SINDBIS病毒基因组的研究

Sindbis病毒经蔗糖密度梯度离心纯化后,用SDS/酚/氯仿/异戊醇处理和乙醇沉淀,制得的基因组RNA具有典型核酸紫外吸收光谱的特征:最大吸收在260nm,最小在235nm。沉降系数为45S。沉降扫描图表明,RNA样品均一,分子完整。 用高渗法在原代鸡胚纤维母细胞测定了基因组RNA的感染性,空斑滴度为5.9×10~4pFU(空斑形成单位)/ml,空斑形态和大小与完整病毒颗粒相同。经核糖核酸酶处理,基因组RNA即丧失感染性,说明了这种生物活性的特异性。     

Universal and strain specific structure features of segment 8 genomic RNA of influenza A virus—application of 4-thiouridine photocrosslinking

RNA structure in the influenza A virus (IAV) has been the focus of several studies that have shown connections between conserved secondary structure motifs and their biological function in the virus replication cycle. Questions have arisen on how to best recognize and understand the pandemic properties of IAV strains from an RNA perspective, but determination of the RNA secondary structure has been challenging. Herein, we used chemical mapping to determine the secondary structure of segment 8 viral RNA (vRNA) of the pandemic A/California/04/2009 (H1N1) strain of IAV. Additionally, this long, naturally occurring RNA served as a model to evaluate RNA mapping with 4-thiouridine (4sU) crosslinking. We explored 4-thiouridine as a probe of nucleotides in close proximity, through its incorporation into newly transcribed RNA and subsequent photoactivation. RNA secondary structural features both universal to type A strains and unique to the A/California/04/2009 (H1N1) strain were recognized. 4sU mapping confirmed and facilitated RNA structure prediction, according to several rules: 4sU photocross-linking forms efficiently in the double-stranded region of RNA with some flexibility, in the ends of helices, and across bulges and loops when their structural mobility is permitted. This method highlighted three-dimensional properties of segment 8 vRNA secondary structure motifs and allowed to propose several long-range three-dimensional interactions. 4sU mapping combined with chemical mapping and bioinformatic analysis could be used to enhance the RNA structure determination as well as recognition of target regions for antisense strategies or viral RNA detection.     

A novel procedure for the localization of viral RNAs in protoplasts and whole plants

Analysis of virus spread using co-expressed reporter proteins has provided important details on cell-to-cell and long-distance movement of viruses in plants. However, most viruses cannot tolerate insertion of large non-viral segments or loss of any open-reading frames, procedures required to detect viruses non-evasively. A technique used to localize mRNAs intracellularly in yeast has been modified for detection of viral RNAs in whole plants. The technique makes use of the binding of the coat protein of MS2 bacteriophage (CPMS2) to a 19 base hairpin (hp). A fusion protein, consisting of the CPMS2, green fluorescent protein (GFP), and a nuclear localization signal (NLS), was nuclear-localized upon transient expression in protoplasts. However, addition of the hp to the 3' untranslated region of Turnip crinkle virus (TCV-hp) and co-transfection of the virus and fusion protein construct into protoplasts resulted in the re-location of GFP to the cytoplasm. Neither the insertion of the hp nor the interaction with the fusion protein impaired any viral functions. Transgenic plants expressing the GFP-NLS-CPMS2 fusion protein were generated, and GFP was detected in nuclei of young plant cells. Foci of GFP cytoplasmic fluorescence were detected in TCV-hp-inoculated leaves at 2 days post-inoculation. Later, GFP was detected in young leaves near the midvein and in the base (support) cells of trichomes in the vicinity of secondary and tertiary veins. In older leaves, cytoplasmic GFP could be visualized throughout many of the leaves. This technique should be amenable for detection of any virus with a transformable plant (or animal) host and may also prove useful for localizing properly engineered host RNAs.     

RNA editing by the host ADAR system affects the molecular evolution of the Zika virus

Zika virus (ZIKV) is a mosquito‐transmitted flavivirus, linked to microcephaly and fetal death in humans. Here, we investigate whether host‐mediated RNA editing of adenosines (ADAR) plays a role in the molecular evolution of ZIKV. Using complete coding sequences for the ZIKV polyprotein, we show that potential ADAR substitutions are underrepresented at the ADAR‐resistant GA dinucleotides of both the positive and negative strands, that these changes are spatially and temporally clustered (as expected of ADAR editing) for three branches of the viral phylogeny, and that ADAR mutagenesis can be linked to its codon usage. Furthermore, resistant GA dinucleotides are enriched on the positive (but not negative) strand, indicating that the former is under stronger purifying selection than the latter. ADAR editing also affects the evolution of the rhabdovirus sigma. Our study now documents that host ADAR editing is a mutation and evolutionary force of positive‐ as well as negative‐strand RNA viruses.     

Identification and functional characterization of the nascent RNA contacting residues of the hepatitis C virus RNA-dependent RNA polymerase

Understanding how the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) interacts with nascent RNA would provide valuable insight into the virus's mechanism for RNA synthesis. Using a peptide mass fingerprinting method and affinity capture of peptides reversibly cross-linked to an alkyn-labeled nascent RNA, we identified a region below the Δ1 loop in the fingers domain of the HCV RdRp that contacts the nascent RNA. A modification protection assay was used to confirm the assignment. Several mutations within the putative nascent RNA binding region were generated and analyzed for RNA synthesis in vitro and in the HCV subgenomic replicon. All mutations tested within this region showed a decrease in primer-dependent RNA synthesis and decreased stabilization of the ternary complex. The results from this study advance our understanding of the structure and function of the HCV RdRp and the requirements for HCV RNA synthesis. In addition, a model of nascent RNA interaction is compared with results from structural studies.     

Genome 3'-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.     

流行性出血热病毒核酸的提取

本文报告流行性出血热R22和A9株病毒培养,同位素标记及核酸提取的初步研究,並获得了该病毒核酸的3个片段即L、M、S、分子量分别约为:3.8、1.9和0.86×10~5道尔顿,不论用20～70％蔗糖密度梯度离心提纯的病毒,或用30％蔗糖垫层离心的粗制病毒,均获同样结果,但多数情况下,用蔗糖密度梯度离心时,除病毒峰外,还发现主要由细胞组份(即线粒体和核糖体等)组成的另一峰,并经常影响病毒RNA的提取,对如何获得纯净病毒及其核酸进行了讨论。     

Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.     

Characterization of a cytoplasmic polyhedrosis virus from Estigmene acrea (Lepidoptera)

A cytoplasmic polyhedrosis virus (CPV) containing a segmented double-stranded RNA genome was isolated from Estigmene acrea larvae by isopycnic centrifugation in a sucrose density gradient. Ten double-stranded RNA segments with molecular weights (MW) from 2.8 to 0.67 × 10⁶ were separated by agarose gel electrophoresis. A total of ten virus proteins ranging from 14,000 to 128,000 MW were detected after separation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A MW of 28,500 was determined for E. acrea CPV occlusion body protein.     

Structural Constraints and Mutational Bias in the Evolutionary Restoration of a Severe Deletion in RNA Phage MS2

A 4-nucleotide (nt) deletion was made in the 36-nt-long intercistronic region separating the coat and replicase genes of the single-stranded RNA phage MS2. This region is the focus of several RNA structures conferring high fitness. One such element is the operator hairpin, which, in the course of infection, will bind a coat-protein dimer, thereby precluding further replicase synthesis and initiating encapsidation. Another structure is a long-distance base pairing (MJ) controlling replicase expression. The 4-nt deletion does not directly affect the operator hairpin but it disrupts the MJ pairing. Its main effect, however, is a frame shift in the overlapping lysis gene. This gene starts in the upstream coat gene, runs through the 36-nt-long intercistronic region, and ends in the downstream replicase cistron. Here we report and interpret the spectrum of solutions that emerges when the crippled phage is evolved. Four different solutions were obtained by sequencing 40 plaques. Three had cured the frame shift in the lysis gene by inserting one nt in the loop of the operator hairpin causing its inactivation. Yet these low-fitness revertants could further improve themselves when evolved. The inactivated operator was replaced by a substitute and thereafter these revertants found several ways to restore control over the replicase gene. To allow for the evolutionary enrichment of low-probability but high-fitness revertants, we passaged lysate samples before plating. Revertants obtained in this way also restored the frame shift, but not at the expense of the operator. By taking larger and larger lysates samples for such bulk evolution, ever higher-fitness and lower-frequency revertants surfaced. Only one made it back to wild type. As a rule, however, revertants moved further and further away from the wild-type sequence because restorative mutations are, in the majority of cases, selected for their capacity to improve the phenotype by optimizing one of several potential alternative RNA foldings that emerge as a result of the initial deletion. This illustrates the role of structural constraints which limit the path of subsequent restorative mutations. [Reviewing Editor: Dr. John Hulsenbeck]     

RNA viruses as vectors for the expression of heterologous proteins

RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure.     

RNA干扰在植物中的作用机理及其应用研究进展

RNA干扰(RNAi)是广泛存在于生物中的一种现象,它是小干扰RNA诱导的转录后基因沉默,是生物抵抗异常DNA的一种保护机制,同时在生物生长发育过程中调控基因的表达.本文综述了近年来有关RNA干扰的发现、作用过程及其机理,分析了它与反义寡核苷酸、核酶、脱氧核酶的小同,并介绍了RNA干扰在植物基因功能、植物抗病毒、作物品种改良等方面的应用,为siRNA干扰的进一步利用提供参考资料.     

RNAi的抗病毒研究进展

RNA干扰(RNA interference,RNAi)是真核生物中的特异核苷酸序列产生的基因沉默现象,被认为有抑制病毒复制的功能。最近的研究表明,通过诱导RNAi可以抑制多种病毒的复制,包括人类免疫缺陷病毒Ⅰ型,乙型肝炎病毒,丙型肝炎病毒,登革热病毒,脊髓灰质炎病毒,流感病毒,口蹄疫病毒和重症急性呼吸综合征病毒等。总结了目前运用RNA干扰技术抑制病毒复制的研究进展,展望基于RNAi技术的抗病毒治疗的可能性。     

The first two nucleotides of the respiratory syncytial virus antigenome RNA replication product can be selected independently of the promoter terminus

There is limited knowledge regarding how the RNA-dependent RNA polymerases of the nonsegmented negative-strand RNA viruses initiate genome replication. In a previous study of respiratory syncytial virus (RSV) RNA replication, we found evidence that the polymerase could select the 5'-ATP residue of the genome RNA independently of the 3' nucleotide of the template. To investigate if a similar mechanism is used during antigenome synthesis, a study of initiation from the RSV leader (Le) promoter was performed using an intracellular minigenome assay in which RNA replication was restricted to a single step, so that the products examined were derived only from input mutant templates. Templates in which Le nucleotides 1U, or 1U and 2G, were deleted directed efficient replication, and in both cases, the replication products were initiated at the wild-type position, at position -1 or -2 relative to the template, respectively. Sequence analysis of the RNA products showed that they contained ATP and CTP at the -1 and -2 positions, respectively, thus restoring the mini-antigenome RNA to wild-type sequence. These data indicate that the RSV polymerase is able to select the first two nucleotides of the antigenome and initiate at the correct position, even if the 3'-terminal two nucleotides of the template are missing. Substitution of positions +1 and +2 of the template reduced RNA replication and resulted in increased initiation at positions +3 and +5. Together these data suggest a model for how the RSV polymerase initiates antigenome synthesis.     

Nuclear alterations in a baculovirus-like infection of midgut epithelial cells in the stick insect, Bacillus rossius

In the epithelial cell nuclei of the posterior part of the midgut of the stick insect, Bacillus rossius, a symptomless virus infection is most commonly found. The virus particles consist of rod-shaped DNP nucleocapsids singly enveloped by a membrane and often packed in a pseudocristalline pattern along their major axis; occlusion bodies of any kind are not formed. On the basis of size, structural characteristics, and localization, the virus is thought to be a baculovirus-like virus. The viral infection, which is asynchronous in different nuclei, causes the formation of electron-light virogenic areas, the swelling of the nucleus, the nuclear chromatin lysis, the production of highly variable numbers of virus particles and, eventually, the extrusion of the whole infected nucleus into the gut lumen. Cellular DNA synthesis does not seem to be stimulated by the virus, RNA synthesis is apparently maintained as long as some cellular chromatin is present in the infected nucleus. Nucleolar segregation invariably occurs at late stages of infection, while major cytoplamic alterations have not been noticed until nuclear elimination and cell death occur.     

RNA病毒利用外泌体促进病毒感染的研究进展

外泌体是一种由细胞主动向胞外分泌的囊泡类小体,因其能在细胞间传递蛋白、脂类和核酸等分子,而被认为是一种新的重要的细胞间通讯方式。RNA病毒,如HIV-1、HCV等,作为一类重要的病原体,一直影响着全人类的健康。近来的研究发现,病毒能够利用外泌体的某些相关功能促进其复制与传播。然而,对外泌体与病毒感染的相关研究才刚刚起步,尚有很多方面并未被详细认知,所要研究的内容还有很多。本文主要总结了外泌体在一些RNA病毒感染中的促进作用及其可能的机制,以期让大家了解RNA病毒与外泌体之间已有的相互关系。