通过基因组定量研究猪瘟病毒在细胞中的增殖特性

应用间接免疫荧光、Real-time PCR和病毒感染滴度(TCID50)测定技术,分别从病毒抗原、病毒基因组RNA复制水平和病毒感染滴度变化3个方面,研究了猪瘟病毒(CSFV)在PK-15细胞中增殖的特点,用猪瘟病毒石门株感染96孔板培养的细胞,1×102个TCID50/孔,间接免疫荧光检测结果显示感染后8h能检测到被荧光抗体染色的感染细胞,随感染时间的延长,出现荧光的细胞数量逐渐增多,在感染后72h,几乎所有细胞均能出现荧光。Real-time PCR结果显示在细胞感染初期的8~24h,病毒的基因组RNA复制呈加速趋势,其拷贝数在感染后72h达到高峰。此外,在感染后8h能检测到病毒基因组负链RNA转录,不过负链RNA在病毒增殖过程中维持在较低的水平。TCID50测定结果表明CSFV的感染滴度增加趋势与基因组类似,在病毒感染8h后能检测到具有感染性的子代病毒,感染滴度在8~20h之间逐渐增长,24~48h之间增长速度稍减慢,在感染后48~52h达到高峰,能在72h之内维持较高的感染滴度。     

Hepatitis C Virus Particle Assembly Involves Phosphorylation of NS5A by the c-Abl Tyrosine Kinase

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr³³⁰ (Tyr²³⁰⁶ in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr³³⁰.     

Increase in intracisternal A-type particles in Friend cells during inhibition of Friend virus (SFFV) release by interferon or azidothymidine.

Interferon or azidothymidine inhibition of Friend virus (FV-SFFV) release in Friend erythroleukemic cells results in a 10–20-fold increase in intracisternal A-type particle number within 3–4 days of treatment. Inhibition of Friend or Moloney helper virus by interferon in fibroblast producer cells does not result in a similar increase in intracisternal A-type particles. Friend cells with marginal FV production but with high levels of A-type particles do not change their A-type particle levels upon exposure to interferon. These data suggest that A-type particle expression in Friend cells may be linked to the presence of the transforming SFFV. No marked increase of viral RNA levels during inhibition of virus release is observed in Friend cells.     

Regulation of hepatitis C virion production via phosphorylation of the NS5A protein

Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to uncouple virus production from RNA replication, as described herein, may be useful in understanding HCV assembly and may be therapeutically important.     

Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles

Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433) impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interaction and virion production, suggesting that phosphorylation of these serine residues is important for virion production. In addition, we found that the alanine substitutions in the serine cluster suppressed the association of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly. These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core interaction.     

Effect of 5'-deoxy-5'-S-isobutyl adenosine (SIBA) on the synthesis of polyoma virus constituents in infected mouse cells

At 100 microM 5'-S-isobutyladenosine (SIBA) inhibits polyoma virus production in infected mouse embryo fibroblasts and in mouse kidney cells, as measured by plaque formation and by haemagglutination assays. SIBA has no significant effect on the synthesis of T and V antigens as well as on viral DNA synthesized in infected cells. Analysis of virus production on CsCl gradients on CsCl gradients showed that in the presence of SIBA reduced amount of heavy viral particles is produced and that part of these particles are pseudovirions containing low density DNA instead of supercoiled viral DNA.     

Effect of butyrate on the expression of the human preprourokinase gene introduced into Chinese hamster ovary cells

A plasmid containing the human preprourokinase gene cDNA under the control of the simian virus 40 early region promoter was introduced into CHO-K1 cells and recombinant cell lines secreting a relatively high level of urokinase were obtained. In the course of studying the effects of various agents on the recombinant cell lines, we found that exposure of recombinant cells to 5 mM butyrate for 24 hours resulted in a 2-3 fold increase in urokinase production. The induction by butyrate was dose-dependent. The half maximal dose was approximately 2 mM; maximal stimulation occurred at 5-10 mM. Cell growth, on the other hand, was inhibited by butyrate concentrations greater than 2.5 mM. The response of cells to butyrate was rapid: a significant increase in urokinase production was observed 6 hours after exposure to 5 mM butyrate. Butyrate treatment increased not only the extracellular level but also the intracellular level of urokinase.     

Inhibition of virus production in peripheral blood mononuclear cells from human immunodeficiency virus (HIV) type 1-seropositive donors by treatment with recombinant HIV-like particles.

We have previously reported on the assembly of recombinant human immunodeficiency virus (HIV)-like particles that contain gag structural proteins and present env glycoproteins gp120 and gp41 on their surfaces (O. Haffar,. J. Garriques, B. Travis, P. Moran, J. Zarling, and S.-L. Hu, J. Virol. 64:2653-2659, 1990). On the basis of their structures, we hypothesized that the recombinant particles would interfere with virus infection and tested our hypothesis in vitro by using peripheral blood mononuclear cells (PBMC) from HIV type 1-seropositive donors. Addition of the recombinant particles to PBMC concomitant with stimulation by anti-CD3 inhibited virus production, as determined by reduced levels of p24 in the culture supernatants. This inhibition of p24 production correlated with lower levels of cell-associated viral DNA. Several lines of evidence suggested that the recombinant particles exerted their antiviral effects primarily by inhibiting virus production from latently infected cells and not by inhibiting subsequent virus spread. Importantly, CD4+ T-cell stimulation by specific antigen or by anti-CD3 was not inhibited by treatment with the recombinant particles. This apparent selective inhibition of virus replication in infected PBMC represents a novel property of the recombinant HIV-like particles.     

RacGTPase-activating protein 1 interacts with hepatitis C virus polymerase NS5B to regulate viral replication

Hepatitis C virus (HCV) is a positive-strand RNA virus responsible for chronic liver disease and hepatocellular carcinoma (HCC). RacGTPase-activating protein 1 (RacGAP1) plays an important role during GTP hydrolysis to GDP in Rac1 and CDC42 protein and has been demonstrated to be upregulated in several cancers, including HCC. However, the molecular mechanism leading to the upregulation of RacGAP1 remains poorly understood. Here, we showed that RacGAP1 levels were enhanced in HCV cell-culture-derived (HCVcc) infection. More importantly, we illustrated that RacGAP1 interacts with the viral protein NS5B in mammalian cells. The small interfering RNA (siRNA)-mediated knockdown of RacGAP1 in human hepatoma cell lines inhibited replication of HCV RNA, protein, and production of infectious particles of HCV genotype 2a strain JFH1. Conversely, these were reversed by the expression of a siRNA-resistant RacGAP1 recombinant protein. In addition, viral protein NS5B polymerase activity was significantly reduced by silencing RacGAP1 and, vice versa, was increased by overexpression of RacGAP1 in a cell-based reporter assay. Our results suggest that RacGAP1 plays a crucial role in HCV replication by affecting viral protein NS5B polymerase activity and holds importance for antiviral drug development.     

Production of avian oncoviral subgroups after multiple infection.

The number of different oncoviral env genes that can be expressed by a single chicken embryo fibroblast was investigated. Fibroblasts were infected with one to three subgroups of Rous-associated virus, which is a nontransforming avian oncovirus, then superinfected with a transforming virus, Rous sarcoma virus, of a different subgroup. The subgroups of viruses released by the resulting clones were analyzed. When two viral subgroups were used for preinfection, all the resulting clones produced transforming virus particles having the subgroup of the superinfecting virus, and most clones produced transforming virus particles of all the infecting viral subgroups. However, when cells were preinfected with three viral subgroups, many of the resulting clones did not produce transforming virus particles having the subgroup of the superinfecting virus, and only 1 of 23 clones produced transforming particles of all the infecting viral subgroups. DNA annealing experiments showed that cells infected with three or four viral subgroups had an additional 8 to 20 copies of proviral DNA per cell. Finally, most clones resulting from cells simultaneously infected with three or four viral subgroups were able to produce virus of all infecting subgroups. It appears that the number of exogenous oncoviral env genes that can be expressed by a single cell is limited, and in the range of 4 to 8-20 per cell.     

MDA5 participates in the detection of paramyxovirus infection and is essential for the early activation of dendritic cells in response to Sendai Virus defective interfering particles

Defective interfering (DI) particles are byproducts of virus replication that potently enhance dendritic cell (DC) maturation by virus infection. DI particles have been reported for many different viruses and are strong inducers of type I IFNs. The cellular mechanisms involved in the response to DI particles are not known. In this study, we show that 1) DI particles are recognized by more than one viral sensor independently of TLRs and type I IFN signaling; 2) The helicase MDA5 participates in the detection of DI genomes as MDA5-deficient DCs respond inefficiently to Sendai virus stocks containing DI particles; 3) DI particles stimulate the expression of IRF3-responsive genes by a uniquely potent mechanism when compared with other prototypic viral stimulus; and 4) the efficient detection of DI particles overcomes virus immune antagonism. These data highlight the outstanding adjuvant capacity of DI particles in stimulating mouse and human DCs. They also offer biological relevance to the previously reported inhibition of MDA5 by different paramyxovirus V proteins. The unique mechanism by which DI particles trigger the maturation of DCs represents a novel strategy that could be further exploited for the development of potent adjuvant molecules.     

高滴度感染力H5N1亚型禽流感病毒假病毒颗粒的制备

利用一个瞬时共转染系统,将H5N1亚型禽流感病毒的血凝素（Hemagglutinin,HA）蛋白与神经氨酸酶（Neuraminidase,NA）蛋白整合到鼠白血病病毒假病毒颗粒表面,包装成表达HA与NA的假病毒颗粒,通过透射电子显微镜形态学观察、感染滴度分析、血凝试验和中和试验研究其生物学特性。研究获得了高滴度感染力的H5N1假病毒颗粒（H5N1 Pseudotyped particle,H5N1Pp）,H5N1Pp的感染力滴度为1E8 Pp/mL,形态、血凝活性及中和特性均与野生H5N1病毒相似,结果为H5N1病毒受体、HA与NA的功能、中和抗体、抗病毒药物开发研究的开展建立了平台。     

Cycloleucine blocks 5'-terminal and internal methylations of avian sarcoma virus genome RNA.

Cycloleucine, a competitive inhibitor of ATP: L-methionine S-adenosyltransferase in vitro, has been used to reduce intracellular concentrations of S-adenosylmethionine and by this means to inhibit virion RNA methylation in chicken embryo cells that are infected with B77 avian sarcoma virus. Under conditions of cycloleucine treatment, where virus production as measured by incorporation of radioactive precursors or by number of infectious particles is not significantly affected, the internal m6A methylations of the avian sarcoma virus genome RNA are inhibited greater than 90%. The predominant 5'-terminal structure in viral RNA produced by treated cells in m7G(5')pppG (cap zero) rather than m7G-(5')pppGm (cap 1). It appears from these results that internal m6A and penultimate ribose methylations are not required for avian sarcoma RNA synthesis and function. Furthermore, these methylations are apparently not required for transport of genome RNA to virus assembly sites. The insensitivity of the 5'-terminal m7G methylation to inhibition by cycloleucine suggests that the affinity of S-adenosylmethionine for 7-methylguanosine methyltransferase is significantly greater than for the 2'-0-methyltransferases or the N6-methyltransferases.     

Glycoprotein enrichment in Moloney leukemia virus structural proteins released from interferon-treated cells

Interferon treatment of Moloney-leukemia-virus-infected cells (3T3/MLV) leads to the formation of virus particles enriched with viral structural glycoproteins, in addition to the inhibition of virus production. A preferential inhibitory effect on incorporation of RNA and proteins rather than glycoproteins was found in the released virus particles from interferon-treated cells. Enrichment in 70,000- and 45,000-dalton glycoprotein (gP-70, gP-45) in these particles was further demonstrated by polyacrylamide analysis of viral proteins pulse-labeled with [3H]-leucine. Viral glycoproteins released as soluble antigens were also determined. A 40% reduction was found in gP-70 and gP-45 released from interferon-treated cells. Radioimmunoprecipitation of pulse-chase-labeled cellular viral proteins showed no effect of interferon on the formation of viral structural 30,000-, 15,000- to 12,000-dalton proteins, and gP-70 and gP-45 from their respective precursors. The uncoordinate effect of interferon inhibition on viral 30,000-dalton protein and gP-70 is discussed.     

Cis effect of the type 5 adenovirus E1A gene enhancer element on cellular transformation

Mutants of type 5 adenovirus that lack all or part of the early region 1A (E1A) gene enhancer element transform rodent embryo fibroblast (CREF) cells at higher efficiencies than wild-type virus. An analysis of viral E1A cytoplasmic mRNA levels in mutant and wild-type virus-infected CREF cells revealed no differences in the levels of the E1A mRNAs. This implies that a decrease in the rate of viral E1A gene expression was not responsible for the transforming properties of the enhancer-less viruses. Unlike wild-type virus, however, the mutant viruses were able to replicate their genomes in the normally nonpermissive CREF cells. This change in viral DNA template concentration further resulted in an increase in early gene mRNA concentrations in mutant-virus-infected CREF cells. These studies suggest several possible mechanisms that could be responsible for the increased transforming potentials of these viruses, including 1) a cis effect of removing the viral E1A enhancer element on the efficiency of viral DNA integration, 2) viral DNA replication, or 3) an increase in the levels of the viral E1A and E1B mRNAs owing to viral DNA replication in the virus-infected CREF cells.     

Effects of temperature and antibody on the cyclid growth of vesicular stomatitis virus.

To see the effects of temperature on the interrelated cyclic production of standard and defective interfering (DI) particles of vesicular stomatitis virus, a temperature-sensitive (ts) G114 mutant was passaged successively at different temperatures and the production of the two types of viral particles as well as the ability of Chinese hamster ovary cells to survive each passage was continuously monitored. When the temperature was nonpermissive for standard virus, the synthesis of both standard and defective interfering particles was inhibited. When revertants appeared in the population, their ability to take over the infection depended on the permissiveness of the temperature for the temperature-sensitive mutant. At permissive temperatures periodic inhibition of both types of standard viruses was maintained by the production of defective interfering particles. Reverents did not become a majority of the population due to this periodic inhibition. When the conditions were nonpermissive for the mutant, revertants became the major standard virus in the population within a few passages. These findings can be understood if conditions of high and low multiplicities are dissected out together with a thorough understanding of the individual properties of each of the viral particles and of the result of interactions between them. In the presence of antiserum which neutralized only 90% of the viral particles, cyclic production of standard virus occurred, with a decline in the total amount of virus produced after each cycle. Therefore, in the presence of limiting concentrations of antiserum, the virus appeared to be able to establish a persistent cyclic growth pattern.     

Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

The triglyceride-synthesizing enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) plays a critical role in hepatitis C virus (HCV) infection by recruiting the HCV capsid protein core onto the surface of cellular lipid droplets (LDs). Here we find a new interaction between the non-structural protein NS5A and DGAT1 and show that the trafficking of NS5A to LDs depends on DGAT1 activity. DGAT1 forms a complex with NS5A and core and facilitates the interaction between both viral proteins. A catalytically inactive mutant of DGAT1 (H426A) blocks the localization of NS5A, but not core, to LDs in a dominant-negative manner and impairs the release of infectious viral particles, underscoring the importance of DGAT1-mediated translocation of NS5A to LDs in viral particle production. We propose a model whereby DGAT1 serves as a cellular hub for HCV core and NS5A proteins, guiding both onto the surface of the same subset of LDs, those generated by DGAT1. These results highlight the critical role of DGAT1 as a host factor for HCV infection and as a potential drug target for antiviral therapy.