Single-base pair unwinding and asynchronous RNA release by the hepatitis C virus NS3 helicase

Nonhexameric helicases use adenosine triphosphate (ATP) to unzip base pairs in double-stranded nucleic acids (dsNAs). Studies have suggested that these helicases unzip dsNAs in single-base pair increments, consuming one ATP molecule per base pair, but direct evidence for this mechanism is lacking. We used optical tweezers to follow the unwinding of double-stranded RNA by the hepatitis C virus NS3 helicase. Single-base pair steps by NS3 were observed, along with nascent nucleotide release that was asynchronous with base pair opening. Asynchronous release of nascent nucleotides rationalizes various observations of its dsNA unwinding and may be used to coordinate the translocation speed of NS3 along the RNA during viral replication.     

A liver-specific enhancer in the core promoter region of human hepatitis B virus

An 88-base pair fragment in the core promoter of the human hepatitis B virus (HBV) contains a functional promoter and a strong liver-specific enhancer. This enhancer functions in human hepatoma cells, where it is much more active than the previously described HBV enhancer in stimulating expression of the linked bacterial chloramphenicol acetyltransferase gene expressed from heterologous promoters. Studies of the role of this enhancer-promoter in HBV may help to clarify mechanisms of gene expression in cells infected with HBV and the role of the virus in the pathogenesis of hepatitis and hepatocellular carcinoma.     

Murine hepatitis virus: effect on liver RNA

After infection of mice with hepatitis virus MHV3, the RNA in the liver undergoes changes. The fraction extracted with phenol at 0 degrees C does not alter. However, the fraction extracted with hot phenol at elevated pH (60 degrees C, pH 8.3) shows a 16S peak on sucrose-density-gradient centrifugation. This fraction shows actinomycin D-resistant incorporation of C(14)-orotic acid in infected but not in control livers-possible evidence of the RNA nature of MHV3.     

Reversible cleavage and ligation of hepatitis delta virus RNA

A 148-nucleotide subfragment of hepatitis delta virus RNA was shown to undergo cleavage and ligation reversibly. The direction of the reaction is determined by the presence or absence of Mg2+ ions, with the presence of Mg2+ favoring the cleavage reaction. Ligation requires specific conformation of the RNA molecules involved and occurs only between two cleaved RNA fragments that are still held together by hydrogen bonds. The ligation reaction occurs rapidly on removal of Mg2+ by EDTA. This represents a new class of RNA enzymes.     

Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase

NS3, an essential helicase for replication of hepatitis C virus, is a model enzyme for investigating helicase function. Using single-molecule fluorescence analysis, we showed that NS3 unwinds DNA in discrete steps of about three base pairs (bp). Dwell time analysis indicated that about three hidden steps are required before a 3-bp step is taken. Taking into account the available structural data, we propose a spring-loaded mechanism in which several steps of one nucleotide per adenosine triphosphate molecule accumulate tension on the protein-DNA complex, which is relieved periodically via a burst of 3-bp unwinding. NS3 appears to shelter the displaced strand during unwinding, and, upon encountering a barrier or after unwinding >18 bp, it snaps or slips backward rapidly and repeats unwinding many times in succession. Such repetitive unwinding behavior over a short stretch of duplex may help to keep secondary structures resolved during viral genome replication.     

Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease

Persistent infections with hepatitis C virus (HCV) are likely to depend on viral inhibition of host defenses. We show that the HCV NS3/4A serine protease blocks the phosphorylation and effector action of interferon regulatory factor-3 (IRF-3), a key cellular antiviral signaling molecule. Disruption of NS3/4A protease function by mutation or a ketoamide peptidomimetic inhibitor relieved this blockade and restored IRF-3 phosphorylation after cellular challenge with an unrelated virus. Furthermore, dominant-negative or constitutively active IRF-3 mutants, respectively, enhanced or suppressed HCV RNA replication in hepatoma cells. Thus, the NS3/4A protease represents a dual therapeutic target, the inhibition of which may both block viral replication and restore IRF-3 control of HCV infection.     

Complete replication of hepatitis C virus in cell culture

Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10(5) infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-alpha and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.     

Cloned fragment of the hepatitis delta virus RNA genome: sequence and diagnostic application

Hepatitis delta virus (HDV) is a replication-defective etiological agent of hepatitis that requires hepatitis B virus (HBV) as a helper. A complementary DNA (cDNA) fragment of the RNA genome of HDV was cloned into the plasmid vector pBR322, and the primary nucleotide sequence and predicted protein products of the cDNA fragment were determined. This cloned cDNA fragment has been used as a sensitive radioactive probe for the detection of HDV RNA in the serum of patients with either acute or chronic HDV infections.     

Human sarcomas contain RNA related to the RNA of a mouse leukemia virus

Labeled DNA complementary to the RNA of the Rauscher leukemia virus was hybridized with RNA from the polysome fraction of human sarcomas. Eighteen out of 25 specimens contained RNA possessing homology to the RNA of the mouse leukemia virus but not to that of the unrelated viruses causing mammary tumors in mice or myeloblastosis in chickens. Further, no normal adult or fetal tissues showed significant amounts of RNA specific to mouse leukemia virus. It appears that human sarcomas contain RNA sequences homologous to those found in an agent related to a virus known to cause sarcomas in mice.     

丙型肝炎病毒抗体ELISA法弱反应性分析及其确认意义

目的分析抗-HCVELISA法弱反应性结果,减少漏诊误诊。方法采用ELISA法检测抗-HCV,对吸光度值/临界值(S/CO)比值在0.75～3.50之间的标本再采用聚合酶链反应(PCR)进行确认。结果在抗-HCV呈弱反应性即S/CO在0.75～3.50之间的24标本份中,0.75≤S/CO<1.0的标本9份,S/CO≥1.0的标本15份;经PCR确认,有5份阳性,其余19份为阴性,其中4份阳性样本的S/CO<1.0,14份阴性样本的S/CO≥1.0。结论对于弱反应性样本,应进一步做确认检测,以防止漏诊误诊。     

HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection

Natural killer (NK) cells provide a central defense against viral infection by using inhibitory and activation receptors for major histocompatibility complex class I molecules as a means of controlling their activity. We show that genes encoding the inhibitory NK cell receptor KIR2DL3 and its human leukocyte antigen C group 1 (HLA-C1) ligand directly influence resolution of hepatitis C virus (HCV) infection. This effect was observed in Caucasians and African Americans with expected low infectious doses of HCV but not in those with high-dose exposure, in whom the innate immune response is likely overwhelmed. The data strongly suggest that inhibitory NK cell interactions are important in determining antiviral immunity and that diminished inhibitory responses confer protection against HCV.     

Induction and suppression of RNA silencing by an animal virus

RNA silencing is a sequence-specific RNA degradation mechanism that is operational in plants and animals. Here, we show that flock house virus (FHV) is both an initiator and a target of RNA silencing in Drosophila host cells and that FHV infection requires suppression of RNA silencing by an FHV-encoded protein, B2. These findings establish RNA silencing as an adaptive antiviral defense in animal cells. B2 also inhibits RNA silencing in transgenic plants, providing evidence for a conserved RNA silencing pathway in the plant and animal kingdoms.     

Isolation of a neurotropic type C virus

A neurogenic paralysis of the lower limb can be induced and serially transmitted in mice by a nontransforming type C virus strain that originated in an embryo of a wild mouse. The virus exerted a neurotropic effect on the anterior horn neurons.     

一株韩国新型DHV的分离及RT-PCR鉴定

从山东省某疑似鸭肝炎发病区分离到1株病毒JFX08,血清中和试验结果表明该分离毒与传统的Ⅰ型鸭肝炎病毒无交叉保护,分离毒回归3日龄雏鸭,可复制出鸭肝炎的典型症状和病理变化。根据已公布的韩国新型(基因C型)DHV的序列设计合成一对引物,进行RT-PCR扩增,得到大小约414 bp的目的条带;测序分析发现,分离毒与传统Ⅰ型DHV之间的相似性较低,而与韩国新型DHV之间的相似性高达93.2%～94.0%;进化分析结果表明,该分离毒与韩国新型DHV的遗传距离最近,属基因C型DHV。     

Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids

Chronic hepatitis B virus (HBV) infection is a major cause of liver disease. Only interferon-alpha and the nucleosidic inhibitors of the viral polymerase, 3TC and adefovir, are approved for therapy. However, these therapies are limited by the side effects of interferon and the substantial resistance of the virus to nucleosidic inhibitors. Potent new antiviral compounds suitable for monotherapy or combination therapy are highly desired. We describe non-nucleosidic inhibitors of HBV nucleocapsid maturation that possess in vitro and in vivo antiviral activity. These inhibitors have potential for future therapeutic regimens to combat chronic HBV infection.