A Ribonucleoprotein Complex Protects the Interleukin-6 mRNA from Degradation by Distinct Herpesviral Endonucleases

During lytic Kaposi’s sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3’ untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3’ UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3’ UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.     

RNase P核酶对人巨细胞病毒UL54基因mRNA体外切割作用

外部引导序列(EGSs)是mRNA靶序列互补并引导RNaseP切割的小RNA片段。我们设计与人巨细胞病毒HCMV(Human Cytomegalovirus)UL54基因mRNA序列互补的EGSs,将其与大肠杆菌来源RNaseP催化核心M1RNA构建成M1GS核酶。通过对UL54基因亚克降片转录产物体外切割研究,证实该核酶具备对UL54 mRNA片段的特异切割能力,可以发展成为一种抗病毒试剂。     

Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses

Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi’s sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman’s disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited in vitro and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome, thus thwarting key cellular defense mechanisms.     

The exonuclease and host shutoff functions of the SOX protein of Kaposi's sarcoma-associated herpesvirus are genetically separable

The Kaposi's sarcoma-associated herpesvirus (KSHV) SOX protein, encoded by ORF37, promotes shutoff of host cell gene expression during lytic viral replication by dramatically impairing mRNA accumulation. SOX is the KSHV homolog of the alkaline exonuclease of other herpesviruses, which has been shown to function as a DNase involved in processing and packaging the viral genome. Although the exonuclease activity of these proteins is widely conserved across all herpesviruses, the host shutoff activity observed for KSHV SOX is not. We show here that SOX expression sharply reduces the half-life of target mRNAs. Extensive mutational analysis reveals that the DNase and host shutoff activities of SOX are genetically separable. Lesions affecting the DNase activity cluster in conserved regions of the protein, but residues critical for mRNA degradation are not conserved across the viral family. Additionally, we present evidence suggesting that the two different functions of SOX occur within distinct cellular compartments-DNase activity in the nucleus and host shutoff activity in the cytoplasm.     

Splice site consensus sequences are preferentially accessible to nucleases in isolated adenovirus RNA.

The conformation of RNA sequences spanning five 3' splice sites and two 5' splice sites in adenovirus mRNA was probed by partial digestion with single-strand specific nucleases. Although cleavage of nucleotides near both 3' and 5' splice sites was observed, most striking was the preferential digestion of sequences near the 3' splice site. At each 3' splice site a region of very strong cleavage is observed at low concentrations of enzyme near the splice site consensus sequence or the upstream branch point consensus sequence. Additional sites of moderately strong cutting near the branch point consensus sequence were observed in those sequences where the splice site was the preferred target. Since recognition of the 3' splice site and branch site appear to be early events in mRNA splicing these observations may indicate that the local conformation of the splice site sequences may play a direct or indirect role in enhancing the accessibility of sequences important for splicing.     

Cellular activity of Rev response element RNA targeting metallopeptides

The cellular chemistry of metallopeptide complexes designed to target and inactivate an HIV Rev response element (RRE) RNA sequence in vivo has been evaluated by use of an efficient cellular fluorescence assay. Transcribed messenger RNA encoding the green fluorescent protein (GFP) that includes a target RNA sequence is sensitive to cleavage chemistry mediated by metal derivatives of GGH(G) _x TRQARRNRR RRWRERQR (x = 0, 1, 2, 4, 6). This results in a significant decrease in expression of GFP that can be quantified by fluorimetry. Optimal inactivation of the target RRE RNA was achieved with linkers where x = 0 or 1. Neither the Rev control peptide (lacking metal-binding or linker sequences) nor the metal-binding motif alone had any significant effect. Consequently, both the cleavage motif and the RNA targeting motif are essential to promote cellular cleavage of the target RRE RNA. However, target inactivation was also observed in experiments with metal-free peptide, consistent with recruitment of intracellular metal ion by the peptide following cellular uptake, with subsequent cleavage of the RRE target RNA. The RRE RNA cleavage activities of metallopeptide complexes were further confirmed by in vitro experiments and mammalian cell assays.     

Cleavage properties of an estrogen-regulated polysomal ribonuclease involved in the destabilization of albumin mRNA.

Previous work from this laboratory [Dompenciel,R.E., Garnepudi,V.R. and Schoenberg,D.R. (1995)J. Biol. Chem.270, 6108-6118] described the purification and properties of an estrogen-regulated endonuclease isolated from Xenopus liver polysomes that is involved in the destabilization of albumin mRNA. The present study mapped cleavages made by this enzyme onto the secondary structure of the portion of albumin mRNA bearing the major cleavage sites. The predominant cleavages occur in the overlapping APyrUGA sequence AUUGACUGA present in a single-stranded loop region, and in AUUGA located within a bulged AU-rich stem. A structural mutation which converted the major loop cleavage site to a hairpin bearing one APyrUGA element eliminated cleavage at the intact site. This confirms that the polysomal RNase is specific for single-stranded RNA. Additional point mutations in the major loop characterized the nucleoside sequence requirements for cleavage. Finally, snake venom exonuclease was used to demonstrate the polysomal RNase generates products with a 3' hydroxyl. Binding of an estrogen-induced protein to a portion of the 3'UTR of vitellogenin mRNA may be involved in its stabilization by estrogen [Dodson,R.E. and Shapiro,D.J. (1994)Mol. Cell. Biol.14, 3130-3138]. The core binding site for this protein bears the sequence APyrUGA, suggesting that stabilization may be accomplished by occlusion of a cleavage site for the polysomal RNase.     

抗HPV11／E2基因Ribozyme对靶RNA的体外剪切研究

为了寻找HPV11型引起的生殖系统感染的治疗途径和探讨HPV的致病机理,本实验以HPV11病毒质粒为模板,扩增出HPVll型E2区644bp片段,采用pGEM-T-Easy Vector为载体,构建pTV-644克隆载体,经筛选得到克隆株,提取质粒测序鉴定。采用上海生化所陈农安教授编制的锤头状Ribozyme设计软件进行计算机分析,选择Ribozyme对靶基因的最佳剪切位点,及进行基因同源性分析和生物学功能分析,选择出针对HPVllE2靶基因的RZ2777,在最适条件下进行体外剪切反应,发现人工合成和体外转录得到的Ribozyme分子均能在相应位点准确切割靶RNA分子,选择合适的反应条件切割效率达到60％以上,Km和Kcat值分别为0.63μmol/L、0.12μmol/L,RibozymeL两端的5′-cis-ribozyme和3′-cis-ribozyme自我剪切释放并未影响切割活性,但靶RNA侧翼序列影响了Ribozyme的剪切活性。实验研究表明,Ribozyme可能成为治疗HPVll型引起的尖锐湿疣的有效手段,并有望在分子水平上开辟出基因治疗HPVll病毒感染的另一新天地。     

结核分枝杆菌ICL mRNA特异性10-23脱氧核酶切割 活性的鉴定及其切割特点

为鉴定结核分枝杆菌异柠檬酸裂合梅(ICL)特异性的10-23DRz在无细胞体系切割ICL mRNA的活性,并探讨其在不同条件下以及联合应用时对靶mRNA的切割特点,采用计算机软件模拟ICL mRNA的二级结构,据此选择适合的待切割靶点并设计针对相应靶点的特异性10-23DRz（DZ1～DZ5）.PCR法扩增获得icl基因并克隆入质粒pET32a+.采用T7 RNA聚合酶体外转录法获取ICL全长mRNA后分别用DZ1～DZ5在无细胞体系中对ICL mRNA进行切割,切割产物经变性聚丙烯酰胺凝胺电泳后用银染法鉴定各DRzs的活性.选择切割活性最强的DZ4考察不同10-23DRz剂量、不同反应时间、不同镁离子浓度条件下及不同错配或突变10-23DRz的切割特点.联合应用DZ1、DZ4及DZ5在无细胞体系中对ICL mRNA进行切割,检测10-23DRz联合应用对切割效率的影响.结果表明,DZ1、DZ3、DZ4及DZ5可在无细胞体系中有效地切割ICL mRNA,其切割效率在30.8%～64.5%之间.对DZ4切割活性的检测发现,其对靶mRNA的切割具有剂量和时间的依赖性;在2～20 μmol/L范围内,DZ4的切割活性与Mg2＋浓度呈正相关;DZ4单侧底物结合臂上含一个不与靶mRNA配对的碱基时其切割效率大大降低,两侧底物结合臂上各含一个不配对的碱基或活性中心域第6位出现碱基突变（G→C）时,DZ4完全丧失切割活性.联合应用2种或2种以上10-23DRz可显著增强对底物RNA的切割效率.10-23 DRz特异、有效地切割结核分枝杆菌ICL全长mRNA并显示一定的叠加效应,有望用于抗结核分枝杆菌潜伏感染的基因治疗.     

RNA recognition by the human polyadenylation factor CstF.

Polyadenylation of mammalian mRNA precursors requires at least two signal sequences in the RNA: the nearly invariant AAUAAA, situated 5' to the site of polyadenylation, and a much more variable GU- or U-rich downstream element. At least some downstream sequences are recognized by the heterotrimeric polyadenylation factor CstF, although how, and indeed if, all variations of this diffuse element are bound by a single factor is unknown. Here we show that the RNP-type RNA binding domain of the 64-kDa subunit of CstF (CstF-64) (64K RBD) is sufficient to define a functional downstream element. Selection-amplification (SELEX) experiments employing a glutathione S-transferase (GST)-64K RBD fusion protein selected GU-rich sequences that defined consensus recognition motifs closely matching those present in natural poly(A) sites. Selected sequences were bound specifically, and with surprisingly high affinities, by intact CstF and were functional in reconstituted, CstF-dependent cleavage assays. Our results also indicate that GU- and U-rich sequences are variants of a single CstF recognition motif. For comparison, SELEX was performed with a GST fusion containing the RBD from the apparent yeast homolog of CstF-64, RNA15. Strikingly, although the two RBDs are almost 50% identical and yeast poly(A) signals are at least as degenerate as the mammalian downstream element, a nearly invariant 12-base U-rich sequence distinct from the CstF-64 consensus was identified. We discuss these results in terms of the function and evolution of mRNA 3'-end signals.     

Crystal structure of a KSHV-SOX-DNA complex: insights into the molecular mechanisms underlying DNase activity and host shutoff

The early lytic phase of Kaposi's sarcoma herpesvirus infection is characterized by viral replication and the global degradation (shutoff) of host mRNA. Key to both activities is the virally encoded alkaline exonuclease KSHV SOX. While the DNase activity of KSHV SOX is required for the resolution of viral genomic DNA as a precursor to encapsidation, its exact involvement in host shutoff remains to be determined. We present the first crystal structure of a KSHV SOX-DNA complex that has illuminated the catalytic mechanism underpinning both its endo and exonuclease activities. We further illustrate that KSHV SOX, similar to its Epstein-Barr virus homologue, has an intrinsic RNase activity in vitro that although an element of host shutoff, cannot solely account for the phenomenon.