Identification and Mutational Analysis of a Rej Response Element in Jaagsiekte Sheep Retrovirus RNA

Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus causing a contagious lung cancer of sheep. JSRV encodes unspliced and spliced viral RNAs, among which unspliced RNA encodes Gag and Pol proteins and a singly spliced mRNA encodes Env protein. In another study we found that JSRV encodes a regulatory protein, Rej, that is responsible for synthesis of Gag polyprotein from unspliced viral RNA. Rej is encoded in the 5′ end of env, and it enhances nuclear export or accumulation of cytoplasmic unspliced viral RNA in 293T cells but not in most other cell lines (A. Hofacre, T. Nitta, and H. Fan, J. Virol. 83:12483-12498, 2009). In this study, we found that mutations in the 3′ end of env in the context of a cytomegalovirus-driven full-length JSRV expression construct abolished Gag protein synthesis and released viruses in 293T cells. These mutants also showed deficits in accumulation of unspliced viral RNA in the cytoplasm. These mutants defined a Rej-responsive element (RejRE). Inhibition of CRM1 but not Tap function prevented nuclear export/accumulation of cytoplasmic unspliced RNA in 293T cells, similarly to other complex retroviruses that express analogous regulator proteins (e.g., human immunodeficiency virus Rev). Structural modeling of the RejRE with Zuker M-fold indicated a region with a predicted stable secondary structure. Mutational analysis in this region indicated the importance of both secondary structures and primary nucleotide sequences in a central stem-bulge-stem structure. In contrast to 293T cells, mutations in the RejRE did not affect the levels of cytoplasmic unspliced RNA in 293 cells, although the unspliced RNA showed partial degradation, perhaps due to lack of translation. RejRE-containing RNA relocalized Rej protein from the nucleus to the cytoplasm in 293 and rat 208F cells, suggesting binding of Rej to the RejRE.Jaagsiekte sheep retrovirus (JSRV) is a betaretrovirus that causes ovine pulmonary adenocarcinoma, an infectious lung tumor of sheep (10, 29). Ovine pulmonary adenocarcinoma has morphological resemblance to a human lung cancer, bronchioloalveolar carcinoma, which is only weakly associated with cigarette smoking. In recent years, complete infectious and oncogenic molecular clones of JSRV have been isolated (30). We and others found that the JSRV envelope (Env) protein also functions as an oncogene in that it can induce morphological transformation of fibroblast and epithelial cell lines in culture and tumors in animals (1, 24, 34). Further studies have demonstrated that amino acids in the cytoplasmic tail of the Env transmembrane (TM) protein are important for transformation, as are multiple domains in the surface (SU) protein (17, 18).The nuclear export of mRNA is a critical step in gene expression. All retroviruses employ unspliced genome-length RNA as mRNA for synthesis of Gag and Pol proteins, while splicing yields mRNA(s) for Env (and other) proteins (15). Thus, genome-length mRNA for Gag and Pol is equivalent to an unspliced precursor for Env mRNA. A key issue for retroviruses is how they transport unspliced genome-length RNA to the cytoplasm. This is accomplished by two general mechanisms. The human immunodeficiency virus type 1 (HIV-1) Rev protein (encoded by a doubly spliced mRNA) specifically binds to a Rev-responsive element (RRE), located in RNA of the env gene. The Rev/RRE complex recruits the cellular CRM1/Xpo1 protein (as well as other cellular proteins), which results in transport of this RNA-protein complex to the cytoplasm (7). Similarly, human T-cell leukemia virus type 1 (HTLV-1) Rex protein binds a Rex-responsive element on viral RNA, resulting in export via the CRM1 pathway (21). The betaretroviruses mouse mammary tumor virus (MMTV) and human endogenous retrovirus K (HERV-K) also encode analogous regulatory proteins (Rem and Rec, respectively) (19, 22, 27).In contrast, the betaretroviruses Mason-Pfizer monkey virus (MPMV) and simian retrovirus (SRV) contain constitutive RNA export elements (constitutive transport elements [CTEs]) that facilitate nuclear export of unspliced RNA (4, 41). The MPMV CTE is located between env and the 3′ long terminal repeat (LTR); it binds to the cellular trans-acting factor NXF1/Tap, which directs nuclear export of the RNA-protein complex to the cytoplasm (14). Rous sarcoma virus and the related avian leukosis viruses contain direct repeat sequences flanking the src gene or in the 3′ untranslated region of their RNA (28). Structure-function analyses of these RNA-exporting elements revealed specific stem-loop structures that are important for activity and for binding of the host cell factors (3).Like other betaretroviruses, JSRV contains the standard genes gag, pro, pol, and env. In addition we recently found that JSRV also encodes a regulatory factor, Rej (17a). Rej is reminiscent of MMTV Rem and HERV-K Rec in that it is encoded in the 5′ end of env and it is required for efficient synthesis of Gag protein. We found that Rej is required for translation of unspliced viral RNA, and in 293T cells it also enhances accumulation of cytoplasmic unspliced viral RNA in the cytoplasm. In the results presented here, we show that JSRV RNA also contains a Rej-responsive element (RejRE) in the 3′ end of env that is required for translation of Gag protein and efficient export or accumulation of unspliced viral RNA in the cytoplasm in 293T cells. Mutational analyses of RejRE based on M-fold suggest that both primary sequences and secondary structures in this region play important roles in nuclear export or accumulation of unspliced viral RNA in the cytoplasm and Gag synthesis. This accumulation is independent of Tap but dependent on CRM1. Moreover, Rej protein was exported from the nucleus to the cytoplasm in cells expressing wild-type JSRV RNA but not RejRE mutants, suggesting binding of Rej protein to the RejRE.     

KAR5 Encodes a Novel Pheromone-inducible Protein Required for Homotypic Nuclear Fusion

KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.     

绵羊肺腺瘤病毒NM株前病毒基因组的克隆与全序列分析

本研究参照GenBank中已发表的绵羊肺腺瘤病毒的基因组全序列,设计合成8对引物,从内蒙古某羊场自然感染绵羊肺腺瘤病的病肺肿瘤组织中提取总DNA为模板,对JSRV-NM株基因组分8段进行PCR扩增,产物分别为8个(531bp, 888bp, 949 bp, 944bp, 1428bp, 947bp, 1836bp, 538bp)基因片段,将其分别克隆入pMD-18 T载体中进行双向测序并拼接序列,获得完整的JSRV-NM株前病毒基因组全序列.结果表明,JSRV-NM株前病毒基因组全长7430bp,有相互重叠的4个较长的开放阅读框(ORF),分别代表gag、 pro、 pol 和 env基因.与绵羊肺腺瘤病毒Ⅰ型即南非代表株(NC-001494)和绵羊肺腺瘤病毒Ⅱ型即美国代表株(AF105220)的核苷酸同源性比较分别为90.4%和90%,推导出的氨基酸同源性分别为90%和89.1%.分析JSRV-NM株基因组结构,发现在LTR的上游和下游都具有外源性exJSRV特有的ScaⅠ酶切位点,在gag基因编码的NC区发现有2个较典型的"胱氨酸-组氨酸序列",可形成锌指结构.在env基因编码的TM区有特异性的"YXXM"基序.用地高辛标记外源性exJSRV特异的JSRV-2片段制成探针,原位杂交法检测自然感染绵羊肺腺瘤病(OPA)的病肺组织中JSRV-NM的 RNA及前病毒DNA,结果表明OPA患羊肺肿瘤细胞的胞浆和核内都有JSRV-2基因mRNA的表达, 说明JSRV-NM株是具有致瘤作用的外源性反转录病毒.这是我国首次报道的绵羊肺腺瘤病毒的基因组全序列.     

绵羊肺腺瘤病毒NM株前病毒基因组的克隆与全序列分析

本研究参照GenBank中已发表的绵羊肺腺瘤病毒的基因组全序列,设计合成8对引物,从内蒙古某羊场自然感染绵羊肺腺瘤病的病肺肿瘤组织中提取总DNA为模板,对JSRV-NM株基因组分8段进行PCR扩增,产物分别为8个(531bp,888bp,949bp,944bp,1428bp,947bp,1836bp,538bp)基因片段,将其分别克隆入pMD-18T载体中进行双向测序并拼接序列,获得完整的JSRV-NM株前病毒基因组全序列。结果表明,JSRV-NM株前病毒基因组全长7430bp,有相互重叠的4个较长的开放阅读框(ORF),分别代表gag、pro、pol和env基因。与绵羊肺腺瘤病毒Ⅰ型即南非代表株(NC-001494)和绵羊肺腺瘤病毒Ⅱ型即美国代表株(AF105220)的核苷酸同源性比较分别为90.4%和90%,推导出的氨基酸同源性分别为90%和89.1%。分析JSRV-NM株基因组结构,发现在LTR的上游和下游都具有外源性exJSRV特有的ScaⅠ酶切位点,在gag基因编码的NC区发现有2个较典型的“胱氨酸—组氨酸序列”,可形成锌指结构。在env基因编码的TM区有特异性的“YXXM”基序。用地高辛标记外源性exJSRV特异的JSRV-2片段制成探针,原位杂交法检测自然感染绵羊肺腺瘤病(OPA)的病肺组织中JSRV-NM的RNA及前病毒DNA,结果表明OPA患羊肺肿瘤细胞的胞浆和核内都有JSRV-2基因mRNA的表达,说明JSRV-NM株是具有致瘤作用的外源性反转录病毒。这是我国首次报道的绵羊肺腺瘤病毒的基因组全序列。     

绵羊肺腺瘤病毒中国NM株部分gag基因的克隆与序列分析

参照GenBank中已发表的绵羊肺腺瘤病毒的基因序列,设计合成一对引物,对绵羊肺腺瘤病毒(Jaagsiekte sheep retrovirus,JSRV)内蒙株的gag基因中主要编码CA蛋白的基因段进行PCR扩增,产物经琼脂糖凝胶电泳分析,呈现一条约897bp的特异条带,将其回收后克隆入pMD-18 T载体中,并进行序列测定与分析.结果表明,与南非株(基因序列号NC-001494)的gag基因序列比较,核苷酸同源性为83%,推导出的氨基酸同源性为84%.与美国株(基因序列号AF105220)的gag基因序列比较,核苷酸同源性为81.5%,氨基酸同源性为83%.这是我国首次报道的绵羊肺腺瘤病毒的gag基因的一段序列,为我国科研工作者进行更深入的研究奠定基础.     

绵羊肺腺瘤病毒中国NM株部分gag基因的克隆与序列分析

参照GenBank中已发表的绵羊肺腺瘤病毒的基因序列,设计合成一对引物,对绵羊肺腺瘤病毒(Jaagsiekte sheep retrovirus,JSRV)内蒙株的gag基因中主要编码CA蛋白的基因段进行PCR扩增,产物经琼脂糖凝胶电泳分析,呈现一条约897bp的特异条带,将其回收后克隆入pMD—18T载体中,并进行序列测定与分析。结果表明,与南非株(基因序列号NC—001494)的gag基囚序列比较,核苷酸同源性为83％,推导出的氨基酸同源性为84％。与美国株(基因序列号AF105220)的gag基因序列比较,核苷酸同源性为81．5％,氨基酸同源性为83％。这是我国首次报道的绵羊肺腺瘤病毒的gag基因的一段序列,为我国科研工作者进行更深入的研究奠定基础。     

离心力和剪应力应答蛋白1是肿瘤坏死因子受体1的新调控因子

离心力和剪应力应答基因1(responsive to centrifugal force and shear stress gene 1,RECS1)被剔除的小鼠易患囊性内侧坏死和动脉扩张症,伴随着血管组织基质金属蛋白酶9表达水平的增强.本室前期研究发现,稳定表达RECS1的小鼠成纤维细胞对肿瘤坏死因子受体2激动性抗体的敏感性被明显弱化,显示RECS1参与肿瘤坏死因子信号的调控.本文研究了RECS1对肿瘤坏死因子受体1（tumor necrosis factor receptor-1, TNFR1）的调控作用.结果显示,RECS1结合TNFR1,并抑制过量表达TNFR1诱导的核转录因子-κB (NF-κB)活化.缺失突变研究发现,RECS1分子上有NPLY和SPEDY两个模体是其抑制TNFR1信号所必需的.免疫共沉淀实验发现,NPLY是RECS1与TNFR1结合所必需的.而SPEDY的缺失不影响RECS1与TNFR1的结合.另外,免疫共染色实验显示,RECS1与TNFR1共定位于细胞内核体.这些实验结果进一步揭示了RECS1负调控肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)信号进而参与调控血管发育与重塑的生物功能及可能机理.     

LIA4 Encodes a Chromoshadow Domain Protein Required for Genomewide DNA Rearrangements in Tetrahymena thermophila

Extensive DNA elimination occurs as part of macronuclear differentiation during Tetrahymena sexual reproduction. The identification of sequences to excise is guided by a specialized RNA interference (RNAi) machinery that targets the methylation of histone H3 lysine 9 (K9) and K27 on chromatin associated with these internal eliminated sequences (IESs). This modified chromatin is reorganized into heterochromatic subnuclear foci, which is a hallmark of their subsequent elimination. Here, we demonstrate that Lia4, a chromoshadow domain-containing protein, is an essential component in this DNA elimination pathway. LIA4 knockout (ΔLIA4) lines fail to excise IESs from their developing somatic genome and arrest at a late stage of conjugation. Lia4 acts after RNAi-guided heterochromatin formation, as both H3K9 and H3K27 methylation are established. Nevertheless, without LIA4, these cells fail to form the heterochromatic foci associated with DNA rearrangement, and Lia4 accumulates in the foci, indicating that Lia4 plays a key role in their structure. These data indicate a critical role for Lia4 in organizing the nucleus during Tetrahymena macronuclear differentiation.     

The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer

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The Hansenula polymorpha ATG25 Gene Encodes a Novel Coiled-Coil Protein that is Required for Macropexophagy

We have isolated the Hansenula polymorpha ATG11 and ATG25 genes, which are both required for glucose-induced selective peroxisome degradation (macropexophagy). ATG11 was identified before in other yeast species and shown to be involved in the Cvt pathway in Saccharomyces cerevisiae and glucose-induced micropexophagy in Pichia pastoris. Our data indicate that HpATG11 is required for macropexophagy. ATG25 represents a novel gene that encodes a 45 kDa coiled-coil protein. We show that this protein co-localizes with Atg11 on a small structure, which most likely represents the pre-autophagosomal structure (PAS). Cells of a constructed ATG25 deletion strain (atg25) displayed relatively slow, continuous degradation of peroxisomes by microautophagy during growth on methanol in the presence of excess nitrogen that also continued after induction of selective peroxisome degradation. This suggests that the processes of selective and non-selective autophagy are dysregulated in atg25 cells.     

The Rad9 Gene of Coprinus Cinereus Encodes a Proline-Rich Protein Required for Meiotic Chromosome Condensation and Synapsis

The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stage of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus.     

Vps13D Encodes a Ubiquitin-Binding Protein that Is Required for the Regulation of Mitochondrial Size and Clearance

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