RNA 干扰技术抑制病毒复制的研究进展

RNA干扰是指dsRNA抑制细胞内同源基因表达的现象,RNA干扰现象自发现以来在短短的几年里,已成功地用于不同种属的生物研究。dsRNA不仅参与内源基因表达调控,而且能够抑制宿主内病原微生物基因的表达,参与构筑生物体的防御机制。近年来,运用RNAi技术在哺乳动物中的研究不断深入,尤其是抑制病毒复制的研究成果令人欣喜,这为人类动物抗病毒治疗提供了新的思路。     

Inferring Host Gene Subnetworks Involved in Viral Replication

Systematic, genome-wide loss-of-function experiments can be used to identify host factors that directly or indirectly facilitate or inhibit the replication of a virus in a host cell. We present an approach that combines an integer linear program and a diffusion kernel method to infer the pathways through which those host factors modulate viral replication. The inputs to the method are a set of viral phenotypes observed in single-host-gene mutants and a background network consisting of a variety of host intracellular interactions. The output is an ensemble of subnetworks that provides a consistent explanation for the measured phenotypes, predicts which unassayed host factors modulate the virus, and predicts which host factors are the most direct interfaces with the virus. We infer host-virus interaction subnetworks using data from experiments screening the yeast genome for genes modulating the replication of two RNA viruses. Because a gold-standard network is unavailable, we assess the predicted subnetworks using both computational and qualitative analyses. We conduct a cross-validation experiment in which we predict whether held-aside test genes have an effect on viral replication. Our approach is able to make high-confidence predictions more accurately than several baselines, and about as well as the best baseline, which does not infer mechanistic pathways. We also examine two kinds of predictions made by our method: which host factors are nearest to a direct interaction with a viral component, and which unassayed host genes are likely to be involved in viral replication. Multiple predictions are supported by recent independent experimental data, or are components or functional partners of confirmed relevant complexes or pathways. Integer program code, background network data, and inferred host-virus subnetworks are available at http://www.biostat.wisc.edu/~craven/chasman_host_virus/.     

Saint Louis Encephalitis Viral Ribonucleic Acid Replication Complex

Pulse-labeled Saint Louis encephalitis viral ribonucleic acid (RNA) is found in the cytoplasm of infected cells associated with a membranous structure which sediments with an average value of 250S. The integrity of the complex is destroyed by detergents and ribonuclease; however, it is stable in ethylenediaminetetraacetic acid (EDTA) which differentiates this structure from cellular polyribosomes. With cultures in which cellular RNA was highly labeled prior to infection, ribosomal RNA could not be demonstrated in the complex isolated from EDTA-sucrose gradients. Single-stranded 43S and the 26S and 20S forms of viral RNA were found in the complex. Viral RNA polymerase activity in sucrose-gradient fractions sedimented in the same region as the fractions which contained the pulse-labeled viral RNA. The polymerase incorporated (3)H-guanosine triphosphate into acid-precipitable material in the absence of added template. It was also found that the replication complex contains viral-specific proteins.     

Cellular Compartmentalization of Herpesvirus Antigens During Viral Replication

HEp-2 cells infected with herpes simplex virus develop five distinct immunofluorescent elements. Three (small nuclear granules, large nuclear granules, and an amorphous mass filling the nucleus) contain antigens which react with a rabbit serum prepared against boiled infected cell debris. A labeled pool of human antibody revealed antigens making up cytoplasmic granules and those responsible for a diffuse cytoplasmic fluorescence. All five immunofluorescent elements are demonstrable with a rabbit serum prepared against unheated infected cell debris. Viral antigens are segregated in the nucleus or in the cytoplasm; within the limits of detection, each antigen accumulates in one compartment only. The antigens responsible for the diffuse cytoplasmic fluorescence and for the amorphous nuclear mass are synthesized early in infection; they are formed in arginine-deprived cells and exist in a form which does not sediment on centrifugation at 79,000 x g for 2 hr. The antigens comprising the nuclear and cytoplasmic granules arise relatively late in infection; they are not formed in arginine-deprived cells, and they are readily sedimented on centrifugation at 79,000 x g for 2 hr. Heating (60 C for 2 hr) confers on one or more cytoplasmic viral antigens a new specificity; the altered antigens are demonstrable with labeled rabbit anti-boiled infected cell serum which normally does not combine with cytoplasmic antigens.     

EWSR1 Binds the Hepatitis C Virus cis-Acting Replication Element and Is Required for Efficient Viral Replication

The hepatitis C virus (HCV) genome contains numerous RNA elements that are required for its replication. Most of the identified RNA structures are located within the 5′ and 3′ untranslated regions (UTRs). One prominent RNA structure, termed the cis-acting replication element (CRE), is located within the NS5B coding region. Mutation of part of the CRE, the 5BSL3.2 stem-loop, impairs HCV RNA replication. This loop has been implicated in a kissing interaction with a complementary stem-loop structure in the 3′ UTR. Although it is clear that this interaction is required for viral replication, the function of the interaction, and its regulation are unknown. In order to gain insight into the CRE function, we isolated cellular proteins that preferentially bind the CRE and identified them using mass spectrometry. This approach identified EWSR1 as a CRE-binding protein. Silencing EWSR1 expression impairs HCV replication and infectious virus production but not translation. While EWRS1 is a shuttling protein that is extensively nuclear in hepatocytes, substantial amounts of EWSR1 localize to the cytosol in HCV-infected cells and colocalize with sites of HCV replication. A subset of EWRS1 translocates into detergent-resistant membrane fractions, which contain the viral replicase proteins, in cells with replicating HCV. EWSR1 directly binds the CRE, and this is dependent on the intact CRE structure. Finally, EWSR1 preferentially interacts with the CRE in the absence of the kissing interaction. This study implicates EWSR1 as a novel modulator of CRE function in HCV replication.     

Replication of the Parvovirus MVM II. Isolation and Characterization of Intermediates in the Replication of the Viral Deoxyribonucleic Acid

The time course of the appearance of intracellular viral DNA has been studied in mouse L cells infected with the single-stranded DNA virus MVM (minute virus of mice) by using a selective extraction procedure. Approximately half of this DNA elutes from hydroxyapatite as single-stranded DNA. It is sensitive to Escherichia coli exonuclease I and shows a sedimentation profile similar to DNA from the virus, suggesting that it is progeny viral DNA. The remainder of the selectively extracted DNA elutes from hydroxyapatite in the position of double-stranded DNA and is resistant to exonuclease I. Most of this DNA has a sedimentation coefficient of 14 to 16S, indicating that its molecular weight is twice that of the viral DNA. Denaturation renders the majority of the double-stranded DNA sensitive to exonuclease I, but a significant fraction renatures spontaneously in a monomolecular fashion, indicating that it has a cross-linked or hairpin structure. Chromatography of the double-stranded DNA on benzoylated diethylaminoethyl cellulose resolves two components, one with duplex structure and one which contains single-stranded regions. A short pulse label late in infection predominantly labels the latter class of DNA, suggesting that it contains replicating intermediates. The possible roles of these various forms of DNA in the replication of the viral genome are discussed.     

Digoxin Suppresses HIV-1 Replication by Altering Viral RNA Processing

To develop new approaches to control HIV-1 replication, we examined the capacity of recently described small molecular modulators of RNA splicing for their effects on viral RNA metabolism. Of the drugs tested, digoxin was found to induce a dramatic inhibition of HIV-1 structural protein synthesis, a response due, in part, to reduced accumulation of the corresponding viral mRNAs. In addition, digoxin altered viral RNA splice site use, resulting in loss of the essential viral factor Rev. Digoxin induced changes in activity of the CLK family of SR protein kinases and modification of several SR proteins, including SRp20 and Tra2β, which could account for the effects observed. Consistent with this hypothesis, overexpression of SRp20 elicited changes in HIV-1 RNA processing similar to those observed with digoxin. Importantly, digoxin was also highly active against clinical strains of HIV-1 in vitro, validating this novel approach to treatment of this infection.     

Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal brain stem encephalitis complicated with pulmonary edema. Several severe EV71 endemics have occurred in Asia-Pacific region, including Taiwan, and have become a serious threat to children’s health. EV71 infection is initiated by the attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between viruses and cell surface receptors, soluble factors may also influence the binding of EV71 to host cells.Galectin-1 has been reported to participate in several virus infections, but is not addressed in EV71. In this study, we found that the serum levels of galectin-1 in EV71-infected children were higher than those in non-infected people. In EV71 infected cells, galectin-1 was found to be associated with the EV71 VP1 and VP3 via carbohydrate residues and subsequently released and bound to another cell surface along with the virus. EV71 propagated from galectin-1 knockdown SK-N-SH cells exhibited lower infectivity in cultured cells and less pathogenicity in mice than the virus propagated from parental cells. In addition, this galectin-1-free EV71 virus was sensitive to high temperature and lost its viability after long-term storage, which could be restored following supplement of recombinant galectin-1. Taken together, our findings uncover a new role of galectin-1 in facilitating EV71 virus infection.     

Simian Virus 40 DNA Replication in Isolated Replicating Viral Chromosomes

Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for "normal" DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of "Okazaki pieces," and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated alpha-(32)P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.     

Aquareovirus NS80 Initiates Efficient Viral Replication by Retaining Core Proteins within Replication-Associated Viral Inclusion Bodies

Viral inclusion bodies (VIBs) are specific intracellular compartments for reoviruses replication and assembly. Aquareovirus nonstructural protein NS80 has been identified to be the major constituent for forming globular VIBs in our previous study. In this study, we investigated the role of NS80 in viral structural proteins expression and viral replication. Immunofluorescence assays showed that NS80 could retain five core proteins or inner-capsid proteins (VP1-VP4 and VP6), but not outer-capsid proteins (VP5 and VP7), within VIBs in co-transfected or infected cells. Further co-immunoprecipitation analysis confirmed that NS80 could interact with each core protein respectively. In addition, we found that newly synthesized viral RNAs co-localized with VIBs. Furthermore, time-course analysis of viral structural proteins expression showed that the expression of NS80 was detected first, followed by the detection of inner shell protein VP3, and then of other inner-capsid proteins, suggesting that VIBs were essential for the formation of viral core frame or progeny virion. Moreover, knockdown of NS80 by shRNA not only inhibited the expression of aquareovirus structural proteins, but also inhibited viral infection. These results indicated that NS80-based VIBs were formed at earlier stage of infection, and NS80 was able to coordinate the expression of viral structural proteins and viral replication.     

The Universal Epitope of Influenza A Viral Neuraminidase Fundamentally Contributes to Enzyme Activity and Viral Replication

The only universally conserved sequence among all influenza A viral neuraminidases is located between amino acids 222 and 230. However, the potential roles of these amino acids remain largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics, and growth kinetics, we found that this sequence could markedly affect viral replication. Additional experiments revealed that enzymes with mutations in this region demonstrated substantially decreased catalytic activity, substrate binding, and thermostability. Consistent with viral replication analyses and enzymatic studies, protein modeling suggests that these amino acids could either directly bind to the substrate or contribute to the formation of the active site in the enzyme. Collectively, these findings reveal the essential role of this unique region in enzyme function and viral growth, which provides the basis for evaluating the validity of this sequence as a potential target for antiviral intervention and vaccine development.     

Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions.     

Herpes Simplex Virus and Human Cytomegalovirus Replication in WI-38 Cells I. Sequence of Viral Replication

A comparison, under standardized conditions, of herpes simplex virus (HSV) and human cytomegalovirus (CMV) revealed differences in viral morphology, in the timing of their infectious cycles, and in several morphological events during those cycles. Structural distinctions between the two viruses included the coating of unenveloped cytoplasmic CMV capsids, but not those of HSV, and a variation in the structure of their cores. Since the two cycles were carried out in the same host cell strain under conditions of one-step growth (input multiplicity = 10 PFU/cell), it was possible to construct time scales locating the major events of each cycle. Comparison of the two showed that HSV replicated and released progeny within 8 h postinfection, whereas CMV required 4 days. These results correlated well with those of concurrent plaque assays. Within the longer CMV cycle, most of the major events appeared retarded to a similar degree, and no obvious limiting step in particle production could be identified. Distinctions between the two cycles included the following: condensation of the chromatin in HSV- but not CMV-infected cells; the greater tendency of HSV to produce membrane alterations; and the appearance of cytoplasmic dense bodies in CMV- but not HSV-infected cells. Identification of these differences even under identical conditions of culture and infection strongly implies that they result from intrinsic differences in the nature of the viruses, and are not caused by variations in experimental conditions.