using antisera raised against peptides, which comprise the N and the C termini. Neither an N- nor a C-terminal peptide is cleaved from p16 during maturation. Neither deglycosylation of p16 by endoglycosidases nor binding of lectin to p16 was detectable. Introduction of typical N-glycosylation sites at the proposed sites of p16 failed in carbohydrate attachment. Flotation experiments with membranes of BDV-infected cells on density gradients revealed that p16 is not an integral membrane protein, since it can be dissociated from membranes. Our experimental data strongly suggest that p16 is a typical nonglycosylated matrix protein associated at the inner surface of the viral membrane, as is true for homologous proteins of other members of the Mononegavirales order.     

The Network Architecture of the Saccharomyces cerevisiae Genome

We propose a network-based approach for surmising the spatial organization of genomes from high-throughput interaction data. Our strategy is based on methods for inferring architectural features of networks. Specifically, we employ a community detection algorithm to partition networks of genomic interactions. These community partitions represent an intuitive interpretation of genomic organization from interaction data. Furthermore, they are able to recapitulate known aspects of the spatial organization of the Saccharomyces cerevisiae genome, such as the rosette conformation of the genome, the clustering of centromeres, as well as tRNAs, and telomeres. We also demonstrate that simple architectural features of genomic interaction networks, such as cliques, can give meaningful insight into the functional role of the spatial organization of the genome. We show that there is a correlation between inter-chromosomal clique size and replication timing, as well as cohesin enrichment. Together, our network-based approach represents an effective and intuitive framework for interpreting high-throughput genomic interaction data. Importantly, there is a great potential for this strategy, given the rich literature and extensive set of existing tools in the field of network analysis.     

The Hepatitis E Virus Open Reading Frame 3 Product Interacts with Microtubules and Interferes with Their Dynamics

Hepatitis E virus (HEV) is the causative agent of hepatitis E, a major form of viral hepatitis in developing countries. The open reading frame 3 (ORF3) of HEV encodes a phosphoprotein with a molecular mass of approximately 13 kDa (hereinafter called vp13). vp13 is essential for establishing HEV infections in animals, yet its exact functions are still obscure. Our current study found evidence showing interaction between vp13 and microtubules. Live-cell confocal fluorescence microscopy revealed both filamentous and punctate distribution patterns of vp13 in cells transfected with recombinant ORF3 reporter plasmids. The filamentous pattern of vp13 was altered by a microtubule-destabilizing drug. The vp13 expression led to elevation of acetylated α-tubulin, indicating increased microtubule stability. Its association with microtubules was further supported by its presence in microtubule-containing pellets in microtubule isolation assays. Exposure of these pellets to a high-salt buffer caused release of the vp13 to the supernatant, suggesting an electrostatic interaction. Inclusion of ATP and GTP in the lysis buffer during microtubule isolation also disrupted the interaction, indicating its sensitivity to the nucleotides. Further assays showed that motor proteins are needed for the vp13 association with the microtubules because disruption of dynein function abolished the vp13 filamentous pattern. Analysis of ORF3 deletion constructs found that both of the N-terminal hydrophobic domains of vp13 are needed for the interaction. Thus, our findings suggest that the vp13 interaction with microtubules might be needed for establishment of an HEV infection.The hepatitis E virus (HEV), the sole member of the genus Hepevirus, is a single-strand positive-sense RNA virus that is the causative agent in endemics and epidemics of acute human hepatitis in many parts of the world (5). Transmitted mainly from contaminated water through the fecal-oral route, HEV infection causes a fulminant form of hepatitis that has a mortality rate of up to 20% in pregnant women (28). HEV infection is considered zoonotic. Swine and chicken HEV strains have been found in the United States (11, 23). A swine strain can infect chimpanzees under experimental conditions, and a human strain that is genetically similar to the swine strain can experimentally infect pigs (22). Direct evidence of the zoonotic nature of HEV infection has been provided in reports of a series of cases of HEV infection in people who ate undercooked deer meat 6 to 7 weeks before the onset of the disease (19, 33, 39). HEV RNA recovered from the leftover deer meat was found to be identical in nucleotide sequence to the HEV RNA recovered from the individuals who became ill (31).The HEV genome is approximately 7.2 kb in length and consists of three open reading frames (ORFs) (32). ORF1 encodes a nonstructural polyprotein that includes the RNA-dependent RNA polymerase. ORF2 encodes the capsid protein, the major structural protein in virion. ORF3 encodes a phosphoprotein that was found to be essential for establishing an HEV infection in macaques and pigs under experimental conditions (9, 12). It has been reported that ORF3 translation initiates at the third in-frame AUG codon, which lies 23 bases downstream of the ORF1 termination codon (10, 12). Propagation of HEV and studies of virus replication still rely upon nonhuman primates due to the lack of an effective cell culture system. As a result, functional study of the ORF3 product in HEV biology and infection is limited.The phosphoprotein encoded by HEV ORF3 has a molecular mass of approximately 13 kDa (hereinafter called vp13) (32). The exact functions of vp13 in HEV infection remain unknown although the findings of a number of studies have shown that it plays a role in cellular signaling pathways (13, 17, 24, 34-36, 40). During subcellular fractionation of COS-7 cells transfected with a vp13-expressing plasmid, vp13 was found to partition with the cytoskeletal fraction (40). Deletion of the N-terminal hydrophobic domain of vp13 abolished the association with the cytoskeleton fraction. The vp13-binding proteins in the cytoskeleton and the nature of this interaction are not known.In this study, we found that the HEV ORF3 product localizes to microtubules and interferes with their dynamics. The filamentous pattern of vp13 distribution in the cell was abolished by a microtubule-destabilizing drug. vp13 led to elevation of acetylated α-tubulin. These results suggested that vp13 interaction with the microtubules might facilitate HEV infection. We further studied the nature of the vp13-microtubule interaction.     

The Footprint of Genome Architecture in the Largest Genome Expansion in RNA Viruses

The small size of RNA virus genomes (2-to-32 kb) has been attributed to high mutation rates during replication, which is thought to lack proof-reading. This paradigm is being revisited owing to the discovery of a 3′-to-5′ exoribonuclease (ExoN) in nidoviruses, a monophyletic group of positive-stranded RNA viruses with a conserved genome architecture. ExoN, a homolog of canonical DNA proof-reading enzymes, is exclusively encoded by nidoviruses with genomes larger than 20 kb. All other known non-segmented RNA viruses have smaller genomes. Here we use evolutionary analyses to show that the two- to three-fold expansion of the nidovirus genome was accompanied by a large number of replacements in conserved proteins at a scale comparable to that in the Tree of Life. To unravel common evolutionary patterns in such genetically diverse viruses, we established the relation between genomic regions in nidoviruses in a sequence alignment-free manner. We exploited the conservation of the genome architecture to partition each genome into five non-overlapping regions: 5′ untranslated region (UTR), open reading frame (ORF) 1a, ORF1b, 3′ORFs (encompassing the 3′-proximal ORFs), and 3′ UTR. Each region was analyzed for its contribution to genome size change under different models. The non-linear model statistically outperformed the linear one and captured >92% of data variation. Accordingly, nidovirus genomes were concluded to have reached different points on an expansion trajectory dominated by consecutive increases of ORF1b, ORF1a, and 3′ORFs. Our findings indicate a unidirectional hierarchical relation between these genome regions, which are distinguished by their expression mechanism. In contrast, these regions cooperate bi-directionally on a functional level in the virus life cycle, in which they predominantly control genome replication, genome expression, and virus dissemination, respectively. Collectively, our findings suggest that genome architecture and the associated region-specific division of labor leave a footprint on genome expansion and may limit RNA genome size.     

Evolutionary Conservation of the PA-X Open Reading Frame in Segment 3 of Influenza A Virus

PA-X is a fusion protein of influenza A virus encoded in part from a +1 frameshifted X open reading frame (X-ORF) in segment 3. We show that the X-ORFs of diverse influenza A viruses can be divided into two groups that differ in selection pressure and likely function, reflected in the presence of an internal stop codon and a change in synonymous diversity. Notably, truncated forms of PA-X evolved convergently in swine and dogs, suggesting a strong species-specific effect.